Rusak 1979

Physiological Reviews
Published and Copyright by

The American Physiological Society Vol. 59, No. 3, July 1979
Neural Regulation of Circadian Rhythms

BENJAMIN RUSAK AND IRVING ZUCKER
Department of Psychology, Dalhousie University, Halifax, Nova Scotia, Cam&; a&

Department of Psychology, University of California, Berkeley, California
I. Introduction ......................................................... 449
A. Background and scope ............................................. 449
B. Historical perspective ............................................. 450
II. Entrainment ......................................................... 452
A. Extraretinal light entrainment ..................................... 452
B. Retinal light entrainment .......................................... 459
C. Nonphotic entrainment ........................................... 463
III. Rhythm Generation .................................................. 466
I
A. Invertebrates .................................................... 466
B. Vertebrates ...................................................... 479
IV. Mammalian Reproductive Cycles ...................................... 500
A. Estrous and menstrual cycles ...................................... 500
B. Male seasonal cycles ............................................... 512
V. Prospectus .......................................................... 513
I. INTRODUCTION
A. Background and Scope
Rhythmicity is a fundamental property of living matter-from single cells

to complex animals, innumerable structures and functions undergo periodic
changes in shape or rate (71). Endogenous fluctuations in motor and sensory
capacity influence the integrative capacities of animals. The role of such rhythms
in temporal organization ranks with homeostasis as a device for increasing
fitness. Circadian rhythms (CRs) have major adaptive significance because they
help synchronize the organism to periodic fluctuations in the external environ-
ment and also facilitate integration of the individual’s internal milieu. To para-
phrase Aschoff (13), CRs establish a mirror of the changing external world in the
internal milieu and thereby prepare the organism for programed or predictable
environmental changes.
0031-9333/79/000000-00$01.25 Copyright 0 1979 the American Physiological Society 449
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Copyright © 1979 American Physiological Society. All rights reserved.
450 BENJAMIN RUSAK AND IRVING ZUCKER Volume 59
Circadian rhythms are endogenous oscillations with a period of approxi-

mately 24 h. They are distinguishable from the majority of other biological
oscillations (e.g., cardiovascular, respiratory, reproductive) by the corre-
spondence of their period to that of a major environmental cycle, the solar
day (13). Not all 24-h biological cycles are CRs. Some daily periodicities result
from regular environmental fluctuations in temperature, light, humidity, etc.;
these exogenously driven biological cycles disappear when the environment is
held constant. The term circadian is restricted here to those approximately
24-h biological rhythms whose cyclicity persists (free-runs) under constant
conditions. The terms diel, diurnal, and nocturnal more accurately describe
24-h cycles whose endogenous nature is in doubt. Our main concern is with CRs,
although we treat die1 rhythms where circumstantial evidence suggests they are
circadian.
The growing interest of both neuroscientists and chronobiologists in the
neural mechanisms underlying circadian phenomena is reflected in the appear-
ance of several recent reviews (51, 62, 239, 251, 296). The present review
includes essentially all animal species for which significant information exists,
but this survey is necessarily limited by the scope of our interests and’ com-
petence. Although we have included data on both invertebrate and vertebrate
species, our emphasis throughout is on mammals. Only a brief historical review
is provided as background to the current literature.
B. Historical Pempective
The acceptance of circadian rhythmicity as a property of living organisms

owes much to the work of eighteenth and nineteenth century plant physiologists.
Their efforts forced the conclusion that at least some 24-h biological rhythms
are not derived from environmental periodicities but rather are endogenous (71).
Investigation of the neural basis for CRs had to await the irrefutable docu-
mentation of similar phenomena in multicellular animals. Richter (314) was
among the f!irst to remark on the persistence of die1 rhythms of activity in a mam-
mal (the common rat) housed in constant darkness (DD). It was, however,
Johnson’s landmark study (171) with wild-caught Peromyscus leucopus that
anticipated future developments by about three decades. His animals were
active during the dark phase of the solar day; in DD they displayed “marked
periodicity of activity but the time of activity no longer corresponded to the
time of darkness outside.” He concluded that the activity rhythm was not directly
dependent on the daily fluctuation of environmental conditions and was an
expression of an internal physiological rhythm. Johnson subsequently suggested
that “the animal has an exceptionally substantial and durable self-winding and
self-regulating physiological clock, the mechanism of which remains to be
worked out” (172, p. 326).
The call for the “working out” of the clock mechanism remained un-
answered from the 1920’s until the 1960’s. Isolated investigations of diurnal
rhythmicity subsequent to various neural interventions were reported in the
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Julg am? NEURAL REGULATION OF CIRCADIAN RHYTHMS 451
intervening decades. Rothmann (cited in 184) maintained that the 24-h rhythm
of sleep-wakefulness survived decortication in a dog. Kleitman and Camille (184)
on the other hand concluded that the diurnal sleep-wakefulness cycle of dogs
depended on the presence of a fu nctionally active cerebral cortex and noted
that decorticate dogs reverted to a polyphasic existence characteristic of young
puppies. Lemkuhl is cited (183) as demonstrating that extirpation of the cerebrum
dimini .shed.activity lev pelsand n.ormal periodicity in pigeons. Removal of various
parts of the nervous system of an insect (Carausius morosus) reportedly
disrupted the 24-h rhythm (100). Mason described disturbances in die1 cortisol
rhythms in monkeys after damage to the hippocampal-fornix system (232). The
daily rhythm in pituitary ACTH content was shown to persist after supratha-
lamic ablation of the mouse brain; lesions that included the thalamus and hypo-
thalamus interfered with this rhythm (123). Neither systematic nor conclusive
by modern criteria, this work did not progress to the stage of answering basic
questions about the role of the central nervous system (CNS) in CRs.
Beginning in the 1950’s the prevailing xeitgeist facilitated acceptance of a
preeminent role for the nervous systemin CR generation. Pittendrigh is quoted
as stating that “the elaborate chronometers of birds, bees, and crustaceans
are all in the nervous system and probably depend on neurological organiza-
tion for some of their unique complexity” (118). In a related vein Halberg et al.
(138) stated that “a hypothalamic oscillator . . . driven from within (by feed-
back) and triggered from without, may well control certain 24-h rhythms of the
body as a whole.” These prophetic speculations did not derive from a substantive
empirical base. One could reasonably defend the position that a localized center
for CRs was an oversimplification (141). The presence of CRs in organisms
without nervous systems and their reported persistence in in vitro preparations
of nonneural mammalian tissue (10) did little to encourage examination of the
CNS as the major source of circadian rhythmicity. Most prevalent perhaps
was the view expressed by Harker (141) that there is a basic 24-h rhythm
present in the cells of all animals and that any cell or group of cells could
constitute a circadian clock.
Most neurobiologists equipped to tackle the problem of neural participation
in CRs were largely unaware of the phenomena waiting to be explained;
unfortunately those chronobiologists conversant with the phenomenology were
not neurally inclined in their experimentation.
The current interest among neurobiologists in circadian phenomena is
attributable to several recent developments: the description of circadian
rhythmicity in isolated single neurons and in isolated eyes of the marine mollusk
Aplysia caZifomica (157, 376) and the identification of the suprachiasmatic
nuclei (SCN) of the mammalian hypothalamus as putative circadian oscillators
(252, 370). The reports of Harker (142, 143), which stipulated the cockroach
subesophageal ganglion as a source of CRs, were subsequently not substantiated
(see p. 471) but did much to stimulate research on the neural substrate of insect
CRs. Richter’s investigations (315,316) had earlier opened the modern era of re-
search on mammalian neural clocks.
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Three major issues currently attract the attention of neuroscientists inter-

ested in circadian clocks: 1) anatomical localization and biochemical characteri-
zation of circadian pacemakers in the CNS, 2) specification of the mechanisms
by which central clocks are linked or coupled with the myriad processes (in
endocrine and skeletal motor systems) they control, and 8) determination of the
neural pathways and chemical reactions by which central clocks are entrained
by the external environment. Since the vast majority of CRs are entrainable
by the light-dark (LD) cycle, the description of the transduction process and
the determination of pathways by which photic information gains access to
central pacemakers are main concerns.
II. ENTRAINMENT
During entrainment the period (T) of a biological rhythm takes on the

period (T) of the entraining stimulus. For a CR to be stably entrained there
must be a daily phase shift equal to the difference in period length between
7 and T: with 7 > T a daily phase advance must be effected; when 7 < T the
CR must be phase delayed (294). The pacemakers underlying CRs are of neces-
sity differentially sensitive to xeitgebers (entraining cues) at different stages of
their cycles. Phase-response curves (PRCs) describe the phase dependence of
xeitgeber effects and help elucidate various features of entrainment.
Light is the principal entraining agent for CRs. In nature the period of the
day-night cycle is constant at T = 24 h although the duration of the light phase
and the intensity of illumination are among several parameters that vary on a
seasonal and daily basis. The overwhelming majority of laboratory studies have
used photoperiods ,with abrupt transitions from light to dark (no dawn or dusk
and no continuous change in light intensity) but there is no reason to doubt the
validity of the main conclusions obtained with these methods. Much has been
learned about the formal properties of the entrainment process (297) but we
remain in relative ignorance of the physiological mediation of entrainment.
The precise receptors for entrainment of a given CR are not known, nor is there
much information on the photopigments that transduce light information into
neural signals.
A. Extraretinal Light Entrainment
Among invertebrates and. nonmammalian vertebrates the existence and

functional significance of extraocular photoreceptors (EOPs) for entrainment of
CRs are well established. Extraocular photoreceptors are widely distributed
taxonomically and have evolved independently several times (1). They are pre-
sumably useful in insects for monitoring the LD cycle during metamorphosis
when larval external receptors are destroyed and replaced with adult structures.
They may play a similar role in amphibians, where they also are of potential
significance for the perception of polarized light (1). Underwood (399) speculated

July 19?‘9 NEURAL REGULATION OF’ CIRCADIAN RHYTHMS 453
that EOPs may have provided the receptors for entrainment of CRs prior to the
evolution of lateral eyes.
I. Invertebrates
Truman (393) and Brady (62) distinguish two classes of insect clocks that
differ in several respects, including the mechanisms of their entrainment by
light. One type is exemplified by the clock gating silkmoth eclosion (see p. 476);
the photoreceptor is extraocular and resides in a brain locus either closely
associated with or identical to that of the driving oscillator (394). The second
type is exemplified by the clock controlling cockroach activity rhythms (see
p. 471); it includes an ocular photoreceptor coupled neurally to a central oscillator
localized in each optic lobe of the brain (270, 271). In cockroaches, unlike silk-
moths, disconnection of the compound eyes from the brain prevents entrainment
(at least in moderate light intensities), so the clock mechanism does not appear
to be directly light sensitive (62).
Reports of extraocular or ocellar-mediated entrainment of behavioral
rhythms in roaches (e.g., 21) and related species may be predicated on inadequate
control procedures (62). However, in one orthopteran species [Ephippiger sp.
(94)] entrainment of activity seems to involve both extraocular (cephalic) photo-
receptors and the compound eyes. In addition, the silkmoths entrain flight
activity rhythms via a cephalic photoreceptor-clock complex, and neither the
eyes nor the ocelli are involved (395). The insects therefore present a diverse
array of entrainment mechanisms that cannot be described by a simple rule
relating to types of rhythm. Information on extraretinal entrainment in insects
has been reviewed in detail by Truman (395a).
Entrained crayf&h (Procambavw clarlcii) have a bimodal activity rhythm;
the activity peak that occurs early in the dark phase (D) is a typical CR entrain-
able via EOPs. Covering the eyes, bilateral ablation of the ommatidia, elimina-
tion of the lamina ganglionaris, or excision of the caudal ganglion are all con-
sistent with photic entrainment (288). Apparently EOPs are localized extra-
caudally and are not confined to the opticlobe, nor is the optic stalk necessary
for entrainment. Higher light intensities are needed to entrain the electro-
retinogram (ERG) when light impinges on the retina than when it falls on the
brain; extraretinally localized light is sufficient for entrainment. The EOPs have
not been localized and ablated, so it is not known whether the retinal pathway
is sufficient for entrainment. Page and Larimer (288) speculate that the EOPs
for locomotion and for the ERG are in the supraesophageal ganglion, although
they do not exclude other CNS regions (optic lobe) from participation. It re-
mains unclear whether there exist multiple extraretinal pathways for entrain-
ment and whether entrainment of different rhythms is mediated by separate EOPs.
Sensitivity of the median eyes to light in the scorpion (Androctones
austraZis) is under circadian control and undergoes a 3-log-unit peak-to-trough
change each cycle. The comparable change in the lateral eyes is only 0.3 log

units (117). In this strictly night-active species the receptors in the two eyes
and their associated visual systems supply the CNS with different afferent
information about the time course of ambient light. Fleissner (117) suggests
that the lateral eyes may provide information about the time course of dusk
whereas the median eyes could mediate dawn and dusk transition phases. Com-
parison of inputs e,manating from these different receptors could be of signifi-
cance for entrainment.
The locomotor activity of intact Aplysia is diurnal, with activity onset
preceding L,, in LD 12:12. Bilateral eye removal eliminates anticipation of the L
phase but still permits synchronization (not necessarily entrainment) of activity
to the onset of low light intensity. This photic response persists for over 90 days
and does not depend on social communication of intact with eyeless animals (212).
The fact that eye removal eliminates anticipation of the L phase suggests that
the eyes are part of the oscillator complex controlling activity (211,212). Photic
driving of activity after eye removal demands extraocular photoreceptors, as
does evidence that the rhythm of neural activity in the eye can be entrained
by an extraocular receptor (48), although the eye’s rhythm can also be entrained
directly by light in vitro (104). Extraocular receptors can also mediate entrain-
ment of activity rhythms in another mollusk, the slug Lirnux wuzximus (117).
The circadian oscillators in the two eyes ofA@ysia are not strongly coupled
(154) and photoreceptors in one eye are not effective in entraining the contra-
lateral eye (211). There are many photoreceptors inApZysia besides those in the
eye; these include receptors in the skin of the mantle, the abdominal and cerebral
ganglia, and the oral tentacles. Lickey et al. (211) suggest that entrainment
of the various circadian oscillators in this animal (and thereby mutual synchroni-
zation) is effected by the same external xeitgeber (light) and that a mechanism
of internal coupling between oscillators need not be invoked.
2. Amphibians
The frontal organ and the pineal body of most amphibian species retain
neural connections with the brain and have elements resembling the photo-
receptive cells of the lateral eyes (1). The pineal is sensitive to visible and ultra-
violet light. The frontal nerve that connects to the pineal and also innervates
the frontal organ responds to chromatic and achromatic light. These organs
are well constituted to serve as EOPs for entrainment (1).
Most amphibians are mainly nocturnal in their activity and in a number of
species CR entrainment persists in eyeless individuals. The frontal organ of frogs
may contain the photoreceptors for entrainment of CRs. In newts either the
EOP resides in the pineal body itself or the pineal is necessary for entrainment
for some other reason (1). Entrainment in some amphibians occurs via ocular
and extraocular receptors. Frogs are one of the very few vertebrates in which
pineal (frontal organ) involvement in entrainment has been established (1).

Jdfj 1979 NEURAL REGULATION OF CIRCADIAN RHYTHMS 455
3. Reptiles
Potential EOPs in reptiles include the pineal organ and the parietal eye.
The pineal has modified or rudimentary photoreceptors whereas the parietal eye
has well-organized photoreceptors that resemble the cones of the lateral eyes.
Both organs give electrical responses to illumination (see review in 401). The
locomotor’rhythm can be entrained in lateral eye-ablated animals of seven dif-
ferent species (399). Entrainment persists in Scelopomcs olivaceus after com-
bined removal of the lateral eyes, parietal eyes, and pineal organ, although the
activity pattern is altered (399,401). At least some EOPs are capable of intensity
as well as wavelength discriminations (see 401). In some lizards lateral eye
removal has no apparent effect, suggesting that for some CRs entrainment may
be exclusively via EOPs. In S. oZivaceus EOPs have been localized to un-
specified portions of the brain (401).
The parietal eye of lizards has an ERG whose sensitivity varies with the
wavelength of the stimulating light. Engbretson and Lent (102) studied a prepa-
ration consisting of the parietal eye, parietal nerve, and pineal of the collared
lizard (Crotaphytus collaris) maintained in a natural photoperiod. Photo- and
chemoresponsiveness was nearly reversed during the two phases of the lizard’s
daily photoperiod. In the L. phase illumination of the eye produced a fourfold
increase in nerve impulse activity (an on-response); responsiveness to light was
modulated by chemical stimulation of the pineal and norepinephrine (NE)-
sensitive neurons in the pineal appeared to feed back to the parietal eye to
augment its light responsiveness. Illumination of the eye during the D phase did
not increase nerve activity, but activity tended to increase when the light
stimulus was terminated (an off-response). Application of NE to the pineal
during the D phase had little effect. Pineal efferent regulation of the visual
pathway has yet to be documented in other vertebrates; the absence of direct
neural connections between the pineal and retina (250) probably rules out
a strictly comparable mechanism in birds and mammals.
4. Birds
Influential early work on extraocular photoreception was generated bY

Benoit (see review in 36), who demonstrated effects of light on the gonads of
orbitally enucleated’ ducks. Studies in which radioluminescent paint was im-
planted into the brains of various avian species (e.g., 153, 281) indirectly sug-
gested that extraretinal photoreception could mediate light effects on theneuro-
endocrine axis. Light-conducting fiber optics implanted in and near the hypo-
thalamus in white-crowned sparrows (Zonotrichia Zeucophrys gambelii )
produced gonadal development in both blinded and sighted birds held in non-
stimulator-y short photoperiods. The region of greatest sensitivity to light was
the ventromedial portion of the hypothalamus, and anterior hypothalamic place-

ments were relatively ineffective (419b). The authors present a cogent discussion
of problems associated with EOP localization studies, including the problem
of light transmission to sites remote from the site of implantation. These observa-
tions are not, sense strictu, relevant to the search for receptors involved in
entrainment but are worth considering because entrained CRs frequently medi-
ate the effects of photoperiods on the hypothalamic-hypophyseal-testicular axis.
The locomotor activity of enucleated sparrows (Passer domesticus) can be
entrained by ambient cycles of low light intensity (240). Photoreceptors in the
eye as well as EOPs in the brain mediate effects of light intensity on 7 of this
rhythm (240). Other work has established dermal light sensitivity in pigeon
embryos and neonates (147) but the role of these receptors in entrainment
remains to be documented.
Ocular photoreceptors contribute to the effects of light on the circadian
system of some birds; the light threshold for entrainment is lower in sighted
than in enucleated sparrows (240). The arrhythmicity observed in Passer in LL
also is dependent on the eyes.
The number of extraocular photoreceptors and their typology, sensitivity
to wavelength, and locus within the CNS are unknown. Studies of gonadal
condition in quail implanted with radioluminescent paint suggest at least some
localization of EOPs in the hypothalamus, optic lobe, and rhinencephalon (153).
Careful studies of CR entrainment after similar procedures are called for and
could substantially increase our understanding of EOP localization and function.
It would be fruitful to test for EOPs in areas of the avian CNS that receive
optic nerve projections. Specifically the anterior hypothalamic region, site of
termination of the retinohypothalamic tract (RHT) in several avian species
(55, 237), would be a likely starting point (see also 280-282).
Photic entrainment of pinealectomized Passer indicates the pineal is at
most one of many EOPs. In these birds light information from the eyes and
from the EOPs summates in producing entrainment (240).
5. Mammals
The specific retinal receptors involved in entrainment are unknown. Rod

receptors of albino rats (Rattus norvegicus) were once thought unnecessary
for photic entrainment of CRs. Long-term low-intensity LL reportedly caused
complete degeneration of the rod cell layer (9). These “rodless” rats were capable
of learning visual discriminations (9) and of entrainment of their corticosterone
rhythms to the LD cycle (97). The hypothesis that “rodless” or “receptorless”
animals have residual visual function can now be rejected. Vertebrate retinas
contain both rod and cone photoreceptors; retinas of nocturnal species such as
the rat have a small number of conelike receptors and diurnal mammals have
a small but functional retinal rod system (168). Some degree of duplex vision
is a distinct possibility even for the most visually specialized mammals and
cones could mediate entrainment in “rodless” retinas. Intense LL first damages

July 1979 NEURAL REGULATION OF CIRCADIAN RHYTHMS 457
the rhodopsin-containing rods (scotopic system) while sparing the photopic

mechanism of the albino rat (79). Cones on the whole seem much less affected
by LL and probably mediate vision in LL-damaged rats. In rats housed in LL
the percent of cones detected increased from 1.5% to 60% (79). Surviving rods
also could subserve entrainment of CRs in light-damaged retinas. Incomplete
sampling and inadequate histological technique may have contributed to claims
of rodlessness in LL-housed animals; LaVail et al. (201) used improved histo-
logical methods on rats with inherited retinal degeneration and detected
photoreceptor cells where previously none had been found. We conclude
that a very small number of rods are capable of mediating entrainment of CRs
and/or cones are sufficient receptors for entrainment in nocturnal as well as
diurnal mammals. A comparable situation occurs in diurnal pigeons, where
LL induces retinal damage, including loss of cone outer segments (228). In these
animals the rods have a higher threshold to LL damage than the predominant
cone receptors. The relative sensitivity and efficiency of rod and cone receptors
for entrainment in animals of different activity types are important problems
awaiting study.
Extraocular photoreceptors, if they exist, are presumed to have no func-
tional significance for entrainment of mammalian CRs (see 334). This conclusion
is based on the failure of blind adult mammals to entrain to LD cycles. To date
only nocturnal species have been studied systematically; the light intensities
used have invariably been far below those available to animals in nature. In most
artificially illuminated laboratory rooms light intensity is between 50 and 100 ft-c
(160-320 W/cm2) (419). Folk (118) estimates that in direct sunlight at noon the
light intensity varies between 4,000 ft-c in midwinter and 9,000 ft-c in midsum-
mer. The visible portion of the solar spectrum can penetrate into all metabolic
compartments of the body (419), including the brains of mammals where EOPs
are likely to reside. Mammals exposed to natural daylight at 1500 h have signifi-
cant amounts of light penetrating their brains; light even reaches d eep sub-
cortical structure 1ssuch as the hypothalamus (402). The amou nt of ligh t reac hing
the brain is reported to be inversely proportional to the size of the animal (402).
Until one can document the absence of photic entrainment in a number of blind
diurnal and nocturnal mammals exposed to light intensitie s approximating those
of direct sunlight, the conclusion that the eyes of adult mammals contain the
only photoreceptors for entrainment must’ remain tentative. Small diurnal
mammals would seem especially good candidates in which to demonstrate en-
trainment mediated via EOPs. Prospects with nocturnal mammals are poor be-
cause they probably do not experience direct bright sunlight in nature.
Extraretinal photoreception is possible in infant rats at 12 days of age (429).
Light coming through the closed eyelids also can affect physiological responses
at the time that the EOPs presumably are functional (429). The significance
for entrainment of the temporarily functional extraretinal system is not clear,
Par titularly for fossorial nocturnal rodents; in the ordinary course of events
the young of such species would not be expected to have exposure to LD cycles
until somewhat later in life.

458 BENJAMIN RUSAK AND IRVING ZUCKER Volu?7@ 59
Light impinging directly on the hypothalamus of enucleated rats apparently

is able to influence the endocrine system. This finding is intriguing because
the putatively responsive tissue has been localized to the SCN (219). The SCN
has been identified as a likely circadian pacemaker in rats and other mammals
(see p. 479). The possibility therefore is raised that clocklike neurons retain
direct sensitivity to light. The Lisk and Kannwischer (219) finding also is inter-
esting because the’ RHT, which is the principal pathway for retinally mediated
entrainment in mammals, terminates in the SCN. This brain region, ifit contains
EOPs, would be ideally situated to integrate photic input from retinal and
extraretinal sources.
The data establishing direct SCN responsiveness to light are far from com-
pelling, however, and suffer from questionable statistical procedures in which
the percentage of estrous vaginal smears across .days is presented for an entire
group of animals rather than for individual rats (219). The reported effect of light
on the hypothalamus could as easily represent . the~ contribution
. of a single animal
as a more gener&l pheno~enm. It is e&&i&l that the main findings of this study
be confir@d-and &tended. Of course, it would be most satisfying to demonstrate
entrainment in blind mammals after selective illumination of different parts
of the nervous system. This is preferable to documenting changes in light-
dependent neuroendocrine responses, which could be affected by light inde-
pendently of the circadian system. A perplexing issue in extraretinal photo-
reception by neural tissue is the nature of the photopigment within the cranium
for transduction of light into neural activity. There is no evidence that light
can act directly on nonpigmented neurons to modulate their activity.
Systematic studies of action spectra and thresholds for entrainment are
generally not available. Green light (A = 530 t 45 nm) is the most potent and
red light (h = 660 t 19 nm) and ultraviolet light (A = 360 t 34 nm) are the
least effective in entraining the rat’s body temperature rhythm (234). Blue light
and green light are also most effective for influencing mouse locomotor activity
(364). Wavelengths in the blue-green spectral region are most effective
for phase-shifting the die1 rhythm of torpor in pocket mice, Perogmzthus
penidlatus (127).
The possibility that different receptors (and photopigments) mediate en-
trainment of different CRs in a given organism has not received extensive
experimental ..attention. Systematic exploration with different wavelengths and
different circadian end points is potentially highly instructive. It could establish
whether a given wavelength is effective only for some rhythmic functions,
thereby documenting the multiplicity of circadian pacemakers in metazoans.
Usually EOPs are not located close to the organs that depend on photic
stimuli; rather they are proximal to or part of the central nervous system (345).
The EOPs that mediate light effects on the gonads, for example, appear to be
in the nervous system. Since these endocrine organs are regulated by the brain
and pituitary, cranial localization of EOPs is quite sensible. Localization of
EOPs in the CNS may represent a first step in the evolution of photoneural-
endocrine systems. Centralization of photoperiodic control is advantageous

July xiv9 NEURAL REGULATION OF CIRCADIAN RHYTHMS 459
because the CNS can effectively integrate visual input with afferent information
from other modalities (345). Perhaps most significant is the economy achieved
in reducing the number of receptors necessary for transducing photic informa-
tion. Where many functions are light dependent, as is true for mammals, a single
set of receptors can effectively and efficiently synchronize all rhythms if light
information is funneled through it to the central oscillator(s) (345).
B. Retinal Light Entrainment
I. Anatomy
a) Receptors. Distribution, receptive fields, and discharge properties of

retinal ganglion cells for entrainment of CRs are completely unknown. With the
advent of newer histochemical methods for tracing neural connections we can
expect progress on this front in the near future. A recent study (72) provides
a useful model. Retrograde transport of horseradish peroxidase (HRP) injected
into visual relay nuclei permitted identification of ganglion cells that project
to the lateral geniculate nuclei (LGN) and to the superior colliculi. Horseradish
peroxidase injected into parvocellular laminae of the LGN labeled virtually
every ganglion cell within the circmnsc-ribed zone of the retina known to project
to that region of the‘ gem culate. .Only the largest ganglion cells of the more
peripheral retina and not all parafoveal cells proj ected to the m.agnocellular
layer of the LGN. Scattered ganglion cells of all sizes projected to the superior
colliculus. Injections of HRP restricted to the suprachiasmatic nuclei should
help identify the retinal ganglion cells that form the retinohypothalamic projec-
tion (see below). However, certain neuronal cell types lack the capacity for
uptake or transport of HRP or are too small or inactive to permit use of this
technique (72). The HRP method may have limited applicability to the study
of the unusually small SCN neurons (5-15 pm).
Lesions restricted to various parts of the rat retina produce diffuse, non-
localized degeneration in the medial terminal nuclei (MTN) (192). The accessory
optic tract (AOT) fibers that terminate in the MTN appear to originate in all
parts of the retina with no apparent topographic specificity in AOT projections
(192). This is consistent with the dispensability of the AOT projection for spatial
visual perception; one might predict similar findings for the RHT projections
to the SCN. Studies of SCN degeneration after restricted retinal lesions are
not feasible with the Fink-Heimer method (215, 255) and await application of
more appropriate histological procedures.
b) Visual projections. Knowledge of the central visual projections of the
albino rat has been summarized by Moore (248). In brief, visual processes from
ganglion cells form the optic nerves that enter the optic chiasm. Of the four paths
leaving the chiasm the largest is the primary optic tract (POT), consisting
of uncrossed and decussating components that terminate in the superior collicu-
lus, pretectal nuclei, and lateral geniculate nuclei. The superior accessory optic

tract (SAOT) runs with the POT and terminates in the midbrain tegmentum.
The inferior accessory optic tract (IAOT) of rats leaves the chiasm and courses
along the ventral part of the brain alongside fibers of the medial forebrain
bundle to terminate eventually in the MTN of the midbrain tegmentum. The
IAOT was originally thought to consist entirely of decussated retinal fibers
but is now known to contain uncrossed components as well (250). The IAOT
was recently described in the hamster (214) after earlier attempts to detect
it had failed (99).
The RHT is a bilateral projection that directly connects the retina with the
suprachiasmatic nuclei of the hypothalamus. The claim that the RHT of hamsters
projects to the ventromedial hypothalamus (301,302) has not been substantiated
(214). All modern anatomical investigations show it terminating only in the SCN.
This projection is discussed on p. 480.
The neurotransmitters involved in retinofugal pathways are not known.
The LGN and superior colliculus have high levels of cholinergic enzymes but
the optic nerves show little or no choline@ activity; it is unlikely that acetyl-
choline is involved in synaptic transmission, of direct optic projections of rats
either at the subcortical or cortical levels (44).
2. Function
Identification of entrainment pathways has been based nearly exclusively

on studies of animals with selective interruption of one or more of the above-
described visual projections. Inferences derived from ablation studies are sub-
ject to many pitfalls (348) and should be tentative pending confirmation with
other methods. It is often difficult to disentangle the effects of neural interven-
tions on entrainment from those on the rhythm-generating apparatus. To facili-
tate this process animals should be tested under constant conditions (LL and DD)
to establish the integrity of the CR. Once normal circadian organization is estab- .
lished deviations from normal synchronization to the LD cycle can more con-
fidently be ascribed to changes in entrainment. Few studies have satisfied these
criteria; the majority report only changes in the distribution of a chosen param-
eter between the L and D phases and are of limited value.
Comprehensive data are available only for nocturnal species; there is, how-
ever, no reason to anticipate that diurnal and nocturnal animals use different
projections to entrain to the LD cycle. Anatomical differences between the
retinofugal pathways of diurnal and nocturnal squirrels, for example, are slight
and probably not a consequence of the activity type (389).
a) primary optic tracts. The role of central visual projections in entrainment
was first assessed by Altman (8), who ablated the superior colliculi or the
striate cortices of hooded rats. The brain-damaged groups continued to be more
active during the D phase of the LD 12:12 cycle but their ratio of D to L phase
activity decreased appreciably. Interruption of the SAOT and POT with large
lesions centered in the LGN did not interfere with expression of corticosterone

July 1979 NEURAL REGULATI0.N OF CIRCADIAN RHYTHMS 461
rhythms of albino rats (251), nor was light entrainment of pineal enzyme rhythms
eliminated by similar lesions (251, 254). Ablation of the LGN also failed to
eliminate the nocturnal drinking rhythms of albino (369) and hooded (371) rats
in LD 12:12. Destruction of the POT and SAOT permitted these animals to re-
entrain their drinking rhythms at least as rapidly as controls after a 12-h phase
shift in the illumination cycle (369). These studies suffer from several shortcom-
ings, the most common being the small number of daily observations used to
characterize the rhythm (usually 3 per 24 h) and the use of data from groups of ani-
mals rather than from individuals to assess entrainment.
More thorough work has confirmed that rats with POT and SAOT transec-
tions entrain their activity rhythms to the LD cycle (91). Similar results have
been found with golden hamsters; steady-state entrainment of wheel running
appeared normal in hamsters bearing bilateral LGN lesions that interrupted
the POT and SAOT and variously damaged the hippocampus, fimbria of the
hippocampus, internal capsule, caudate nucleus, and putamen. However, the
rate of reentrainment after a 12-h phase shift of the LD cycle was far slower
than for neurologically intact controls (427). This first suggestion that the POT
might play some role in entrainment was confirmed and extended in a more
complete investigation (332) in which hamster POTS and SAOTs were lesioned
as they rose laterally out of the posterior border of the chiasm; the chiasm
itself was damaged, as were the supraoptic nuclei and portions of the lateral
hypothalamic area, amygdala, and hippocampus.
Some of the hamsters with complete bilateral POT damage had unusually
long active phases that extended into the L phase of the LD cycle or showed
a long phase lead of activity onset relative to the D phase. Reentrainment
of the activity rhythm after a 4-h delay in the LD cycle was much slower in the
POT-lesioned animals than in controls. In addition, the period of the free-
running activity rhythm in constant dim illumination was unusually long for the
lesioned animals. The long 7 in 0.2-1x illumination suggests that the ambient
illumination may have been perceived as more intense by the brain-damaged
animals. Damage to the POT frequently produces near total pupillary dilation
(mydriasis) and could cause retinal flooding with light (414). Other tests indicate
a seemingly paradoxical sensitivity to light after destruction of the POTS (369,
371). The 7 of the activity rhythm in POT-damaged hamsters was not tested
in DD to determine whether the oscillator or its sensitivity to light was affected
(332). Some hamsters with apparently comparable and complete POT lesions
entrained normally to the LD cycle and free-ran with normal 7 values in dim LL
(332). The basis for these individual differences is not known.
After POT lesions light may be less efficient at phase shifting either the
dawn or dusk components of the circadian activity rhythm. According to one
model (296) these components ordinarily are driven toward each other by light
striking their respective sensitive phases. The hypothesized change could ac-
count for the phase leads, late activity offsets, slow phase shifts, and long
active phases of the POT-damaged animals (330, 331).
All studies agree that “normal” steady-state entrainment can occur to a

greater or lesser extent after POT damage; all investigators so far also report
a complete loss or gross impairment in the POT-damaged animals’ ability to per-
form ,relatively simple discriminations based on light-intensity differences (78,
248, 332,427). Thus the intensity discrimination involved in entrainment appears
to be separable from that involved in learning other behavioral discriminations
based on intensity differences. A recent study (204) suggests that information
on light intensity and pattern may be coded in separate pathw ‘WS relative and
absolute intensity discriminations depend on different visual systems (204).
Normal entrainment of POT-damaged rats to LD cycles employing bright light
and absolute darkness does not imply that these animals would entrain normally
to all illumination cycles. In natural or artificial photoperiods where intensity
differences between the L and D phases are relative rather than absolute the
differences from controls might well be substantial.
b) Inferior accessory optic tracts. Bilateral transection of the IAOT was
reported to eliminate the response of the pineal enzyme hydroxyindole
0-methyltransferase (HIOMT) to light (248). This parameter varies with photo-
period but is not a true circadian rhythm; it provides a useful index of retinally
mediated light input to the pineal. Moore subsequently failed to replicate his
earlier findings (250); the pineal HIOMT response to light persists even when
the IAOT and POTSAOT are destroyed. Bilateral lesions of the MTN do elimi-
nate the HIOMT response to light (250). However, the latter effect is unlikely
to be due to interruption of the IAOT visual projection; more probably it reflects
destruction of descending sympathetic fibers to the pineal (250).
Reports that steady-state entrainment and phase shifting of rat drinking
rhythms survive interruption of the IAOT (369) also must be reevaluated. The
surgical procedures used in the earlier studies to accomplish bilateral IAOT
interruption were predicated on the then-current belief that the IAOT of the rat
is a completely decussated pathway. The demonstration of a clear uncrossed
IAOT component (250) means that the animals studied had one uncrossed IAOT
component intact. It is remotely possible that elimination of this remaining
tract would affect entrainment.
Lesions of the accessory optic system, with the qualification noted above,
do not interfere with the rat’s ability to perform discriminations based on visual
cues (427). In rhesus monkeys (Macaca mulatta) bilateral destruction of the
accessory optic system does not affect performance of a response based on dis-
crimination of total luminous flux. Visual input from the accessory visual system
is essential for these discriminations in monkeys subjected to visual cortical
ablations (290). Comparable data on species other than primates are lacking.
In primates, pathways such as the AOTs, in conjunction with the POTS, may be
sufficient for entraining circadian rhythms. Even in rodents destruction of the
RHT is consistent with some degree of entrainment (331), presumably mediated
by the POT/AOT projections.
c) Binocular mechanisms. Adult rats with one eye removed or occluded
show significantly lower D-to-L ratios in wheel-running activity and drinking
and are slower to reentrain these behaviors after a phase shift in the photo-

July mw NEURAL REGULATION OF CIRCADIAN RHYTHMS 463
period (369,427). The magnitude of the differences between one-eyed and two-
eyed rats is exaggerated when the contrast between the L- and D-phase illumina-
tion intensity is reduced (427). Rats were maintained in a constantly illuminated
room; during alternating 12-h periods an opaque contact lens occluded first the
left eye and then the right eye. The drinking rhythm of these animals did not
entrain to this manipulation; although each eye received temporally patterned
visual input (12 h of light exposure followed by 12 h of darkness during occlu-
sion), the inputs to the two eyes were 180’ out of phase. This suggests central
algebraic summation of total luminous flux reaching the retinas occurs during
entrainment of CRs. Support for this conjecture comes from observations that
the drinking rhythms of rats can be entrained when one eye is constantly
illuminated and the second eye is exposed to a simulated LD cycle produced
by covering and uncovering the eye at 12-h intervals (427).
Each SCN nucleus receives RHT input from both eyes, with the majority
of RHT fibers originating in the contralateral eye (148, 247). There are no
functional asymmetries when animals process light with both eyes; however,
the input to the two SCN after unilateral enucleation is asymmetrical and this
may account for the decreased rate of reentrainment in unilateral enucleates
after photoperiod phase shifts (427). Removal of one eye at birth produces a
nearly equal distribution of remaining RHT fibers to the two SCN (366) and is
correlated with an elimination of entrainment differences between one- and two-
eyed animals (363).
C. Nonphdic Entrainment
Primacy of the LD cycle as a xeitgeber has long been acknowledged. The

efficacy of nonphotic xeitgebers and their ability to override the effects of con-
current LD cycles have recently been established (170, 197, 198). A number
of questions areraised by these studies: I) How many distinct circadian oscil-
lators does a given mammal possess? 2) Which nonphotic xeitgebers entrain CRs
under natural conditions, in contrast to artificial laboratory settings? 3) What is
the relation of photic to nonphotic xeitgebers? 4) By what transduction process
do nonphotic xeitgebers gain access to their oscillators? 5) How do nonphotic
stimuli influence light-sensitive pacemakers?
1. Temperature
Ambient temperature fluctuations do not entrain CRs in endotherms,

although they are effective in ectotherms (307). The endotherm’s homeostatic
defense of deep-body temperature ensures that external temperature cycles
are not transduced into temperature changes in the milieu of the central pace-
makers. A true test of the temperature compensation or independence of mam-
malian clocks therefore would depend on documentation of temperature-related
changes within the presumed pacemakers. Activity rhythms of bats tested at

several constant temperatures showed temperature compensation of period.

Body temperatures were not reported but tissue temperature in these hetero-
therms probably reflected changes in ambient temperature (307).
Lindberg and Hayden (217) monitored arousal from torpor in a facultative
heterotherm Perognathus Zongimembris and observed entrainment to tem-
perature cycles in DD. The threshold for entrainment was approximately 1.5”C,
although changes as large as 3, 5, or 9OC did not produce truly stable entrain-
ment. A PRC for temperature pulses was described but the 7 and PRC values
do not accurately predict the phase angles of the entrained state. Studies of the
related species Perognathus intewnedius failed to document temperature
entrainment (375).
2. Social stimuli and handling
White-footed mice handled briefly during the active phases of free runs
manifested a 40.min delay in activity onset the next day (307). In isolation in LL
each of two monkeys had its own free-running feeding rhythm; when they were
placed together there was a high correlation in their feeding times (323).
Conspecific song acted as a weak xeitgeber for house sparrows (Passer
domesticus) (238a).
The activity of a family of beavers (Castor canadensis), presumed to be
in darkness under snow and ice cover, free-ran with 7 = 27 h (57). This may
indicate social entrainment of the group as a whole (mutual phase synchroniza-
tion) but all individuals may have had very similar free-running 7 values.
Again, this form of entrainment, if it occurs, is presumably most effective in the
absence of an LD cycle. However, separate male and female groups of red wolf-
coyote hybrids living under natural illumination showed differences in their
activity patterns that were largely eliminated when the mixed group was
reunited (324).
In an isolation chamber an LD cycle per se often is not sufficient to entrain
all human CRs (15). The addition of auditory cues produces entrainment; differ-
ent ranges of entrainment are possible for the activity and temperature rhythms
(15). Some blind people have a sleep-wakefulness cycle with 7 = 24.9 h (243);
some individuals complain of periodic sleep disorders that could reflect break-
aways from nonphotic entrainment (243).
However, there is also evidence that LD cycles do influence entrainment
of some human CRs (54). Lack of entrainment of isolated humans to LD cycles
may have resulted from a less rigorous application of the illumination cycle in
human than in animal studies (89a). Note also that the apparently unusual
sensitivity to social cues reported in humans may simply reflect inadequate
information on other social species (see above and discussion in 334). Humans
are unique in having the ability to increase light exposure beyond that available
during the solar day, but whether our entrainment mechanism has come to
differ significantly from that of other soci al mammals remains to be established.

July 1979 NEURAL REGULATZON OF CIRCADIAN RHYTHMS 465
3. Food availability
The efficacy of various food-restriction schedules for synchronization of CRs

is well established; we cite but a few of the many relevant studies. Liver
glycogen and serum glucose rhythms are strongly influenced by feeding schedules
in rats fed for 4 h/day at different phases of an LD 12:12 cycle (291). Entrainment
of corticosterone rhythms and rhythms in several enzymes reflects an inter-
action of the LD and feeding cycles, but the cornea1 mitotic index remains
synchronized to the LD cycle (291). Rats offered a high-fat palatable diet
during the L phase and a nutritionally adequate routine diet in the D phase
were transformed from nocturnal into diurnal feeders; there was an attenua-
tion but not an elimination of the normal circadian body temperature rhythm
and a shift in water intake, but wheel-running activity was unaffected (289).
The conclusion that internal processes of rats do not mandate nocturnal rhythms
of food intake when palatable food is available in daytime is valid for this
experimental situation (289). In a field setting, photic entrainment of internal
clocks does not preclude the animal’s taking advantage of short-lived, unpre-
dictable resources that occur at the “wrong” time of day. Such consummatory
activity, elicited by nonphotic stimuli, does not imply that the oscillator to
which this function is ordinarily coupled has been phase-shifted either before
or as a result of opportunistic and unscheduled feeding activity.
Individual rats restricted to eating and drinking for 2 h/day during L demon-
strate a 12-h phase shift in plasma corticosterone and body temperature rhythms
and a similar shift in time of peak plasma protein and albumin (197). In this
case the feeding schedule overrides the LD cycle. Food-restriction schedules
withT = 24 h can entrain the free-running drinking rhythm in LL (56); <with re-
turn to ad libitum feeding the original free run in drinking is restored. However,
the phase of the free run is not determined by the phase of the prior feeding
schedule (56). The phase data suggest that the pacemaker for drinking is free-
running throughout the restricted feeding schedule and that there is “temporary
uncoupling of the behaviors from their pacemaker(s), allowing ingestion to be
passively driven” (56); i.e., the feeding schedule synchronizes the overt rhythm
without entraining the underlying circadian oscillator. Similar results were ob-
tained in a study in which blinded rats were active during a l-h period of access
to water, but continued simultaneously to generate free-running circadian ac-
tivity rhythms (256a). The conclusion that behavior synchronization does not
imply an effect on an oscillator is reinforced by the finding that rats may
synchronize several rhythms to imposed 22- or 28-h LD cycles while simultane-
ously expressing free-running circadian rhythms of the same parameters (97a).
The data and conclusions from these studies of rats are at odds with those
from studies of squirrel monkeys (Saimiri sciureus). Squirrel monkeys fed
ad libitum in LL generate a drinking rhythm with 7 = 25 h; when feeding is
restricted to 3 h/day 7 for drinking is 24 h. Most drinking ordinarily occurs
prandially and in this case during the 3-h period of food availability, so this
finding does not necessarily imply entrainment in the usual sense. The phase

of the drinking free run when the animals are fist returned to ad libitum feeding
is reportedly determined by the phase of the previous deprivation cycle (380).
The data supporting this conclusion are not compelling and the instability of en-
trainment produced by the fmd-restriction schedule is notable (especially for
the urine-volume and K+-excretion rhythms). It remains to be established that
the feeding schedule itself is a xeitgeber with status equal to that of the LD
cycle. The persistence of urinary rhythms in monkeys deprived of food and
water or trained to eat every 2 h in LD 12:lZ (257) indicates that the LD cycle
may be sufficient for entrainment of the rhythms under study.
The LD cycle effectively and ordinarily entrains eating and drinking
rhythmsinallmammals so f%r studied (e.g., 334,380,425,428). Food-deprivation
schedules clearly are capable of influencing the expression of various rhythms
but the exotic nature of these manipulations makes it unlikely that they do so
normally. Free-feeding squirrel monkeys eat by day (317,257) and free-feeding
rats mainly by night (425). Neither species eats all its daily food in a single 3- to
4-h interval. Since eating and drinking rhythms are always entrained by the
LD cycle the ultimate control of all food-related rhythms is by the illumina-
tion cycle.
Temporally coded stimuli generated by food and water intake are secondary
xeitg&rs whose utility for temporal integration derives from their isomorphic
relation with the photoperiod. The presumed unidirectional nature of this rela-
tion (food intake cannot alter the LD cycle) argues for the primacy of photo-
period as xeitgeber.
Illumination cycles may influence the circadian system of mammals via
several routes (332) and the system itself comprises a multiplicity of oscillators
(296, 331). However, the present evklence does not convincingly demonstrate
the existence of multiple, independent, nonphotic xeitgebem affecting the
circadian system. Rather, the illumination cycle appears to entrain a neural
oscillatory complex coupled to other oscillators that are not influenced directly
by photic input. The fact that a central oscillatory complex has exclusive access
to light information that it transmits to other systems does not necessarily
imply a strictly hierarchical coupling mechanism (258); feedback effects of
peripheral structures on central oscillators have been clearly demonstrated (426a).
III. RHYTHM GENERATION
A. Invertebmtes
Neural control of circadian rhythmicity has been studied in species Tom

several invertebrate classes. Behavioral repertoire, hardiness, and large size
have influenced the choice of experimental animal. The species used in the great
majority of studies are:

July 1979 NEURAL REGULATJON OF CIRCADIAN RHYTHMS 467
Mollusca: several species of Aplysia, especially A. califowzica; Limax

maximus
Arthropoda: Crustacea: Procambamcs sp., Cambarus sp., Carcinus
sp., Uca sp., Gecarcinus lateralis, Limulus
polyphemus
Insecta: Dictyoptera: Periplaneta americana, Leucophuea
maderae, Blaberus craniifer
Orthoptera: Acheta (Gryllus) dornesticus,
Teleogqllus commodus ,
Romalea microptera,
Ephippiger sp.
Lepidoptera: several Saturniid silkmoths,
especially Antheraea sp.,
Hyalophora cecropia
Coleoptera: Blaps gigas
I. Aplysia
a) RX R15, a large neuron in the parietovisceral ganglion of Aplysia,

shows a characteristic pattern of electrical activity both in vivo and in vitro
(376). In DD the spontaneods activity of R15 generally includes one or two
strong peaks of activity; earlier suggestions that this represented a true circadian
rhythm of electrical activity (213,376,377) have not been substantiated (18,34).
The Aplysia in the latter studies manifest a single peak of activity from R15
in DD; the phase of the peak is determined by the time of dissection during
the LD cycle to which the ApZysia had been exposed. Apparently R15 is differ-
entially sensitive during the LD cycle to the phase-shifting effects of an un-
identified agent (dissection trauma, temperature pulses, or light exposure dur-
ing dissection).
R15 in vitro is very sensitive to temperature and can be driven by tempera-
ture cycles of very small amplitude (34, 211, 360, 376). In cultures maintained
at constant temperature R15 can sustain ultradian (1-4 h) but not circadian
rhythms (34, 211).
Electrical activity of whole-brain cultures of the mollusk Limax maximus
can also be driven by temperature cycles, and here too there is no evidence
of a persistent circadian rhythm in constant conditions (190).
b) Eye. The excised Aplysia eye survives several days in culture medium
and produces spontaneous bursts of electrical activity (compound action poten-
tials: CAPS) that can be recorded from the cut end of the optic nerve. The
frequency and amplitude of CAPS vary with a circadian periodicity that is sus-
tained for the life of the preparation (157, 161, 162).
Several conclusions about the Aplysia eye rhythm seem securely estab-
lished. 1) The CAP rhythm is generated by a set of secondary neurons whose

axons run in the optic nerves; these tend to cluster near the base of the eye.
2) Centrifugal influences are exerted on each eye by the CNS of the intact
animal; this brain efference appears to include output from the contralateral eye.
3) Inhibitors of ribosomal protein synthesis phase-shift or disrupt rhythmicity
depending on dosage and pattern of administration. 4 ) The availability .of mono-
and divalent cations influences rhythm generation. Evidence to support these
conclusions is outlined below and in a recent review (211).
I) SITE OF GENERATION. The period of the CAP rhythm was reported
to decline in an apparently graded fashion as greater portions of the eye were
excised; when 2% of the eye remained, only short, ultradian rhythms were
expressed (165). Jacklet and Geronimo (165) concluded that the CAP circadian
rhythm reflected coupling among a large population of (ultradian) oscillators.
However, another study (352, 377, 378) reported no reduction in period when
only a very small portion of the base of the eye remained intact.
Jacklet (38, 160, 163) has reviewed his own and Sener’s (352) results and
has indicated some technical inadequacies in the performance and especially
the analysis of Sener’s experiments. Published records from these studies (378)
show the apparent persistence of a single circadian cycle of CAP frequency
in the grouped data obtained from 13 eyes reduced to a very small size. Results
presented in this way are of doubtful relevance to the present issue; at minimum,
it is premature to dismiss the claims of Jacklet and Geronimo (165) on the basis
of these results. Benson and Jacklet (39) provide additional indirect evidence
for the existence of a population of coupled oscillators on the basis of apparent
cold-induced splitting of the population rhythm.
Strumwasser (377,378) suggested that a small number of cells at the base
of the eye are specialized for circadian rhythm generation; these were claimed
to couple neurochemically to the selcondary neurons whose axons run in the
optic nerv ‘es and generate the CAPS. The hypothesis was based on the assertion
that spontaneous CAP generation is disrupted when neurosecretion is blocked
(16, 17). This conclusion is not supported by other studies that show a per-
sistence of rhythm generation and phase-shifting in culture media that should
block neurochemical transmission (105, 106, 161). Recordings in the studies
by Audesirk (16,17) were restricted to only brief sessions during the first cycle
after dissection; they do not provide substantial contradictions to the findings
of E&in and Jacklet. Although neurochemical transmission is not required for
generation of the CAP rhythm, a serotonergic mechanism appears to play a role
in phase control of the rhythm; addition of serotonin to the culture medium
can phase-shift the eye’s rhythm (85a).
Benson and Jacklet (38-41) have recently reported on the effects of DzO,
light, and temperature on properti .es of rhythms generated by isolated Apl ysia
eyes. These studies exemplify the value of the Aplysia eye preparation for the
analysis of both formal and physiological properties of rhythm generation;
we have not discussed them further because they relate mainly to formal prop-
erties and models of rhythm generation beyond the scope of this review.
II) CENTRAL INFLUENCES. Neural activity recorded from the eye in vitro

occurs in a more regular burst pattern against a lower background level of ac-
tivity, has a longer 7, and responds more slowly to phase shifts of the LD cycle
than the activity of the in vivo eye (104). Although the slower phase shift and
longer 7 are an effect of the culture medium (50, 104), the regular bursting
pattern against a quiet background is a consequen.ce of the loss of cerebral
efference via the optic nerve. Selective cooling of the cerebral ganglion to inhibit
its efference modifies these features of the in vivo preparation in the direction
of the in vitro eye (104).
Attached and isolated eyes entrain to white-light cycl .es, but only the at-
tach .ed eye en trains to red -light (RL) cycl es; optic nerve efference is crucial
for entrainment of the intact eye to RL cycles. This finding demands at least
an RL-sensitive extraocular receptor, but it could also be explained by the
existence of an extraocular RL-sensitive receptor-oscillator complex (48, 211).
The source of the efference to the eyes is not known; it sometimes differs
slightly in the two optic nerves. Stimulation of other cerebral nerves may inhibit
the efference, and very-low-intensity light restricted to one eye may inhibit
the efference in the contralateral optic nerve (104). Cerebral efference plays
a role in resetting the phase of eyes exposed to L + D transitions (300a).
In an intact Aplysia selective exposure of one eye to an LD cycle sufficient
to entrain its activity fails to entrain the activity of the contralateral unstimulated
eye; thus, the two eyes are not strongly coupled (154, 158, 211). However,
some experimental conditions reveal a functional interaction between the eyes.
Intact Aplysia were allowed to free run in DD or dim LL for variable lengths
of time, and the phase relation of the two eyes was then assessed bY excising
the eyes and recording independently from each in vitro. Pairs of eyes excised
after a few days in constant conditions were roughly synchronized; and even
after 8-28 days most pairs of eyes were still O-4 h out of phase; however,
a few at this time were lo-12 h out of phase. By 29-62 days 61% of pairs
were 9.5-12 h out of phase and the majority of the remainder were still 1.5-4.5 h
out of phase; none were separated in phase by 4.5-7.0 h (154). These findings
suggest that the eyes in vivo are weakly negatively coupled and tend to drive
each other into an antiphase relation when they are not subject to synchroniza-
tion by a common xeitgeber. Such a hypothesis is also consistent with prior
evidence that the eyes are not strongly, positively coupled (158, 211).
III) CHEMICAL INFLUENCES. Macromolecular synthesis and ion distribu-
tions are implicated in the generation of the circadian rhythm of CAPS by the
isolated Aplysia eye. Treatment for 12 h with either cycloheximide or puromycin
produced dose-dependent phase delays; 6-h pulses of puromycin yielded a phase-
response curve similar in form to that obtained with l-h light pulses, including
both delays and advances. These drugs reversibly inhibit protein synthesis;
actinomycin D and aflatoxin Bl both block RNA synthesis, and their application
produced an irreversible loss of the CAP rhythm in most eyes (325).
Jacklet (164) has applied 6-h pulses of lo- 5 M anisomycin, another inhibitor
of protein synthesis, to generate a PRC similar to that obtained with puromycin.
When tonically present in the medium at the same concentration, anisomycin

permitted CAP generation for 3 days at relatively low frequencies, but with no
evidence of a circadian rhythm. A lower concentration of anisomycin permitted
the expression of a few circadian cycles before the rhythm damped out, and a yet
lower one permitted continued circadian rhythmicity but with relatively long
7 values of 28 h rather than 26.5 h. Protein synthesis at ribosomes thus is of
considerable significance for rhythm generation, but it is not necessarily part
of the time-keeping mechanism. Proteins may well be required for the continued
operation of a circadian oscillator even if the synthetic. process itself is not
integral to the oscillation (164).
The isolated Aplysia eye offers an excellent opportunity for the manipula-
tion of the ionic milieu of a circadian oscillator and for tests of the proposition
that circadian timekeeping involves ionic flux across cellular membranes (274).
Increasing K+ concentrations in the culture medium for 6 h produced phase
shifts in the rhythm of CAP production whose amplitude and direction depended
on the phase of application. The resultant PRC for high K+ concentration
resembled that for l-h light pulses; moreover, phase shifts could be generated
similarly in a milieu that did not permit neurochemical transmission. E&in
(105) suggests that increased extracellular K+ might phase-shift neural oscil-
lators directly by depolarizing them.
Treatment of in vitro eyes with pulses of either lithium chloride or manga-
nese ions produced all-delay PRCs; treatment with a divalent ionophore, which
equalizes transmembrane distributions of divalent cations, produced a similar
all-delay PRC (106, 107). The phase-shifting capacity of the ionophore was
not affected by manipulating the extracellular concentrations of either Ca2+ or
w 2+, so transport of these ions across the external plasma membrane is not
critical. E&in and Corrent (107) suggest that the critical ion-distribution
changes occur at intracellular membranes, perhaps at the mitochondria. Chemi-
cal treatment to inhibit cellular respiration or other aspects of cellular energy
metabolism also produced all-delay PRCs, but treatment with an inhibitor of the
Na+-K+ pump produced a PRC similar to that produced by high concentrations
of K+. Ion distributions and energy metabolism, probably at an intracellular
membrane, therefore are implicated in rhythm generation; it is not yet possible
to discriminate among several possible models for ionic involvement in circadian
rhythmicity (106, 107).
c) Activity rhythms. Locomotor activity of Aplysia is predominantly
diurnal in LD cycles and free-runs with a circadian period in constant conditions
(159, 200, 378). Removal of the eyes (and their circadian oscillators) does not
eliminate the predominantly diurnal pattern of Aplysia activity in an LD cycle
(49), but it is not certain if this represents true entrainment or a form of
“masking’ (212). These findings are consistent with a “masking” effect and
do not prove that true extraocular entrainment is possible. However, the clear
evidence that at least some eyeless Aplysia generate circadian activity rhythms in
DD establishes the existence of extraocular oscillator(s) for activity (49, 211,212).
The role of the eye in rhythm generation is not clearly defined; Lickey et al.
(212) suggest that the eye is one of several oscillators involved in the control

Jti& 1979 NEURAL REGULATION OF CIRCADIAN RHYTHMS 471
of locomotion. It is instructive to compare the effects of eye removal inA@@z

to the effects of SCN destruction in rodents (see p. 488). The latter also leads
to a disruption of normal entrainment, although some foim of synchronization
to the LD cycle is still possible in many animals. Additional common features
are the increase in activity du,ring the erstwhile “inactive” phase and the loss
of normal circadian rhythms in constant conditions. Significant differences do
exist: entrainment is more resistant to eye removal in Aplysia than to SCN
destruction in rodents, and SCN-lesioned rodents have not been found to gener-
ate circadian rhythms in constant conditions, whereas some eyeless ApZysia
“free-ran as vigorously as the most vigorous intact ones” (212, p. 121). The
analogy is easily stretched too far, but the assertions that 1) a single structure
is involved in both entrainment and generation of rhythms, 2) there are alter-
nate routes for synchronization to LD cycles, and 3) several structures are
likely to form the oscillatory complex normally generating activity rhythms
are all supported with reference to findings in eyeless Aplysia and SCN-
lesioned rodents (see p. 488; 212, 331).
2. Insects
A lack of uniform nomenclature in descriptions of insect nervous systems

is a potential source of confusion to the general reader. Our usage corresponds
generally with that in standard texts (69, 77).
Several reviews are available on the subject of neural control of insect
rhythms (60, 6‘1, 320, 342); especially recommended is the cogent review by
Brady (62) on the larger topic of the physiology of insect circadian rhythms.
Without recapitulating the detailed evidence available elsewhere, we summariz
the conclusions reached in these reviews, as modified somewhat by more recent
findings.
a) &?tyopha and &tkplkTtz. I) SUBESOPHAGEAL GANGLION. Interest
in the cockroach subesophageal ganglion (SEG) stems from claims of intra-
specific transfer of rhythmicity via SEG transplants and from the proposal that
SEG and corpora cardiaca secretions are critical to rhythm generation (142,143).
These claims <have not been supported bystudies in other laboratories (5& 59,
89,114,319). The SEG appears to tonically excite motor activity via the thoracic
ganglia, but neither its secretions nor those of the corpora cardiaca-allata
complex play a significant role in circadian rhythm generation in cockroaches.
An implanted SEG may transiently influence the appearance of rhythmicity,
and parabiosis may permit transfer of a substance that affects activitylevel
in headless roaches. Nevertheless, the evidence indicates that the SEG is not
the site of a driving oscillator for activity rhythms, that rhythmicity cannot
be transferred between roaches by SEG implantation or parabiosis, and that
hormonal coupling is not critical to normal rhythmicity in cockroaches (62,89,319).
One entirely aberrant finding merits reinvestigation. Fingerman et al. (114)
reported normal entrainment of circadian activity rhythms in brainless grass-

hoppers (Romalea microptera). If confirmed, this would suggest that another

structure (e.g., the SEG) might generate and entrain rhythms in this species.
In this species SEG extirpation produced low activity levels throughout the LD
cycle, which were not restored to normal levels by SEG implants. Only the mean
activity levels of groups of insects maintained in LD cycles were reported;
in the absence of.further reports on this species, we do not know which features
of the study contributed to the unique result of behavioral rhythmicity surviv-
ing brain extirpation.
II) PARS INTERCEREBRALIS. Bisection of the cockroach protocerebrum
destroys the median pars intercerebralis (PI) and some of the neurosecretory
cells located there; it was claimed that this intervention produced arrhythmicity
(319). Others have assessed whether the medial (or other) neurosecretory cells
of the protocerebrum are critical to rhythm production. Microcautery of the
medial neurosecretory cells spared rhythmicity in most roaches even though
staining with paraldehyde-fuchsin (PF) revealed few or no neurosecretory cells
in many rhythmic animals (58). Excision of a triangular portion of the PI left
many roaches rhythmic, and some arrhythmic, both temporarily and perma-
nently (269). Many rhythmic animals still had PF-staining material in the lateral
group of neurosecretory cells (pars lateralis); the significance of this fact is
doubtful because the brains of the few arrhythmic roaches examined had all
suffered damage to the corpora pedunculata, through which input from the optic
lobes normally reaches the rest of the brain.
Nevertheless, Nishiitsutsuji-Uwo et al. (269) concluded that the medial and
lateral neurosecretory cells of the protocerebrum are responsible for rhythmi-
cally inhibiting activity in cockroaches and that partial damage permits regen-
eration of neurosecretory units and hence of rhythmicity. This interpretation
was offered with caution since they recognized that disruption of other functions
of neurosecretory cells might secondarily affect rhythm generation.
The absence of PF-staining material in the PI is open to significantly
different interpretations; it may mean either that all neurosecretory cells have
been destroyed or that the surviving cells are secreting at so high a rate that
it prevents the act umul .ation of stainable material in the somata (62). A further
complication is the identification on the basis of ultrastructural evidence of
neurosecretory cells in the optic lobes of roaches that do not stain with PF or
other basic stains (32).
Parallel studies of the PI of crickets have yielded similarly ambiguous
results. Brain bisection of A&eta domesticus produced high levels of apparently
arrhythmic activity; the critical area was localized by lesions to a region of the
protocerebrum lateral to the PI and near the corpora pedunculata. Neck liga-
tures that might be expected to block only humoral communication between
brain and nerve cord failed to eliminate rhythmicity, so a neural, rather than
a humoral , mech .anism was implicated (20 ,398). Destruction of portions of the PI
produced a lack of both rhythmicity and stainable neuros lecretory cells in some
animals; crickets whose rhythms recovered had stainable material present (87).
The objections already raised to similar studies in roaches apply here as well;

in addition this study suffered from a lack of tests in constant conditions, the
presence of a temperature cycle in LD, and some uncertainty as to the ar-
rhythmicity of crickets so labeled (see 62).
Both locomotor activity and stridulation were studied in Teleogryllus com-
modus after cautery of the PI; crickets with residual stainable cells generated free-
running circadian rhythms, but with modified .7 values. Among those without
stainable material some were arrhy thmic in ac tivity and did not stridulate,
others were i nactive but stridulated rhythmically , and one cricket stridulated
rhythmically but locomoted arrhythmically (363). Since some animals with no
neurosecretory cells visible were rhyth .mic in one measure, the activity of these
cells does not seem to be necessary to rhythm generation; nor, on the basis of
evidence discussed below, does neurosecretion appear to be sufficient for ex-
pression of rhythmicity. The issue of neurosecretion aside, neural activity
of other cells in the PI may influence rhythm generation (221); the role of the
cricket protocerebrum isdiscussed in the next section.
Another approach to this question has been to correlate changes in the
anatomy or physiology of neural structures with rhythms in behavior. Since
acti vity and inn umerable other physiological functions vary with a daily rhythm,
the correlation is bound to be high; however, causal relations between various
rhyth ms have not been establishe d and the meaning of the correlation is far from
clear. Some ill ustrative examples include the rhythm of nu clear diameter in cells
in the nervous system of Droso@Za wzelarmgaster (312), peaks of neural activity
recorded from the PI of A. domesticus just prior to the anticipated peak
of locomotion (20), and a peak in RNA synthesis in neurosecretory cells of the PI
during the L phase in the same’species (88).
A further step has been taken in a study of Apis mellifica. The rate of
protein synthesis in the PI normally reaches a peak before the time of foraging
activity. When foraging was restricted to a brief period at an unusual time,
the rhythm of protein synthesis also shifted to anticipate activity (407). This
does not prove that the PI generates the rhythm or even that it is uniquely
involved in preparation for activity, since a parallel rhythm of low amplitude
is found in the corpora allata. However, the proliferation of such correlations,
if shown to be specific and to persist in constant conditions, would provide
evidence suggesting a role of the PI in generating the activity rhythm; such
evidence does not now exist.
III) OPTIC LOBES. Considerable evidence suggests that the optic lobes
contain the driving oscillators controlling circadian activity rhythms in cock-
roaches and crickets. Section of the optic nerves in cockroaches produced free-
running rhythms that could not be entrained (270), whereas separation of the
optic lobes from the protocerebrum by transecting the optic tracts rendered
Leucophaea mudenze arrhythmic (271). One finding suggested a humoral cou-
pling mechanism from the brain to the thoracic effecters: section of the circum-
esophageal connectives (CEC), which should interrupt the direct neural path
from brain to thoracic ganglia, failed to block rhythm generation. This claim
was later withdrawn (295); subsequent studies clearly demonstrate that a neural

link from brain to thorax is essential to the generation of circadian rhythmicity

(62320,321). Ball and Chaudhury (22) claimed that Blaberus craniifm could be
entrained by implantation of either a whole brain or the optic lobes behind
a “window” in the abdomen, thereby implicating a humoral mechanism. How-
ever, the experiments as described are seriously flawed and provide no sub-
stantial evidence for humoral coupling in cockroaches (see 62, 320).
Roberts (320) confirmed the importance of the optic lobes for rhythm
generation in L. maderae and Petiplaneta americana. External layers of the
lobes were removed without blocking activity rhythms, but transection between
the medulla and lobula or large electrolytic lesions near the medulla produced
arrhythmicity. Roberts (320) concluded that a single intact medulla attached
via the optic tract to the protocerebrum and thence to the thoracic ganglia
is both necessary and sufficient to maintain circadian rhythmicity in roach
activity. Sokolove (362), using microlesions in the optic lobes, generally con-
firmed Roberts’ findings, but he also indicated that the most effective lesions
were those in the cell-body region near the second optic chiasm between the
medulla and lobula. Although some lesions in the lobula were effective,
Sokolove (362) concluded that cell bodies near the lobula were more im-
portant for rhythm generation than the more prominent lobula neuropile.
The bilaterally symmetrical optic-lobe clocks apparently interact to gener-
ate circadian activity rhythms, and each eye can entrain both optic-lobe clocks
(284). By contrast the circadian electroretinogram rhythms of the lateral eyes
of tenebrionid beetles (particularly Blaps @gas) are remarkably independent.
In constant conditions each eye’s ERG rhythm free-runs with a different period,
and an illumination cycle applied through fiber optics can entrain one eye while
the other free-runs (189a). These ERG rhythms probably are not controlled by
the bilaterally integrated optic-lobe clocks that regulate a variety of behavioral
rhythms. In ApLysia the eyes also lack positive coupling, but show some nega-
tive coupling (see p. 469; 158, 211). It is instructive to compare these results
to those obtained in crustaceans; their circadian systems appear to be multiply
controlled, and ERG rhythms may persist even after excision of the entire
brain (see p. 477-479; 26).
Optic-lobe lesions disrupted the circadian rhythm of stridulation in the
cricket T. commodes (220), and optic-tract cuts eliminated rhythmicity in ac-
tivity, stridulation (363), and spermatophore production (221). In Ephippiger
destruction of the optic lobes to the level of the medulla produced stridulation
at all times of day, but the intensity of stridulation was modulated by light
acting via an unidentified cephalic photoreceptor. Th ,ese insects could also be
entrained by LD cycles after destruction of ocelli, compound eyes, and the outer
layers of the optic lobes (94), by contrast with other orthopteran species.
In a further study of T. commodus, stridulation was arrhythmic in both LD
and LL after optic-lobe excision; however, a 12:l2 temperature cycle entrained
the stridulation rhythm in these animals (311). The occurrence of transients
and the failure to entrain stridulation to a 1515 temperature cycle suggest that
the 12:12 temperature cycle produced true entrainment of an oscillator and not

July 1979 NEURALREGULATIONOFCIRCADIANRHYTHMS 475
just passive driving of the system. Two hypotheses are offered (311) to account
for these results: 1) there is a damped oscillator in the brain (protocerebrum?)
that can be entrained by a temperature cycle or coupled to a light-entrained
oscillator in the optic lobes or 2) a self-sustained oscillator in the brain depends
on some contribution of the optic lobes for its ability to express circadian
rhythms and for entrainment by light. Confirmation of the findings of this study
would require modification of the generally accepted model (described below)
for control of insect circadian rhythms.
The existence of a circadian mechanism outside the optic lobes is also im-
plied by a recent report describing the persistence of a circadian rhythm of endo-
cuticular growth in bilobectomized cockroaches (BLabems fuscus) maintained
in constant conditions (222a). The rhythm is reported to persist after either
optic-lobe extirpation or decapitation, both of which should render activity
arrhythmic. Interpretation of these findings must await a more complete report
demonstrating the absence of all daily environmental cycles (including tempera-
ture and relative humidity) and the persistence of a cuticular growth rhythm
in lesioned animals whose activity is simultaneously shown to be arrhythmic.
rv) GENERAL COMMENTS. Evidence on the role of neural and neurosecretory
systems in rhythm generation has been summarized in a model (61, 62). It de-
scribes a relatively simple wiring diagram for rhythm generation that is heuristi-
cally useful, but may not reflect the full complexity of contributions by inter-
mediate neural levels to circadian organization (62, 320). The model includes
a pair of light-entrained oscillators in the optic lobes, proximal to the medulla,
whose output reaches the protocerebrum via the optic tracts. The efference
from the protocerebrum is by way of the CEC, whose activity disinhibits the
spontaneously active SEG; the resultant excitation is conveyed via the ventral
nerve cord to the thoracic ganglia to release locomotion and stridulation. The
model can now be elaborated to include interactions between the optic-lobe oscil-
lators that influence the rhythm’s period (284) and the possible existence of
(damped) neural oscillators outside the optic lobes (222a, 311).
The model adequately accounts for the data; however, reservations about
the interpretation of lesion studies in mammals (p. 486) apply here as well.
The meaning of correlations between neural damage and behavioral deficits is
not self-evident. Localized neural damage may modify the function of neural
tissue remote from the lesion site in ways that may not be anatomically obvious
(348). Additionally, loss of rhythmicity might result from damage to systems
that are not themselves oscillators, but whose function is crucial to rhythm
generation (269). Finally, specific functions may be wrongly ascribed to anatomi-
cally salient neuropiles or tracts in the vicinity of effective lesions; the correla-
tion between anatomical and functional prominence may not be perfect (61,362).
A further complication in these anatomical analyses is the difficulty of deducing
the presence and functional status of neurosecretory cells from histological
preparations (see p. 472; 62).
Appropriate description and analysis of results obtained in studies of insect
behavioral rhythms pose a second challenge. Some physiological interventions

radically alter the level of activity so that rhythmicity cannot be meaningfully

assessed; assessment of rhythm integrity is no less a problem when activity
levels stay within the normal range. In the majority of studies reviewed here
activity patterns were classified with such colorful descriptors as “diffusely
rhythmic” and “doubtfully rhythmic,” the criteria being either unspecified or
unique to each author. Agreement on criteria for determining that a particular
deficit has in fact been produced would be an important step in resolving
ambiguities in the lesion-behavior literature. The use of statistical methods to
describe and analyze rhythms (e.g., 94, 284, 311) is an appropriate first step,
but there is as yet no agreement on the proper statistical tools to use in the
analysis of short time series that may contain weak, unstable rhythms against
a noisy background.
The generality of the model presented for other insects and other behaviors
remains unknown. Studies of activity and stridulation in representatives of two
closely related orders form the basis of this model (but see 94, 114), which
already does not apply well to locomotor rhythms in one extensively studied,
phyletically distant order and is quite inappropriate to the circadian eclosion
rhythm in these insects (see below). In addition, experiments in which a single
behavioral end point is studied may yield results that are not applicable to other
circadian rhythms in the same individuals. Some discrepancies between the
effects of lesions on simultaneously recorded locomotion and stridulation
rhythms in individual crickets illustrate this point (363), as does the reported
persistence of a physiological rhythm in roaches after optic-lobe extirpation
(222a). Elucidation of such discrepancies will depend on future neurophysiologi-
cal studies involving multiple behavioral and physiological end points.
b) Lepidopteru. Truman (392, 394, 395) has elegantly investigated the
neural control of pupal eclosion and adult activity rhythms in several species
of giant silkmoths (Saturniidae).
The motor patterns of eclosion proceed relatively normally in an insect
whose brain has been excised, but the light-entrained circadian rhythm of eclo-
sion is lost and moths emerge at all times of day; reimplanting the whole brain,
in the head or the abdomen, permits normal circadian rhythmicity and entrain-
ment of eclosion (392). Both the motor pattern and phase of eclosion are species
specfic, but the hormone signaling its onset is similar across species. Implanting
a heterospecific brain transfers species-typical timing characteristic of the
brain whereas the details of the eclosion motor patterns are determined by the
abdominal ganglia of the host animal (392, 396). The brain contains the mecha-
nism for generation of a circadian rhythm, the receptor for its entrainment,
and the source of a hormone that signals the onset of eclosion activity, but is
not responsible for the particular eclosion pattern.
Lesion and extirpation studies have further localized the clock; removing
the eye imaginal discs, corpora allata, corpora cardiaca, frontal ganglion, and
SEG or cutting the optic nerves, CEC, or ventral nerve cord failed to disrupt
normal gating of eclosion in relation to the LD cycle (394). Both brain bisection
and excision of the optic lobes also permitted normal rhythmicity. The driving

oscillator is not in the optic lobes and the brain does not couple to the abdominal
effecters via neural connectives. These conclusions are different from those
reached in studies of dictyopteran and orthopteran locomotor and stridulatory
rhythms (see p. 475).
Implantation of the (proto)cerebral lobes exclusive of the optic lobes per-
mitted rhythmic gating of eclosion in brainless pupae, although the gate was
relatively broad. These and earlier results indicated an important role for the
neurosecretory cells of the PI; implantation into brainless pupae of a wedge
of tissue containing these cells did not induce substantial rhythmicity, however.
On the other hand, implantation of the remainder of the brain permitted only
random emergence (394). A hormone originating in median neurosecretory cells
of the PI appears to trigger the sequence of events leading to eclosion; the clock
and the photoreceptor for this rhythm seem to reside in the protocerebrum.
Transfer of the median neurosecretory cells unfortunately does not resolve the
question of whether the clock is also localized in these cells, because the absence
of induced rhythmicity may reflect the absence of a photoreceptor (located else-
where in the protocerebrum) or surgical trauma to the cells (394).
Do the differences between the silkmoth clock mechanism and the cockroach
and cricket clocks represent phyletic differences or difference between clocks
that control daily rhythms (locomotion, stridulation) and unique, gated events
(eclosion) (62, 393)? Truman’s (395) study of flight rhythms in giant silkmoths
provided a partial answer to this question. Section of the CEC, excision of
the brain, and implantation of the excised brain into the abdomen produced high
activity levels and arrhythmicity. Extirpation of the SEG or section of the
ventral nerve cord caudal to the SEG produced essentially inactive moths.
Clearly, locomotor rhythmicity depends on a neural, rather than a humoral,
link from brain to effecters, as is typical of other species.
Entrainment of activity in silkmoths depends on neither the eyes nor the
optic lobes. Unilateral excision of the brain and removal of the contralateral
optic lobe still permit normal entrainment. The photoreceptors for entrainment
and the rhythm generator are in the protocerebrum. Activity in the saturnid
moths, unlike activity in crickets and cockroaches, may be controlled by a proto-
cerebral clock that is directly sensitive to light, yet the coupling of brain to
thoracic effecters is not humoral, but neural, in both groups (395). The silkmoth
eclosion clock, while also localized in the protocerebrum (PI?) and directly
entrained by light, is linked hormonally to the abdominal ganglia (394).
3. Crustaceans
Both secretory structures (sinus glands, X organs) and vision-related struc-

tures of the crustacean eyestalk (ES) have been implicated in the control of
circadian rhythms. Rhythms studied include locomotor activity, chromatophore
pigment dispersal, and eye pigment migration, manifested as eye glow and ERG
rhythms.

478 BENJAMIN RUSAK AND IRVING ZUCKER Volurnt? 59
Interpretation of these studies is complicated by several factors: 1) removal

of the sinus gland induces molting only in some species, and molt-related
behavioral changes may mask other effects of ablations (47); 2) there are ex-
treme seasonal changes in rhythmicity and behavior (267); 3) tidal (12.4 h) and
circadian rhythms may be confused in some species, although they may be
regulated independently (63). Brady (62) proposed that existing information
was largely compatible with the cockroach optic-lobe model (see above) as ap-
plied to the analogous crustacean structures. More recent findings do not con-
form to this or any other explicit model.
Eyestalk ablation in the crayfish Potamobius astacus resulted in low levels
of activity and arrhythmicity (174); the loss of rhythmicity was confirmed in
several crayfish genera, but ablation was reported to increase activity (322,344).
Schallek (344) suggested that the effects of ablation were due to the interrup-
tion of neural efferents to the ES from the brain; constant high activity was
reported in crayfish whose optic nerves alone had been sectioned. Eyestalk
removal also increased activity and attenuated rhythmicity in the crab
Gecarcinus lateralis, but this effect was confounded by the intense activity
associated with the induction of the premolt growth stage (47). The tidal
rhythm of Carcinus wuzenus survived ablation of the ommatidia, but ES removal
increased activity and eliminated rhythmicity (267). Preliminary data indicate
a loss of rhythmicity in LD cycles after ES ablation in the lobster Ne@rops
norvegicus; the data are equally consistent with a loss of entrainment or with
a loss of rhythmicity (12).
Only scattered early reports were at variance with these relatively consis-
tent findings: one G. Lateralis remained rhythmic after ES ablation (47), and the
02-consumption rhythm persisted in eyestalkless Uca pugilator in LL; these
same crabs reportedly lost their tidal rhythms (63). A reexamination of the role
of the ES in rhythm generation in Procambarus clarkii has yielded quite differ-
ent results. Removal of the ommatidia and the portion of the ES containing
the lamina ganglionaris did not eliminate entrainment or phase shifting to the
LD cycle (286). Complete ablation of the ES increased activity levels, but many
crayfish remained rhythmic; by contrast, severing the CEC (128) or the ventral
nerve cord made the legs arrhythmic, although these operations should not have
affected humoral influences from the ES (285, 286). Page and Larimer (286)
concluded that rhythmicity is generated in the brain and transmitted neurally
both to the thorax to control locomotion and to the sinus glands to control
their secretions. Their ES-ablated crayfish entrained, phase-shifted, and free-
ran abnormally, in a manner consistent with the loss of only the phase-advance
portion of their PRCs; this intriguing result deserves careful examination and
replication. These findings do not indicate equivalent roles for the neuropiles
of the crayfish ES and the cockroach optic lobes.
Injection of sinus-gland extract into Procambarus bartonii in D produced
pigment migration in the eyes characteristic of L; Welsh (412) stressed that
release of hormones from the sinus glands is normally under the control of brain
efferents. The brain has been implicated in the control of ERG rhythms in

Julg zm? NEURALREGULATJONOFCIRCADIANRHYTHMS 479
P. bouti& (12, 27), but a full report of these studies indicated that low-
amplitude ERG rhythms persisted even after excision of the entire brain (26).
An earlier report (287) indicated that the ERG rhythm of P. clarkii survived
CEC section but was eliminated by optic-tract interruptions or lesions in the
midline of the supraesophageal ganglion. The importance of distinguishing
generation of rhythms from responses to ambient illumination is emphasized
by the finding that the proximal eye pigment showed no circadian migration
rhythm in DD after optic-tract section, but still responded appropriately to LD
cycles (287). The circadian rhythm of lateral eye ERG in Limulus polyphemus
is also under the control of optic nerve efferents from the brain; section of the
nerves eliminated the rhythm (24).
Pigment-dispersal rhythms in chromatophores of autotomized legs re-
portedly persisted in ES-ablated Uca pugilator in LL (409); in Processa edulis
the pigment-dispersal rhythm persisted with low amplitude for one cycle in LD,
but not in DD (275). The critical significance of test procedures cannot be
overemphasized.
The strongest generalizations permitted by the data are offered in Arechiga’s
(11) recent review. He concluded that the brain is important for the genera-
tion of some rhythms and for the control of sinus-gland secretions that may
in turn coordinate rhythms throughout the body; there seem to be several
independent sites of rhythm generation in the crustacean body remote from the
brain. Particular rhythms are likely controlled at several levels, but the rela-
tions among these mechanisms and their precise loci have yet to be elucidated.
B. Vertebates
I. Suprachiasmatic nuclei
After many unsuccessful attempts to eliminate circadian rhythmicity in rats

by various endocrine and neural ablations, Richter (316) reported the elimina-
tion of rhythms in eating, drinking, and activity in rats bearing hypothalamic
lesions. He did not, however, indicate the locus of the successful lesions except
to implicate the “ventral median” portion of the hypothalamus (316). Inde-
pendent lines of research (252, 3’70) on neural control of mammalian circadian
rhythms have focused attention during the last 5 years on the SCN of the
anterior hypothalamus. A detailed understanding of the anatomy and physiol-
ogy of the SCN is essential to the analysis of neural involvement in mammalian
circadian rhythms.
a) Anatomy. I) GROSS AND ULTRASTRUCTURAL ANATOMY. The supra-
chiasmatic nuclei are a pair of ovoid nuclei ventrolateral to the base of the third
ventricle in the anterior hypothalamus and just dorsal to the caudal border of
the optic chiasm (135). The somata of the neurons comprising the SCN are
unusually small (5- 15 pm) and densely packed (133, 196, 379, 385); cell counts
indicate that each nucleus in the rat contains about lo4 neurons packed in a

480 BENJAMINRUSAKANDIRVINGZUCKER Volume 59
volume of about 0.05 mm3 (133, 318). The highest cell density is in the ventro-
medial portion of the nuclei; dorsally, laterally, and caudally a gradual decline
in cell density produces quite indistinct boundaries in these directions (133,318).
The somata of SCN cells have scant amounts of cytoplasm surrounding the
nucleus and few dendrites, each branching only once or twice (196, 255, 385);
the densely packed somata are not separated by glial wrapping, at least in the
mouse (413). Dendrites of ventrally situated cells tend to be oriented parallel
to the chiasm, whereas those of more dorsal cells tend to be oriented parallel
to the ventricle (318,385). In Golgi stained sections axons may be seen to leave
the nuclei dorsocaudally, bending into the periventricular region and coursing
caudally along the ventricle (196, 385; see below).
The SCN contain a broad array of synaptic types and various synaptic
vesicle sizes and types, probably representing a variety of neurotransmitters
(133, 134, 223, 379). Most synapses are Gray type II (GTII: symmetrical,
axodendritic); another large proportion are asymmetrical GTI (axodendritic).
According to current conceptions of synaptic structure-function relations, about
one-third of the synapses in the SCN should be excitatory and two-thirds in-
hibitory (133). There also are a considerable proportion of axosomatic synapses
(GTII) and a small percentage of dendrodendritic synapses in the ventral half
of the nuclei (133, 134). Guldner (133) estimates that up to 2% of synapses
are dendrodendritic; using his most conservative estimate of 300 synapses/
neuron, 1% of synapses in the SCN will still represent a substanti al number
(~6 x 104). Some of these dendrodendritic synapses apparently are formed
by fine collateral branches of dendrites that loop back to terminate on their
dendrites of origin; their function is unknown (134a).
In the ventral portion of the nuclei Giildner (133) describes a complex
consisting of synapses in close proximity, not separated by glial wrapping,
but surrounded overall by astroglial lamellae. The SCN neurons contain special-
ized me mbrane structures, described as stacked or folded cisternae of the endo-
plasmic reticulum and lyin g near the cell mem brane (133, 223). The function
of these structures is not known; they have also been described in the arcuate
nucleus (223). The dendrites and somata of the SCN cells in females contain
more multivesiculated bodies than do those of males (133). Additional informa-
tion about the gross and fine anatomy of the SCN is given by Krieg (196),
Szentagothai et al. (385), and Guldner (133); ultrastructural development of the
SCN is described by Lenn et al. (210).
The pattern of vascularization of the suprachiasmatic region may be signifi-
cant to studies of SCN function. Major blood vessels enter this region from the
anterior margin of the optic chiasm and course caudally; few vessels pass dorsally
through the chiasm into the region of the SCN (207). Knife cuts or lesions
anterior to the SCN therefore may interfere with their vascular supply;although
revascularization eventually may occur, irreversible tissue damage could
already have been produced.
II) AFFERENTS AND EFFERENTS. Three major afferent pathways to the
SCN have been described. One, a projection from the midbrain raphe nuclei,

July am NEURAL REGULATION OF CIRCADIAN RHYTHMS 481
accounts for the high levels of serotonin found in the SCN (2,122,336). A second
projection from the ventral lateral geniculate nuclei (VLGN) reaches the SCN
either via the medial lemniscus or via the optic tract; the projection is bilateral
but heaviest to the ipsilateral side (313, 384). The third.major projection origi-
nates in the retina and reaches the SCN via the optic nerves and chiasm (148,
255). This retinohypothalamic tract has been extensively described and dis-
cussed in recent reports. Unlike other projections and anatomical features of the
SCN that are known almost exclusively from studies of rats, the RHT has been
described in representative mammals from many orders (76, 99, 148, 247, 388,
389,390); a retinohypothalamic projection is found in all vertebrate classes (98).
The common features of the mammalian RHT include a bilateral projection
that rises out of the chiasm, entering the SCN from its ventrolateral aspect,
terminating preferentially in the ventral and caudal portions of the nuclei,
where it forms both GTI and GTII synapses (134b). Interestingly, the projec-
tions from the raphe nuclei and the VLGN also terminate in the ventral portion
of the SCN. Possibly these several inputs are represented in the multisynaptic
complexes that are also confined to the ventral portion of the SCN (133). There
are unequal projections from each eye to the ipsilateral and contralateral SCN,
the contralateral side receiving the heavier projection in all species studied
except the chimpanzee (247, 390).
The RHT generally has been described as consisting of fine, unmyelinated
fibers rising out of the chiasm through the ventral SCN (244, 251); however,
one report describes the fibers as “myelinated” (413), and another describes
caudally entering fibers as turning to run rostrally into the ventromedial SCN
(230). Some RHT fibers have been traced to a region just posterior to the SCN
in the rat (230, 318) and in the chimpanzee (390); these may innervate SCN
dendrites that extend caudal to the SCN border as defined by cell type and
density of somata (251). The RHT may be formed by fine collaterals that branch
in the chiasm from larger, myelinated ganglion cell axons that continue in the
classic optic tracts (231, 244).
The ventrolateral cells that receive the heaviest RHT projection are larger
than and structurally different from the small, densely packed medial cells.
The ventrolateral portion receives a heavy homogenous RHT projection as as-
sessed autoradiographically, whereas the medial portion receives a patchy input
suggestive of encapsulated synaptic complexes (224). The ventral portion of the
medial SCN is also the site of the multisynaptic complexes described by
Guldner (133).
Several other projections to the SCN have also been tentatively identified.
The medial corticohypothalamic tract may carry a projection from the hippo-
campus to the SCN (196; Field, cited in 133), and anterior hypothalamic cells
lateral to the SCN may project into it (318). In addition, a variety of midbrain
and limbic structures may project to the SCN via the stria terminalis and the
periventricular tract (196, 225, 266, 385), and SCN dendrites may receive a
brainstem noradrenergic projection that surrounds the SCN (251). Some of
these supposed projections have been identified only in preparations with re-

duced-silver staining used to mark degenerating axons; this technique is

notoriously prone to both artifact and failure to show delicate fiber tracts,
particularly in the hypothalamus (see discussions in 148,385). Other techniques
are necessary to verify these projections; autoradiographic analyses and electron-
microscopic identification of degenerating terminals in the SCN after interrup
tion of presumed afferent pathways would be especially convincing.
The development of the retinal projection to the SCN has been studied
autoradiographically. Significant labeling of the SCN after injection of labeled
protein precursor into the eye was found by days Z-4 of life in rats (11, 366).
The cobalt-filling technique revealed fibers dorsal to the chiasm by day S and the
appearance of swellings that may represent synapses in the lateral SCN by
day 7 (231). Reduced-silver labeling of degenerating axons after optic enuclea-
tion revealed a projection dorsal to the chiasm in lO- to 19.day-old hamsters
(Mesoticetus a~&.&. This projection was heavier ipsilateral than contra-
lateral to the enucleated eye, and it appeared to be anterior to the developing
SCN (210a). An autoradiographic study of the developing RHT in opossums
(DideZphis marsupialis aurita) yielded similar results: at 17 days of age the
RHT appeared to project to the entire SCN, with the ipsilateral projection
being heavier. By days Ss-42 the projection had become restricted to the
ventral SCN and was heaviest contralateral to the injected eye, as in the adult
(75a). These developmental changes may result from selective cell death in the
retina or from lability of axonal projections to the SCN; alternatively, the
apparent shifts may simply represent slower growth and later arrival of fibers
forming the contralateral projection (75a).
In a strain of congenitally anophthalmic mice neurons in the SCN develop
abnormally; basal dendrites are structurally aberrant and underdeveloped,
perhaps because of a loss of RHT afferents (355). Silver (355) also reports work
in progress showing a high correlation between a loss of behavioral rhythmicity
and the degree of structural abnormality in SCN neurons. These findings may
be independent of the failure of eyes and RHT to develop in these mice, since
blinding rats on day 1 of life, before RHT development (366), produces no signif&
cant morphological changes in the SCN up to 5 wk of age (210). The epigenesis
of both structural and behavioral abnormalities in these mice deserves further
detailed study.
Injection of the surviving eye after unilateral enucleation on day 2 results
in ratios of 1. l- 1.5:1 in autoradiographic labeling of the contralateral over the
ipsilateral SCN, compared with ratios of 2-3:l in controls; there may be either
partial redirection or overgrowth of fibers from the remaining eye to the rela-
tively denervated nucleus ipsilateral to it (366). Bilateral ablation of the SCN
does not induce development of projections to other hypothalamic areas; partial
ablations result in hyperinnervation of the remaining SCN tissue, but the RHT
retains a strong tendency to innervate only the caudal and ventrolateral portions
of the nuclei (261). On the basis of these initial reports the RHT appears to be
quite conservative (i.e., topographically specific) compared with other visual

pathways whose plasticity has been documented (see 347); the plasticity of other
SCN afferents has not been studied.
Very little is known about the efferents of the SCN. The fine axons of SCN
neurons are difficult to follow in Golgi preparations; they are seen to take a dorso-
caudal, periventricular route but their terminations have not been identified
(196,385). The difficulty of producing a highly localized microinjection of labeled
precursor into the tiny, deep-lying SCN has limited the use of autoradiography
for tracing its efferents.
Swanson and Cowan (383) radioactively labeled a single suprachiasmatic
nucleus in one rat and trace d the efferent pathw rays. Al .l projections w ‘ere ipsilat-
era1 to the injection site; none were rostra1 to the nucleus. One projection
coursed medially along the periventricular tract, dorsocaudal to the nucleus;
a second traveled along the ventral aspect of the hypothalamus, passing ventro-
lateral to the ventromedial nucleus. The latter pathway projected to the capsular
zone surrounding the ventromedial, dorsomedial, and arcuate nuclei, and some
fibers appeared to reach the internal lamina of the median eminence. Some of
these projections may represent fibers of passage, since no evidence was pre-
sented for the actual termination of fibers in these areas. The intensity of label-
ing in fine, branching axons falls toward background levels, making the distal
portion of the projection difficult to delineate (383).
General features of this projection were confirmed with the use of a reduced-
silver stain for degenerating fibers after SCN ablation in the “woodmouse”
(Apodemus?) (110). However, Buijs et al. (68) presented preliminary evidence
for thin, vasopressin-containing fibers that project rostrally from the SCN into
the periventricular preoptic area. Portions of the caudal projections have also
been confirmed physiologically (194, 225); in one study approximately 15%
of 200 SCN neurons recorded from by glass micropipettes could be activated
antidromically by stimulation in the hypophysiotropic area near the arcuate and
ventromedial hypothalamic nuclei (343).
b) Physiology. Nishino and colleagues (272,273) have used a transpharyn-
geal approach to record the electrical activity of single neurons in the rat SCN.
Spontaneously active units fired at rates that ranged from <1 to 20 spikes/s in
recording sessions of several hours duration. The firing of a number of single
units oscillated spontaneously from low, irregular rates to high, sustained rates
with periods in the range of 3-5 min. Single electrical pulses delivered to the
optic nerves did not influence the spontaneous activity of SCN neurons; the
optimal rate of optic n.erve stimulation was 5-10/s, and even then the latencies
of the units’ responses were considerabl .e. These results provide strong evidence
that small-caliber, unmyelinated fibers in the optic nerves form the retinal
projection to the SCN.
Optic nerve stimulation excited 40% of the units tested and inhibited 24%;
neurons lying as little as 150 pm apart could be oppositely affected by identical
stimulation parameters (273). Sawaki (343) reported that, although single pulses
delivered to the optic nerves affected few SCN units, trains of pulses at a rate

of about l/s were more effective. The majority of responsive units were excited
by the stim ulation regardless of whether it was delivered to the ipsilateral or
contralateral optic nerve (343). Light flashes to the eyes could also either increase
or decrease firing rates of SCN units, and again most units were excited by the
stimulation (273, 343).
Other studies with light stimulation of the eyes have confirmed that SCN
units have a high response threshold, show long latencies, and are more fre-
quently excited (37%) than inhibited (20%) by light (132a, 215a). It is not clear
whether the long latencies represent slow transmission in the RHT or a poly-
synaptic input reaching the SCN via a POT-VLGN-SCN circuit (334). Several
rats with LGN lesions were tested for SCN responses to light; nearly 60% of
cells recorded from intact rats responded to light, whereas less than 10% were
responsive in LGN-lesioned rats (132a). Two possible explanations for this loss
of responsiveness are that responses to light may in fact be mediated in large
part by a VLGN-SCN projection or that LGN lesions may cause degeneration
of axons forming the POT and of the fine collaterals to these main trunks that
form the RHT (see 231, 244). The condition of the RHT after LGN ablation
has not been described.
A few reports describe the effects of neurotransmitter application on SCN
activity. Both spontaneous and glutamate-induced electrical activity were de-
pressed after iontophoretic application of serotonin to neurons of the cat SCN.
Electrical stimulation of the median raphe also inhibited SCN neural activity (53).
Nishino and Koizumi (272) found that the majority of units tested were in-
hibited by the application of monoamines, with serotonin being more effective
than norepinephrine or dopamine; most cells were also excited by acetylcholine
and to a lesser extent by glutamate. Units that were excited by optic nerve
stimulation were generally more sensitive to the inhibitory effects of mono-
amines than were those inhibited by such stimulation.
The influence of neural activity in the SCN on the activity of other parts
of the nervous system has also been studied; electrical stimulation of the SCN
at high rates (20/s) produced strong inhibition of neural activity in the superior
cervical trunks (which innervate the pineal gland), whereas lower rates of
stimulation were less effective (273). Stimulation of the SCN at high rates also
inhibited spontaneous activity of lateral hypothalamic cells. Stimulation at a
lower rate did not inhibit firing of such units but did reduce the frequency with
which they oscillated spontaneously between low and high rates (191).
These studies establish the existence of connections between the SCN and
other neural structures; interpretations of the functional nature of these connec-
tions, based on such neurophysiological data, must be tentative. First, only
SCN neurons that are spontaneously active under particular experimental con-
ditions (e.g., urethan anesthesia) have so far been studied. It is not known
whether these neurons are typical of the SCN or constitute a functionally
distinct subset of the entire population. Second, the spontaneous activity of
neurons elsewhere in the mammalian nervous system varies systematically with
time of day (25, 169, 191, 346); in addition, the responsiveness of neural tissue

July 1i.m NEURAL REGULATION OF CIRCADIAN RHYTHMS 485
to various stimuli changes with time of day (191, 227, 346). Circadian rhythms
of spontaneous activity and responsiveness of SCN neurons have yet to be de-
scribed, but they very likely exist and modify the responses obtained under
particular recording conditions. In most studies the circadian phase at which
SCN neurons were studied was not reported.
Finally, the responsiveness of SCN neurons to stimuli such as light may
vary with other experimental conditions, including: the state of adaptation of
the eye, the phase of the neuron in its short-period oscillation in firing rate (191),
and the nature and duration of the test. The last condition might be especially
important; a system involved in the control of circadian rhythms might be
expected to respond differently to brief light flashes and to extended light inter-
ruptions at various circadian phases. Therefore, it remains an open question
whether the effects of light as an entraining agent acting on the SCN parallel the
effects of light on SCN neurons as so far described neurophysiologically. Con-
siderable work will be required to provide even partial answers to the questions
raised by these investigations.
Because the suprachiasmatic region has been implicated in the regulation
of reproduction (see below), there has been considerable interest in establishing
whether the SCN contain neurosecretory neurons. The identification of SCN
projections into the hypophysiotropic area and the median eminence of rats
(194, 343, 383) suggests a role for the SCN in reproduction but does not itself
imply neurosecretory activity in SCN neurons. Studies of female rabbits (reflex
ovulators) indicated an elaboration of neurosecretory material in some SCN
neurons after copulation (81). The only class of neurons that responded to the
copulation-derived stimuli was found near capillaries; in their active state these
neurons resembled classic neurosecretory units (magnocellular neurons with
active Golgi apparatus and dense-core vesicles). Ovariectomy of rabbits also
resulted in anatomical signs of increased activity of SCN neurons that may
represent the synthesis of gonadotropin-releasing hormone (80).
In male rats only a few scattered cells, also near capillaries, were Gomori
positive and neurosecretory; such cells, located peripherally in the SCN, were
also found scattered throughout the anterior hypothalamic region (223). The
parvicellular units comprising most of the SCN proper did not appear to be
neurosecretory and showed little or no response to either long-term castration
or colchicine treatment. The authors concluded that only the magnocellular units
were likely to secrete luteinizing hormone-releasing hormone (LHRH) and that
the role of the parvicellular units of the SCN in LHRH control must be indirect
(223). Other studies with immunohistological methods have also demonstrated
LHRH-containing neurons in the SCN of guinea pigs (28, 353) and female rats
(353). The SCN of the albino mouse has been described as made up of “secretory
neurons of the peptidergic type” (413).
There is other evidence for neurosecretion by SCN cells; vasopressin and
its carrier neurophysin have been localized in the rat and mouse SCN (68, 382,
405, 406, 422). There was great variability in the number of neurophysin-
containing cells in one study; single 5-pm sections through the SCN contained

few or up to 65% of cells that had responded to the immunoreactive technique

for identification of neurophysin (423). Neurophysin and vasopressin were
heavily concentrated in the medial and dorsal portions of the SCN (405, 406);
the neurons of this area are structurally different from those of the ventro-
lateral SCN (224). Although an earlier report indicated no evidence of neuro-
physin in the SCN of humans and rhesus monkeys (423), a recent study has
localized both vasopressin and neurophysin in the same neurons of the human
SCN; no oxytocin was found in the SCN (92). Melatonin of unknown origin has
also been localized in the SCN (67) and shown to reach maximum levels at the
time of peak pineal melatonin content (152a).
The general metabolic activity of the rat SCN fluctuates with a daily rhythm
that may reflect a true circadian rhythm; nearby brain regions are not similarly
rhythmic (350). The rhythm appears at or soon after birth (120a). The technique
used measured glucose utilization during 45 min at two daily phases, one early
in the L and one early in the D phase. Glucose utilization, which was highest
in the L phase, was not diminished by turning out the lights prior to the 45min
test. By contrast, turning on the lights during the D-phase test elevated glucose
utilization to normalL-phase levels. These effects are highly reminiscent of the
differential responsiveness to light found in other physiological rhythms (19,
294). It is unknown which aspect of SCN activity is responsible for this pattern
of glucose utilization, but it is clearly not a passive response to light mediated
by the RHT (350). Other features of SCN physiology are also rhythmic; rat SCN
dissected at various phases of an LD cycle showed a daily variation in serotonin
uptake rising to a peak near the L + D transition phase (241); this rhythm
also showed seasonal modulation under unchanging lighting conditions (242).
Whether these or any other physiological rhythms in the SCN persist in con-
stant conditions or in SCN tissue cultured in vitro has not been determined.
c) Function. Functional studies of organs or tissues have classically
included observation of physiological or behavioral sequelae to destruction or
extirpation of the tissue in question. On this basis, attempts are made to guess
the contribution of the tissue to the normal function of the organism. The
procedure is fraught with logical and technical difficulties, particularly when
applied to neural tissue (348), but it has been the most frequently used technique
in the study of SCN function. Conclusions about SCN function based exclusively
on this procedure should be regarded with caution until confirmed by other
techniques (253).
There are several reasons for this caveat. First, the loss of a particular
function after a lesion does not necessarily indicate that the lesioned structure
is uniquely responsible for the generation of that function; lesion studies alone
are inherently ambiguous as to interpretation and mere consistency of effects
does not remove the ambi .guity. Studies of feeding behavior after lateral hypo-
thalamic lesions Pro lvide an instructive illustration of this point (120, 300).
Second, the generality of conclusions reached is limited by the few species that
have so far been studied ? most of the reported studies having been confined
to laboratory rats. Third, in most studies the observational and analytic tools

used were appropriate only to determine that the dependent variable was not
normally rhythmic. This restricted view of the consequence of lesions, combined
with a limited assessment of neural damage, can generate misleading structure-
function correlations predicated on inadequate descriptions of both the struc-
tural and functional changes produced. These cautions should be considered in
evaluating the results and conclusions summarized below.
I) ADRENAL CORTICOSTERONE.Corticosterone levels in the blood of groups
of laboratory rats sacrificed at intervals throughout an LD cycle peak near the
time of the L + D transition; in blinded rats the corticosterone rhythm appears
to free-run with a constant phase relation to the activity cycle (124). This daily
rise in corticosterone release can be eliminated by knife cuts posterior to the
chiasm (and SCN) but not anterior to the SCN (5,132,252). Electrolytic lesions
centered on the SCN have effects similar to those of postchiasmatic cuts (198,
252, 304). Several possible effects of the lesions could produce such results:
1) The corticosterone peak could be phase-shifted; it may occur at unpredictable
phases in each rat or it may be shifted in all rats to a phase that is not sampled
by the experimental proced ure. 2) Entrainment might be lost so that cortico-
sterone peaks free-run with circadian periods that are obscured bY the grouped
data. 8) Entrainment might be lost so that the rhythm free-runs with a non-
circadian period. 4) Corticosterone might be secreted randomly or at a steady
low rate in all rats.
Support for hypothesis 3 is provided by Wilson and Critchlow (416); a knife
was used to separate the medial-basal hypothalamus from the chiasmatic region,
and repeated blood samples were taken from the tail veins of individual rats
at 4-h intervals for 44 consec utive hours . When corticosterone values were
grouped for each phase a flat CWV ‘e was generated at an intermediate level
of corticosterone. However, only one rat showed a flat individual curve over 44 h;
others produced excursions of corticosterone levels that were as great as those
found in controls. The conclusion (416) that individual rats do not show a free-
running circadian rhythm of corticosterone secretion is premature given that
the observation period was brief (by circadian standards) and that lesioned
animals were maintained in an LD cycle. Individual records showed single or
multiple daily peaks of corticosterone secretion that are consistent with free-
running ultradian rhythms or even with free-running circadian rhythms ob-
scured by exogenous effects associated with the LD cycle.
A second study (95, 96) also used tail-vein sampling Tom SCN-lesioned
rats at 4-h intervals over 24. or 36-h periods. Again, excursions in cortico-
sterone levels of normal amplitude were reported, and these were described
as “asynchronous and devoid of circadian rhythmicity.” However, it still re-
mains possible that these rats under constant conditions would show free-
running circadian or ultradian rhythms or randomly timed peaks. These alterna-
tive hypotheses can only be discriminated by further improvement in measure-
ment techniques and by the application of appropriate descriptive and inferential
statistics to the data.
There is no firm evidence for an exogenous “masking’ effect of the LD cycle

on corticosterone, but there is evidence for other exogenous effects on cortico-

sterone after SCN lesions. Restriction of food and water intake to a brief
period in the early L phase rephases corticosterone secretion to early L; this
effect persists after SCN lesions, at least in an LD cycle (198). The contributions
of food restriction and the LD cycle to this phenomenon have not been dis-
criminated; the p.ossibility of exogenous influences on the pattern of cortico-
sterone secretion should be considered in future studies. Such evidence for a
capacity to synchronize a particular function to an exogenous agent after SCN
damage is not unique (see below); it is critical that such effects be distinguished
from the capacity for endogenous generation of a circadian rhythm. Hamsters
with SCN lesions may synchronize activity to particular phases of an LD cycle
but be incapable of generating circadian activity rhythms (330, 331).
Disruptions of corticosterone rhythms by CNS damage may be transient.
Fornix transections produce a loss of normal corticosterone entrainment but
this effect is not permanent (206). The loss of normal corticosterone rhythmicity
in LD 14:lO seen after knife cuts posterior to the chiasm may also be transient;
by 30 wk after surgery rats with cuts showed corticosterone rhythms that were
indistinguishable from those of controls (209). These findings, however, require
confirmation since data were obtained only from groups of animals and no histo-
logical results are presented. Since these studies were performed on rats ex-
posed to LD cycles, they directly address only the issue of entrainment and not
of rhythm generation.
II) PINEAL IV-ACETYLTRANSFERASE. N-acetyltransferase (NAT) is the
enzyme controlling the rate-limiting step in the production of melatonin by the
pineal gland of the rat; the daily rhythm in pineal levels of NAT has been
assayed by the sacrifice of groups of rats at several phases of an LD cycle (181).
This is probably a true circadian rhythm, although it is not technically possible
to demonstrate a free run in a single animal (182). The daily variations in the
level of NAT activity can be eliminated by knife cuts posterior to the chiasm
(253, 254) or by lesions of the SCN (253, 254, 304), but not by cuts anterior
to the SCN (253, 254); however, cuts posterior to the SCN may only partially
attenuate the daily peak of NAT activity (256). Interpretation of such studies
is difficult because of the necessary use of group data, the few time points
sampled, the presence of LD cycles, and group housing of rats. The suprachias-
matic region and the RHT apparently play a role in the entrainment and perhaps
generation of pineal NAT rhythms; there are insufficient data to support any
stronger conclusion.
Recent studies have implicated cholinergic mechanisms in the phase control
of pineal NAT rhythms; intraventricular injections of carbachol, a choline@
agonist, produce both advances and delays in the NAT rhythm (420a). Whether
the cholinergic mechanism involved is in the SCN has not yet been established.
III) WHEEL-RUNNING ACTIVITY. Most studies of activity rhythms after
SCN destruction have focused on the question of whether normal entrainment
and/or circadian rhythmicity can survive such lesions. Few studies have ex-
amined the activity patterns that do survive SCN destruction and none has

adequately assessed the correlation between behavioral effects and the physio-
logical and anatomical sequelae of the lesions (cf. 348). The degree of “nocturnal-
ity” that survives SCN destruction does not itself provide any evidence for a
role for the SCN in generating circadian rhythms; only continuous, long-term
recordings in constant conditions can provide adequate evidence on this point.
The original report of the effects of SCN lesions on wheel-running activity
rhythms of rats described a reduction of total activity and a redistribution of
activity throughout the LD cycle (370). The loss of nocturnality has since been
confirmed in other studies in which the SCN of rats were destroyed or isolated
(132, 304, 367), but no further assessment has been attempted of the nature
of the changes in activity rhythms produced by SCN destruction.
Parallel studies in golden hamsters that have included several months of ac-
tivity recording in constant conditions (dim LL) after SCN lesions have yielded
some similar results and some that differ considerably from those obtained with
rats. Some hamsters with complete or near-complete lesions of the SCN distrib-
uted their wheel-running in brief bursts throughout the day; power spectral anal-
ysis revealed no significant periodicities in the data. Other hamsters produced very
noisy activity records that contained significant ultradian periodicities (ca. 8- and
12-h rhythms) as established by power spectral analysis. Still other lesioned
hamsters generated ultradian rhythms that were easily detected both by eye
and by statistical methods (330, 331). Another report has described SCN-
lesioned hamsters as “arrhythmic” under constant conditions; this assessment
was based on visual inspection of records and was not supported by any further
analysis. The assessment is also not well supported by the two published activity
records from this study (373).
It is agreed that normal circadian rhythms of activity do not occur in rats
or hamsters after SCN lesions, but it does not follow that such animals are
“arrhythmic.” In fact, many lesioned hamsters generate very prominent
ultradian rhythms that may provide clues to the normal function of the SCN.
In some cases ultradian rhythms develop out of the dissociation of previously
coupled rhythmic elements. The normal function of the SCN may be to provide
an internal source of integration for a variety of rhythmic components; apparent
“arrhythmicity” may be the result of a complete dissociation of these elements,
whereas ultradian rhythms may reflect partial dissociation and perhaps mutual
coupling among components. Conditions that permit the expression of ultradian
rhythms or of arrhythmicity are not known; perhaps very large lesions that
damage regions adjacent to the SCN are conducive to arrhythmicity. In one
study the quality of entrainment surviving SCN destruction correlated posi-
tively with the degree of preservation of tissue at the anterior SCN-preoptic
suprachiasmatic (POSC) border (330, 331). The largest lesions destroyed both
the POSC and the SCN regions; these produced some of the most severe disrup-
tions of entrainment and rhythmicity. The involvement in rhythm generation
of the suprachiasmatic region immediately adjacent to the SCN proper deserves
further study (see p. 502).
Neural control of circadian organization may vary between rats and hamsters,

but since published studies are not strictly comparable this question must await
clarification. The possibility that detailed, long-term studies will yield similar
results across species is reinforeed by several recent reports. One described
8-h activity rhythms in mice (A&s muscuZus) after SCN destruction (403).
Other reports have described ultradian rhythms in several behaviors after SCN
lesions in rats (see below). Finally, one study has found circa 8- and 12-h
rhythms in wheel-running activity as well as other behaviors in SCN-lesioned
rats (387a).
IV) SLEEP CYCLES. The sleep-wakefulness cycle of nocturnal rodents can
be described as a sequence of episodes of waking, slow-wave sleep (SWS),
and paradoxical sleep (PS) that is repeated throughout the day, but with more
sleep episodes occurring in L and more waking occurring in D periods. This
pattern may be partly under exogenous control (“masking”) since D periods
can trigger PS episodes (218) whereas L periods may suppress PS (115). Never-
theless, there apparently is a true circadian rhythm in the distribution of sleep
episodes that persists under constant conditions (97a, 245).
The effects of SCN manipulations on sleep cycles have been studied in rats
entrained to LD cycles, with results not completely consistent across studies.
The L-versus-D difference in total sleep time was abolished immediately after
SCN ablation (156). Blinded rats developed an apparently free-running sleep
waking cycle that retained normal internal sleep structure; total sleep time and
relative proportions of sleep stages were both normal in blinded and in lesioned
rats. This last point was also made by Coindet et al. (84), who reported that PS
was distributed evenly in L and D periods after an SCN lesion. In their study
SWS remained concentrated in L after SCN lesions, although the L:D sleep
ratio was reduced compared with control levels. Interpretation of these findings
is difficult because results for SCN-lesioned rats were pooled on the grounds
that they were all “in phase”; the meaning of that phrase is not made clear.
Stephan and Nunez (367) attempted to isolate the SCN using a “core knife”
that produced considerable damage to many parts of the brain; interpretation is
difficult because control recordings yielded unusual sleep cycles (apparently
because of inadequate adaptation to the recording apparatus). In lesioned rats
the L:D distribution of sleep approached 50%, but 3 of 6 lesioned rats clearly
concentrated PS in the D phase. Whether this residual PS rhythm or the residual
SWS rhythm (84) is endogenous or imposed by the LD cycle has not been
established.
Another study recorded sleep cycles of female rats for 8-10 days in LD
14:lO after SCN lesions. The sleep records showed a flat distribution of SWS
across the LD cycle, but PS seemed to peak late in the ,L phase. A larger
anterior hypothalamic lesion that destroyed the SCN flattened both rhythms
in a single rat; no statistical analyses of the data were presented (419a).
Interestingly, lesions in other neural structures often produced sharp peaks
of PS in the D phase (419a); the extensive neural damage caused by the “core
knife” used in the study by Stephan and Nunez (367) also resulted in PS con-
centration in the D phase in some rats. However, the many procedural and

analytic differences among these several studies make it difficult to reconcile

or explain their inconsistent results.
In the most complete study of sleep, power spectral analysis was applied
to the sleep records of rats studied for up to 3 mo after SCN ablation (155).
Rats with lesions that spared the SCN, rats with POT lesions, and peripherally
blinded rats also were studied. Lesions of the POT did not affect the L:D dis-
tribution of sleep; unilateral lesions and those that missed the SCN caused slight,
transient attenuations in rhythmicity. Rats in constant conditions showed clear
freerunning circadian rhythms initially, but over many weeks ultradian pe-
riodicity in the 4- to 7-h range was enhanced while the circadian component weak-
ened (97a, 155; cf. mice, 245). Lesions of the SCN produced an immediate redis-
tribution (i.e., equalization) of PS and SWS between L and D phases and also
enhanced ultradian rhythms of PS and SWS with periods in the 2- to 4-h range.
These findings suggest that the circadian modulation of sleep cyclicity in rats
and mice is rather weak and subject to gradual diminution in constant conditions.
The RHT apparently is critical to the maintenance of L:D differences in sleep
in rodents exposed to an obligatory LD cycie.
V) OTHER CIRCADIAN RHYTHMS. Several other behavioral and physiological
rhythms have been studied, generally in LD cycles, after SCN lesions or
hypothalamic cuts near the SCN. Water intake of rats was distributed evenly
throughout an LD cycle, and there was no indication of a free-running rhythm
after large lesions centered on the SCN (370), “isolation” of the SCN with a
core knife (367), small lesions restricted to the SCN (404), or semicircular cuts
posterior to the SCN (279). In hamsters, SCN lesions also eliminated the usual
nocturnality of drinking in LD cycles and the generation of circadian rhythms
in constant conditions (330,331). Nocturnal&y of eating was also reported to be
eliminated by SCN lesions (265,404) or semicircular cuts posterior to the SCN
(279), but a recent study has indicated persistent nocturnality of food intake
after SCN lesions (V. Critchlow, personal communication). Rats with partial
SCN damage showed a reduction in the nocturnality of feeding and drinking
as assessed by autocorrelation, and some rats with >50% SCN damage gener-
ated 8-h feeding or drinking rhythms (404; cf. 387a).
Daily temperature cycles of rats in LD have been reported to be
eliminated by SCN lesions (198, 337a, 338) and by SCN “isolations” (367),
but another report has indicated that a daily temperature peak in the D phase
persists after SCN ablation (95,96). These studies generally used only grouped
data or infrequent sampling; preliminary results indicate that continuously
monitored body temperature of individual hamsters fails to show normal
rhythmicity in LD after SCN lesions (Rusak, unpublished observations). Tumor-
induced increases in third ventricular pressure can disrupt human body tem-
perature rhythms; relief of the pressure can restore normal cyclicity (283).
These Endings suggest a role for the SCN in the control of body temperature
rhythms, but the very sparse data available and the fact that adjacent neural regions
are known to be involved in temperature regulation (140) make stronger con-
clusions premature.

Daily rhythms of hippocampal norepinephrine content (256) and of heart

rate (338) were disrupted by lesions or knife cuts in the SCN of rats. Large
anterior hypothalamic lesions that included the SCN eliminated entrainment
of growth hormone rhythms to the LD cycle (313a, 415a), while the 3-h rhythm
of hormone release persisted as in unlesioned rats (415a). Rhythms in the
activity of hepatic and renal enzymes involved in gluconeogenesis were also
eliminated by SCN lesions (265a). In squirrel monkeys (Saimiri sciureus )
lesions aimed at the SCN reduced the stability of entrainment and the amplitude
of circadian variation in LL of several rhythms, but the rhythms appeared
to survive these lesions relatively intact (121); whether these lesions actually
damaged the SCN has not been reported. Rats normally show cyclic retention
deficits with a 24-h period after training in a one-trial avoidance task (152).
Lesions of the SCN eliminated the retention deficits at 18 h and 30 h after train-
ing that are presumed to be under circadian control, without affecting the deficit
at 6-h posttraining (366a, 408). Finally, the behavioral sensitivity of female
rats to estradiol (measured by the lordosis quotient) is rhythmic in an LD cycle;
this rhythmicity was abolished by SCN ablation (139).
VI) GENERAL COMMENTS. The growing catalogue of rhythmic functions
disrupted by damage to the RHT-SCN and environs attests to the unique role
this neural complex plays in the temporal organization of mammalian (or at least
rodent) physiology and behavior. A further proliferation of similar studies
examining other functions and other species would permit assessment of the
generality of this role, but would add little to our understanding of the function
of the RHT-SCN complex in mammalian temporal organization. The published
data are a rich source of unanswered questions whose study would add sorely
needed analytic depth to the observational breadth already available. In all
such studies it will be absolutely essential to maintain the distinction between
the presence of exogenously synchronized or light-driven rhythms and the pres-
ence of endogenously generated circadian rhythms; failure to do so muddles
both formal analysis and physiological reality. The following are questions we
consider of particular importance in elucidating the role of the SCN in rhythm
generation.
1) Is there a functional differentiation of cell types in the SCN (224, 405)
or projections from the SCN? There is evidence that lesions that would not
disrupt behavioral rhythms can block testicular regression in hamsters exposed
to short photoperiods (373; Rusak, unpublished observations), that eating and
drinking rhythms might be influenced by slightly different substrates (404),
and that corticosterone and temperature rhythms may be partially dissociated
(V. Critchlow, personal communication).
2) How do observed anatomical and microstructural features relate to pre-
sumed mechanisms for entrainment and rhythm generation? For example, the
presence of dendrodendritic synapses and multisynaptic complexes might be
related to coupling among SCN oscillatory units or to the integration of rhythms
generated elsewhere in the nervous system.

July n.-m NEURAL REGULATION OF CIRCADIAN RHYTHMS 493
3) To what degree do lesion effects reflect destruction of SCN neurons

as opposed to disruption of adjacent pathways and nuclei?
4) How does light influence SCN neural activity at various stages of the
circadian cycle, and which are the receptors responsible for transducing these
influences?
5) By what pathways do light and other external agents influence rhythmic-
ity after destruction of the RHT-SCN complex? What are the targets of these
influences?
6) How do neurotransmitters, and their agonists and antagonists, as well
as electrical stimulation influence the SCN’s ability to generate circadian rhythms
and to respond to light stimuli?
7) To what extent are neurosecretory cells in the SCN (LHRH; vasopressin/
neurophysin) functionally equivalent to similar neurons elsewhere in the hypo-
thalamus? Do these units have a special role in relation to neurosecretory
rhythms, and are they functionally related to nonsecretory SCN neurons in-
volved in rhythm generation?
8) To what extent do observed alterations in function after SCN destruc-
tion permit conclusions about normal SCN function?
9) What objective criteria should be used to assess the existence and
parameters of rhythms? This question refledts the need both for more sophisti-
cated analyses of results and for improvement in the methodology of data
collection.
IO) Does the loss of normal entrainment and rhythmicity after SCN de-
struction have unfavorable consequences for individuals? What are the empirical
“real-world” penalties for disruption of circadian organization?
2. Other brain regions
a) Lateral hypothalamus. Lateral hypothalamic area (LHA) lesions exag-

gerate the nocturnal character of rat eating and drinking rhythms. After LHA
lesions rats fed ad libitum consume less than 1% of their daily water intake
during the 12-h L phase compared with a value of 9% for control rats (328).
Unlike neurologically intact animals, many of the brain-damaged rats injected
with NaCl during the L phase postpone drinking to the D phase. After a fast,
recovered LHA rats, unlike control animals, wait until the D phase to begin
drinking even when restoration of food and water commences during L. Blinded
animals recovered from LHA lesions and injected with NaCl at the beginning
of the inactive phase wait approximately 12 h to ingest most of their water,
although they demonstrate the appropriate behaviors with short latencies if
treated during the active phase (328). Lesions producing these effects damaged
substantial parts of the far LHA, producing extensive degeneration in the POTS
and disruption of the ascending nigrostriatal dopaminergic and dorsal adrenergic
bundles. Rats bearing such lesions are unable to respond to certain stimuli

494 BENJAMIN RUSAK ANI? IRVING ZUCKER Volume 59
during their inactive phases; the consequences of these stimuli persist and are
acted on in the succeeding active phase.
Free runs in eating and drinking rhythms in LL were documented for some
recovered LHA rats and occurred as well in blind LHA rats (327). The
redistribution of behavior to the D phase thus is not due to destruction of a
circadian alimentation clock. Nor is changed sensitivity to light a plausible
explanation, since redistribution of behavior to the active phase occurs in
peripherally enucleated rats with LHA damage (328). The most likely conclusion
is that postdeprivation food and water intakes and responsiveness to injected
NaCl are “modulated according to the phase of an endogenous oscillator” (328).
What remains to be discovered is the nature of the change induced by the lesions
that can alter the influence of the circadian system and decrease plasticity of
regulated behaviors.
b) Ventromedial and dorsomedial hypothalamus. Rats fed ad libitum gain
weight during D, lose weight during L, and undergo D- and L-phase cycles
of lipogenesis and lipolysis (173, 205). Damage to the VMH reverses the die1
weight rhythms and interferes with the lipogenesis-lipolysis cycle (173, 205).
The nocturnal drinking rhythm of rats is attenuated but not eliminated
by VMH damage (125, 173). A nearly equal distribution of food intake is re-
corded in the L and D phases after VMH damage (42, 173); this contrasts with
the robust nocturnal feeding observed in neurologically intact rats (425).
Integrity of the VMH may be required for the expression of the die1 feeding
rhythm. It is highly unlikely that the disturbances are in any way directly
related to impaired entrainment to the LD cycle. Hyperinsulinemia and pe-
ripheral metabolic changes consequent to VMH destruction (120, 205) most
likely override the normal circadian organization and entrainment of feeding.
In addition VMH lesions may interrupt critical SCN efferents to motor systems
involved in feeding.
Lesions of the dorsomedial hypothalamus also attenuate the nocturnal
feeding rhythms of rats (42). Both VMH and DMH damage eliminate diurnal
fluctuations in plasma corticosterone concentrations (35). The lack of multiple
sampling from individual animals and the possibility of free runs in hormone
secretion during exposure to the LD cycle are recognized by several authors
as procedural shortcomings (e.g., 35); in these studies, rhythm elimination
might also reflect interruption of SCN efferents (381, 383).
c) Miscellaneous structures. Slusher (359) was among the first to report
elimination of diurnal differences in rat plasma corticosterone levels after
hypothalamic damage; her effective lesions were in the anterior periventricular
zone and appeared to include the SCN or its immediately caudal efferents.
Damage to the fornix was originally thought to eliminate the rat die1 cortico-
sterone rhythm (246), but subsequent work demonstrated that the effect was
transitory and normal entrainment was recovered by the third postoperative
week (206). Fornix transection, hippocampal ablation, and destruction of the
septal nuclei each allows retention of the entrained corticosterone rhythm

July 19?‘9 NEURAL REGULAT-ION OF CIRCADIAN RHYTHMS 495
in individual lesioned animals (416, 417). The septum and fornix cannot be the
source of the anterior connections to the medial basal hypothalamus necessary
for the expression of entrained corticosterone cycles in rats, although the fornix
may play a more significant role in primates (113, 232).
Corticosterone content of the rat preoptic area, hippocampus, mesencepha-
lon, amygdala, and anterior pituitary undergoes die1 variation similar to that
of plasma corticosterone (208). Hypothalamic corticosterone content also varies
rhythmically with peaks and troughs out of phase with those of the plasma and
of the other brain regions measured. Frontal deafferentation eliminates the
rhythms in corticosterone content of the hypothalamus and preoptic area (208).
Allen and Allen (4) report an absence of plasma corticosterone rhythms after
transection at T3 of the rat spinal cord. This surprising finding may reflect
nonspecific postsurgical trauma rather than involvement of the spinal cord
in entrainment or mediation of adrenal corticosterone secretion.
The midbrain raphe nuclei are sites of serotonin-containing perikarya that
project to the SCN (2) and presumably are responsible for these nuclei having
high serotonin concentrations (122, 336). Raphe lesions substantially reduce
forebrain (including SCN) serotonin content (193, 199, 222). A transitory de-
crease in the L:D ratio of locomotor activity was observed after dorsal or com-
bined dorsal and medial lesions of the raphe nuclei (178). Block and Zucker (52)
also noted that an increased proportion of daily wheel running occurred during
the L phase in LD 12:12 after damage to these nuclei. The lesions were less
effective in interfering with the normal L:D distribution of drinking behavior (52)
and also failed to alter the rhythm of pineal NAT activity (254) or of adrenal
corticosterone (326). The assessment of reentrainment of wheel-running activity
after a phase shift in the photoperiod was complicated by the loss of a well-defined,
stable, phase-reference point after raphe damage (52). Clearly raphe lesions
neither eliminate nor drastically impair steady-state entrainment and phase
shifting of several CRs. It is less certain whether this type of neural damage
affects more subtle parameters of circadian organization and entrainment.
In constant dim light, raphe-lesioned rats were more active at all phases than
controls but still generated free-running circadian rhythms with periods com-
parable to those of control rats (52). Large reductions in forebrain serotonin
and the elimination of the raphe projection to the SCN are therefore consistent
with the generation and expression of CRs; such evidence supports the claim
that the SCN constitute an endogenous neural oscillator.
Removal of the superior cervical ganglion (SCG) eliminates entrained
rhythms in pineal NAT and serotonin. This is a consequence of pineal denerva-
tion and is not attributable to interference with visual pathways for entrain-
ment (250). The behavior of the rat feeding rhythm after SCG removal is less
readily explained in terms of pineal denervation, since SCG ablation but not
pinealectomy retards the rate of phase shifting from an LL free run to entrain-
ment to an LD cycle (30). Baum (30) suggests that postganglionic fibers from
the SCG modify neural activity in hypothalamic regions that synchronize

the feeding rhythm. The paradigm used in this study (shifts from LL to LD)
permits only the most tentative conclusions since the animals’ circadian phases
were not equated in the experimental and control groups at the time the LD
cycle was imposed.
d) NewaL rhythms. We have emphasized the role of neural structures
in regulation of CRs and have neglected the many neural parameters that
themselves undergo diurnal fluctuations. The list of CNS rhythms is long and
growing rapidly. Ziegler and co-workers (421) cite studies showing anterior and
lateral hypothalamic, spinal cord, midbrain, caudate, brainstem, and frontal
cortex variations in norepinephrine content and report a diurnal rhythm of
norepinephrine in monkey cerebrospinal fluid. Significant diurnal variations in
serotonin occur in the amygdala and midbrain of rats (358) and in the telencepha-
lon of hamsters, gerbils, and guinea pigs (292). Many rat brain regions show
cyclic fluctuations in dopamine content (358).
These many rhythms tell us little about the neural bases for circadian
rhythms. They represent additional parameters that may facilitate study of
neural regulatory mechanisms for CRs. As such they have various drawbacks;
they are less easily quantified than any of a number of behavioral variables
and do not lend themselves to long-term study in individual animals. The vast
majority of these rhythms must reflect oscillations in remotely located neural
pacemakers and are not likely to be part of the basic oscillatory network.
Various neural structures generate rhythms of spontaneous electrical
activity and of responsiveness to neural input. The lateral hypothalamus is a
particularly well-explored area: single neurons in the rat LHA cycle spon-
taneously over several minutes from low to high firing rates; stimulation of
other neural structures may change the firing rate, eliminate the oscillation,
or simply slow the oscillation without changing the mean firing rate (191).
Neurons in the LHA also show longer period fluctuations in activity (346);
single units respond with opposite sign to identical stimulation of the splanchnic
nerve at different phases of the daily cycle (191). Another study demonstrated
either increases or decreases in food intake of rats depending on the time of day
at which norepinephrine was injected into the LHA (227).
Such examples could be multiplied; they raise intriguing questions about
the kinds of information conveyed by cycles of neural activity to other portions
of the nervous system. If the instantaneous firing rate carries information in the
nervous system, how do ultradian and circadian changes in spontaneous firing
rate (346), chemical sensitivity (227), and synaptic efficacy (25) influence infor-
mation transmission? These interesting observations do not bear on the issue of
rhythm generation, however, unless the observed rhythms can be shown to
persist in isolation or to be significant to generation of circadian rhythms else-
where. In one study, isolating a hypothalamic island, including the SCN, from
the rest of the brain eliminated circadian rhythms of multiple-unit activity in
several brain regions; rhythms within the hypothalamic island survived the
operation (180b). It will be important to determine if rhythms in other parts
of the hypothalamus persist after separation from the SCN.

July 19?‘9 NEURAL REGULATION OF CIRCADIAN RHYTHMS 497
3. Hormonal injbences on circadian clocks
Circadian pacemakers underlying the activity cycle were long thought

to be independent of hormonal regulation. Richter (315, p. 21) states that
“removal of the endocrine glands -adrenals, gonads, thyroid, pineal, pancreas,
pituitary -had no effect” in speeding up or slowing down or entirely stopping
the clock. Recent studies of birds and mammals indicate that hormones, through
their actions on the neuroendocrine system, regulate the circadian system.
The period of the mouse (A4us musculus) activity rhythm lengthens
gradually after castration (90). Implants of testosterone decrease 7 by 0.22 h,
whereas control procedures have no effect. A Citellus lateralis that did not
become torpid in its winter phase shortened its 7 for activity as body weight
decreased and increased 7 as body weight recovered (264). As in Mus, shorter
7 values were associated with active reproductive organs and presumably with
elev vated serum androgen levels.
In female golden hamsters exposed to an LD cycle the time of onset of
activity fluctuates, beginning earlier on days 3 and/or 4 of the estrous cycle
(259,260,426). This phase shift coincides with increased estradiol secretion (23).
The phase angle of activity onset becomes more negative after ovariectomy
and can be restored to its original value by implants of estradiol benzoate (EB).
The free-running 7 of blind ovariectomized (OVX) hamsters can be shortened
by EB treatment (260); estradiol may help maintain internal synchrony among
oscillators (258a). Sex differences in responsiveness to EB also have been
established: EB either has no effect or lengthens the 7 of activity rhythms
of adult castrated male hamsters. This sex difference is subject to perinatal
sexual differentiation; female hamsters masculinized by a single injection of
testosterone propionate early in life respond as males do and do not shorten
their 7 values when treated with EB in adulthood (426a). Hypophysectomy
substantially lengthens the activity rhythm 7 of male hamsters in LL and DD
and of females in DD (426a).
Collectively, these studies establish that mammalian hormones regulate
the circadian system, most likely via direct actions on central time-measur-
ing systems.
Studies of birds reinforce this conclusion and extend the range of effective
hormones. Sparrows ( Passer domesticus) treated with melatonin shortened
their activity rhythm T in DD or became continuously active (397). Annual
changes in the amount and periodicity of activity of Sturnus vulgaris were
correlated with seasonal changes in hormone secretion (136). Activity was re-
corded for these starlings in dim LL and in DD for 3-mo intervals after transfer
from outdoor aviaries at four different times of the year; 7 was longer during
the reproductive season than at other times. The implication that increased
androgen secretion was responsible for this finding was not supported by an
experiment that failed to demonstrate effects of castration or. testosterone
treatment on 7 (136).
Hormones of the adrenal cortex can set the phase of other daily rhythms,

and they can influence rhythm amplitude and sensitivity to other stimuli (236).
The presence of corticosterone, for example, can determine physiological and
behavioral responses to prolactin in all major vertebrate classes (236). The most
striking example is the phase relation between corticosterone and prolactin as
a determinant of the onset of gonadal development, migratory restlessness,
and direction of migratory flight in Zonotrichia a&co2Eis (236). In adrenalec-
tomized monkeys cortisol entrains the urinary K+-excretion rhythm without
changing feeding rhythms (258). Since the LD cycle ordinarily entrains corticoid
rhythms in humans and other animals (236), it ultimately entrains most if not all
bodily rhythms in complex multicellulars. As noted by Scharrer (345), this is a
parsimonious solution to a potentially difficult problem of synchronization of
many oscillators with different or even identical xeitgebers.
4. Pineal gland
In some vertebrate classes the pineal complex may function as a biological

clock. The evidence is strongest for avian species but falls short of conclu-
sive proof.
The pineals of fish, amphibians, and reptiles are photosensitive; in the
course of evolution the pineal is transformed from a photosensory and endocrine
organ into one that retains only endocrine functions (305). There is no electro-
physiological evidence of direct conventional photoreception in bird or mammal-
ian pineals (305), although recent work with Japanese quail suggests that light
affect&he pineal via retinal pathways (149).
a) Birds. In Passer domesticus pinealectomy abolishes free-running CRs
of locomotion (perch hopping) and of body temperature (238). In white-throated
sparrows (Zonotrichia albicohs) pineal removal eliminates the nocturnal CR
of migratory restlessness in spring and fall and the CR in summer activity (235).
Synchronized activity and body temperature can be restored to the arrhythmic
pinealectomized (PINX) birds by imposing an LD cycle (235, 240).
Pineals transplanted into the eyes of arrhythmic PINX Passer reestablish
locomotor circadian rhythmicity (424) Zimmerman and Menaker (see 239) also were
able to influence the phase of locomotor rhythms by transplanting pineals from
donor birds housed in different photoperiods. The chicken pineal in vivo exhibits pro
nounced circadian rhythms in NAT content (306). Technical problems may
earlier have prevented observation of a persistent NAT rhythm in vitro (45),
but a recent study demonstrated persistence of the rhythm in chicken pineals
mai .ntained in tissue culture for several days in DD (18Oa). Taken tog&her
these observations support Menaker’s claim (238) that the pineal is either a
master circadian oscillator or a coupling device between such a pacemaker and
damped light-sensitive oscillators.
Neural input to the avian pineal is from the SCG; the only efferent path-
way is a tract of fibers of unknown termination that leaves the pineal through
its stalk (306, 424). Interruption of this pathway does not abolish the free-
running CR of locomotion in Passev domesticus (424). The conclusion suggested

July 1979 NEURAL REGULATJON OF CIRCADIAN RHYTHMS 499
by these findings is that the pineal drives remotely based CRs through hormonal
rather than neural channels (240,306). Implants of melatonin, a putative pineal
hormone, produce arrhythmicity or significant changes in 7 in the Passer loco-
motor rhythm (397), ftiher implying a hormonal link between the pineal and
other oscillators.
The role of the pineal as an avian clock is brought into question by the ob-
servation by Rutledge and Angle (335) of normal free-running circadian organi-
zation in the perch hopping of Sturnus vulgaris recorded 6 mo after pinealec-
tomy. Histological confirmation of the total absence of pineal tissue is claimed;
if this can be confirmed, it would establish that the pineal is not the oscillator
in this species.
The preliminary report of a disruption in the Passer locomotor rhythm
after lesions in the vicinity of the SCN (386) creates some problems for a pineal-
clock hypothesis, although the pineal could still function as a driving oscillator
whose coupling with driven oscillators is mediated by the hypothalamus. This is
plausible because the SCN and other parts of the hypothalamus are prime
target tissues for melatonin of pineal origin (306). Rhythms in melatonin secre-
tion (306) may entrain nervous structures to generate a variety of bird CRs.
b) Reptiles. Removal of the pineal and parietal eyes of the lizard SC-
ohvaceus exposed to LL produced large changes in 7 in about half the animals,
arrhythmicity in most of the remaining animals, and, in a single animal, splitting
of the activity rhythm into two components with different 7 values (400).
Pinealectomy and not parietalectomy appears responsible for these effects. The
persistence of circadian rhythmicity in pinealectomized lizards housed in DD
(400) argues against the view that the pineal is a master oscillator; however,
Underwood’s study (400) supports the more conservative claim that the pineal
is a coupling device or part of the effector apparatus for circadian rhythmicity.
c) Mammals. The pineal functions as a neuroendocrine transducer, con-
verting neural into hormonal signals; it receives neural input from superior
cervical postganglionic fibers that terminate in the proximity of pinealocyte
processes (250). After superior cervical ganglionectomy pineal rhythms are
abolished, including the L:D variation in the number of granulated pinealocytes
(37). Transection of the human spinal cord above C8 eliminates diurnal variations
in melatonin excretion (185) and pineal rhythms can also be abolished by a variety
of surgical procedures within the CNS, each probably the equivalent of a central
sympathectomy (250, 340). These arrhythmicities, far from establishing the
pineal as a clock, suggest that its secretory rhythms are dependent on periodic
input originating elsewhere.
Although the pineals of a number of mammalian species exhibit CRs (329)
in no instance has pineal removal interfered with normal circadian organization.
The pineal does not function as a master circadian clock in the few mammals
where this proposition has been tested (303). The pronounced effects of the
pineal on reproductive rhythms of photoperiodic mammals are more readily
attributable to pineal antigonadal activity (309) than to pineal-induced changes
in circadian organization.

IV. MAMMALIAN REPRODUCTIVE CYCLES
In this concluding section we consider the neural and circadian organization

of mammalian reproductive cycles. The material illustrates the value and limita-
tions of the biochronometric approach and the complexity of the issues waiting
to be resolved in this classic area of regulatory biology.
Circadian rhythms mediate measurement of day length and hence phase the
seasonal breeding cycles of some temperature-zone birds and mammals; CRs
also determine the precise timing of reproductive cycles of female mammals
within each breeding season. For 30 years the neural bases of estrous cycles
have been investigated intensely. During this time substrates for photoperiodic
time measurement have not received the experimental consideration warranted
by the importance of the subject.
A. Estrous and Menstrual Cycles
I. General features
Nonpregnant spontaneously ovulating mammals undergo repeated estrous

cycles in which the interval between successive recurrences of ovulation or be-
havioral receptivity (estrus) is relatively constant and species specific. For
example, the estrous cycles of the hamster, guinea pig, and rhesus monkey
are approximately 4, 16, and 30 days long, respectively. The suggestion that
the length of the cycle may be of secondary importance and not subject to great
selection pressure (354) is difficult to reconcile with the functional significance
of short estrous cycles for short-lived species; short cycles promote breeding
after a nonpregnant cycle, after interruption of pregnancy, or after departure
of the young, and they facilitate production of multiple litters during a single
breeding season. Evidence reviewed below suggests that neural control of
anterior pituitary hormone rhythms is different in species with long estrous
cycles compared with those having short cycles. Regardless of cycle length,
the occurrence and timing of ovulation are important to reproductive success;
they constitute the proximate event that determines the time of birth. Few mam-
malian species, with the notable exception of those capable of delayed implanta-
tion (337), can control the length of gestation after the ovum has been fertil-
ized (354).
The sequence of events in the ovary, pituitary, and brain during the rat
estrous cycle is typical of other mammals (177,268): a cohort of ovarian follicles
is selected for development during each cycle. Under the influence of pituitary
follicle-stimulating hormone (FSH) the follicles grow, secreting estrogens.
When estrogen levels attain a predetermined value they inhibit further FSH release
via negative-feedback mechanisms and eventually exert a positive-feedback effect
on the secretion of luteinizing hormone (LH). This culminates in the preovulatory
surge of LH and ovulation. Progesterone also facilitates the LH surge (see 268)

July 19?‘9 NEURAL REGULATION OF CIRCADIAN RHYTHMS 501
and acts synergistically with estrogens to induce behavioral estrus. Corpora

lutea then secrete increasing amounts of progesterone; in the absence of mating
the rat’s corpora lutea regress, progesterone secretion declines, and the cycle
is repeated. Rat corpora lutea are nonfunctional-i.e., they secrete insufficient
progesterone to permit a decidual reaction in the unmated animal; in other
species (e.g., guinea pig, monkey, human) the corpora lutea secrete enough
progesterone to support a decidual response and there is a prolonged luteal
phase that lengthens the estrous or menstrual cycle.
The secretion of FSH and LH is regulated by releasing factors synthesized
within the nervous system. In particular, luteinizing hormone-releasing hormone
(LHRH) manufactured by hypothalamic neurosecretory cells reaches the
pituitary via the hypophyseal-portal vasculature to influence the timing of the
critical preovulatory LH surge. In some polycyclic rodents (e.g., rats, hamsters)
a CNS circadian clock appears to regulate timing of the ovulatory surge of
gonadotropins (6, 233). A minority of investigators suggest that estrous-cycle
periodicity occurs only in response to feedback effects of ovarian steroids and
reflects the time taken to develop a new crop of ovarian follicles to the point of
adequate estrogen production. In this scheme periodicity is inherent in the time
lags of the cycle’s component parts and not in a neural clock (118,354). Evidence
introduced in the next sections indicates that this hypothesis is viable for some
species but must be rejected for others.
2. Biological clock control
Welsh (411) speculated in 1938 that “the same internal mechanism which
is responsible for maintaining, in the rat, an estrous cycle of definite dura-
tion . . . may be, in part responsible for the maintenance of 24 h cycles of
muscular activity.” Observations by Everett and Sawyer (109) also suggested
that a neural signal generated at circadian intervals is essential to ovulation.
Drugs injected during a critical period, which in individual rats is as short as
30-45 min daily (130), delay ovulation by 24 h. The positioning of the daily
critical period is completely controlled by the LD cycle, further implicating the
circadian system (7).
Exposure to LL lengthens the period of the A&s musculus estrous cycle (74)
and shortens cycle length in the baboon Papio cynocephalus (137). Although
free runs were not established for these species, the findings are in accord with
the circadian rule for nocturnal and diurnal species, respectively, and provide
circumstantial evidence for clock control of estrous and menstrual cycles,
Alleva et al. (6) first demonstrated that a mammalian estrous cycle is regu-
lated by a biological clock. Hamsters exposed to an LD 16:8 photoperiod had
estrous cycles with 7 = 96 h; in LL the onset of heat free-ran with 7 significantly
greater than 96 h. The latter finding has been replicated with hamsters main-
tained in constant dim illumination (116).
More recent work has definitively established a circadian link to the hamster
estrous cycle. The period of the estrous cycle under free-running conditions

502 BENJAMIN RUSAK.AND IRVING ZUCKER Volume 59
is four times the period of the concurrently recorded circadian activity rhythm.
Disruptions of the circadian system produced by exposing the animal to LL
invariably also interfere with normal estrous cycles (116). These findings compel
one of two conclusions: either the hamster estrous cycle is subject to regulation
by the same circadian clock that times the activity rhythm or the two rhythms
are generated by separate but closely coupled oscillators.
Fitzgerald and Zucker (116) suggest that the stimulus for the ovulatory
surge of gonadotropins in this species is generated by a circadian system that
includes the SCN. Evaluation of this hypothesis is dif%ult because the estrous
cycle is dependent on many variables involving different levels of organization,
some entirely unrelated to biological clocks. Interpretation of findings involving
brain lesions, the main source of relevant data, is hazardous because of the
compactness, close proximity, and overlap of the neural systems that 1) manu-
facture gonadotropin-releasing hormones (GnRH), 2) generate circadian
rhythms, and 3) serve as target tissues for positive-feedback effects of ovarian)
steroids. Because of notable species differences we consider the involvement
of the SCN separately for each of several mammals. A complete catalogue of
relevant studies is beyond the scope of this review; our bias has been to cite
recently published reports that contain references to the older literature.
3. Neural substrates
a) Rats. I) SUPRACHIASMATIC NUCLEI. C&&low (86) reported that lesions

of the SCN produced estrous acyclicity and was ahead of his time in suggesting
that entrainment of the estrous cycle by the photoperiod was mediated by
a direct retinohypothalamic tract that terminated in the SCN. His findings
challenged the then-prevalent idea that the preoptic area (POA) contained the
substrate that generated the neurogenic stimulus for ovulation. Recent work
has confirmed the importance of the SCN as the probable site of the neurogenic
trigger for LH surges (65, 131, 304). Knife cuts that separate the SCN from
the medial basal hypothalamus (MBH) lead to anovulation; if the cuts are rostra1
to the SCN, however, ovulation or luteinization is maintained (146). Approxi-
mately 90% of rats with large SCN lesions cease ovulating (304); lesions are
effective regardless of the extent of damage to other neural structures. Ovariec-
tomized rats with lesions restricted to the SCN show a virtual absence of LH
surges when treated with estradiol and progesterone (131), although the
negative-feedback response to these steroids is intact and serum LH values
are within the normal range (65, 131). These rats manifest increased LH secre-
tion when treated with LHRH (131). This suggests that the lesion interferes
with rhythmic release or transport of LHRH to the pituitary; alternatively,
elimination of rhythmical SCN influences exerted on the pituitary via hormones
other than LHRH may preclude the normal pituitary response to LHRH. Recent
studies that differentiate between the SCN and the adjacent medial preoptic
nucleus (MPN, apparently equivalent to POSC; see p. 489) identify the latter

nucleus as critical to the production of progesterone-induced LH surges (415),

to spontaneous ovulation, and to ovulation in response to electrical stimulation
of the medial preoptic area (MPOA) in pentobarbital-blocked rats (38713).
Lesions restricted to the adjacent SCN are reported not to prevent spontaneous
ovulation (387%).
II) PREOPTIC AREA. Damage to the POA is neither necessary nor sufficient
for anovulation (64, 364). Lesions restricted to the MPOA induce repeated
pseudopregnancies but normal cyclicity can be reinstated by treatment with
a dopamine agonist that inhibits prolactin secretion (82). This result has been
confirmed and extended; destruction of the sexually differentiated part of the
MPOA (64) causes acute blockade of ovulation and a high incidence of immediate
and delayed pseudopregnancy but does not produce long-term ovulatory failure.
Goodman (126) challenges the conclusion that the MPOA is not necessary for
spontaneous ovulation on the grounds that some MPOA lesions did not extend
far enough laterally (83); more caudal lesions (e.g., in the SCN) are presumed
to be effective because the neural system responsible for the LH surge con-
verges as it approaches the MBH (126). This criticism does not apply to a study
in which total destruction of the MPOA inhibited but did not eliminate the
positive-feedback effects of ovarian hormones on phasic LH release (131).
Differences among the various studies (there are many reports and differences
we have not cited) may be resolvable on the basis of lesion size, vascu .lar
damage (207), type of lesion (radiofie lquency vs. electrolytic) , and type of
preparation studied (acute vs. chronic). The use of different end points (spon-
taneous ovulation vs. ability to elicit an LH surge with exogenous steroids)
also contributes to the seemingly discrepant findings. For example, the positive-
feedback mechanism may show effects of MPOA damage (131) that are never-
theless consistent with ovulation (64). The conclusion that the SCN are the
“primary neural centers for regulation of reproductive cyclicity” (82) is chal-
lenged but not disproved by the observation that estradiol implants in the POA
effectively induce LH surges whereas similar implants in the SCN are in-
effective (126).
III) REGULATION OF LH RHYTHMS. There is no direct neural innervation
of the pituitary secretory cells that manufacture and release LH and FSH (298).
Rhythmic hormone secretion therefore reflects either the clocklike nature of
pituitary cells or the rhythmicity of blood-borne substances (releasing hor-
mones, gonadal steroids) reaching the pituitary (298). Short-term, pulsatile
secretion of gonadotropins may occur in monkey pituitaries isolated from
hypothalamic influences (112), but available evidence indicates that neither
pulsatile nor circadian rhythmicity originates in the rat pituitary (298, 349).
Neural and circadian control of ovulation likely is effected via humoral agents
acting on the adenohypophysis.
Several caveats must be raised. Release rates of gonadotropins from the
pituitary are mainly controlled by releasing hormones but there is little basis
for supposing a high correlation between the content of these hormones in the
brain and their release rates into the systemic or portal circulation (298, 361).

The most meaningful data are the concentrations of releasing hormones in the
portal vasculature during the stages of the estrous cycle. The dynamic methods
of assessing LHRH content currently in use often yield inconsistent or contra-
dictory information. For example, despite very substantial elevation of serum
LH in OVX rats and the greater LH-releasing activity in their portal plasma,
Eskay et al. (103) failed to observe an increase in LHRH concentration in portal
plasma of long-term OVX rats. Some of these discrepancies may reflect sam-
pling difficulties (failure to detect temporally restricted LHRH surges) and others
surely are due to the conditions under which blood sampling is accomplished
(surgical trauma and long-term deep anesthesia). The single most germane
observation is that of Sarkar et al. (341), who report a proestrous LHRH
surge in portal blood of rats. The anesthetic used in their study blocks neither
the spontaneous LH surge nor ovulation; nor does it stimulate LH release.
These data indicate that the LH surge begins shortly after the GnRH surge
is detectable in portal plasma and suggest that LH surges are at least in part
dependent on estrogen-stimulation of LHRH release.
Luteinizing hormone-releasing hormone has been localized in the organum
vasculosum lamina terminalis (OVLT), in the median eminence, and in other
hypothalamic nuclei (see 66, 101, 186). The OVLT is a circumventricular organ
present at the most rostra1 part of the third ventricle in the rat (see 410).
Its medium-sized neurons with neuropile richly vascularized by a capillary
plexus are surrounded by catecholaminergic neurons in the immediately adja-
cent periventricular nucleus and in the diagonal-band nucleus (249). Lesions
restricted to the OVLT (339), which presumably do not disturb general circadian
organization, completely block the estrogen-plus-progesterone-induced FSH
and LH surges. Samson and McCann (339) suggest that rostra1 LHRH-
containing elements are necessary for the positive-feedback effects of steroids
on LH release and that axonal transport of LHRH from the OVLT to the median
eminence (ME) constitutes a significant portion of ME LHRH. Anterior
hypothalamic deafferentation either through or caudal to the SCN decreases
LHRH content of the medial basal hypothalamus and increases LHRH content
in the POA (176). Perikarya containing LHRH have also been localized to the
SCN region (66,353). S&al6 et al. (353) suggest that LHRH in the ME originates
from cells within the SCN. This hypothesis is not consistent with the small
decreases in ME LHRH after SCN lesions (175), nor with the report that
LHRH was not detectable in the SCN of females killed on the morning of
proestrus (189). Very few LHRH-positive nerve fibers are found within hypo-
thalamic islands after complete MBH isolation; the arcuate nucleus of deaffer-
ented animals has no positive-staining LHRH cells (353). It appears likely that
rhythmic release of LHRH is eliminated in deafferented animals and that the
amount of LHRH reaching the portal vasculature is greatly reduced after
lesions that interfere with the OVLT and perhaps other rostra1 hypothalamic
structures. The LHRH of rostra1 hypothalamic origin denied access to the portal
system may be secreted into the general circulation; its dilution in the plasma
would diminish or eliminate its significance for LH release. This sequence

of events could account for the discrepancy between normal serum LHRH
levels and depressed serum LH and FSH levels (176).
IV) INFLUENCE OF ESTROGENS.Estrogen treatments that induce diurnal
surges of serum FSH and LH have no significant effect on preoptic-region
GnRH content of control or hypothalamically deafferented rats (176). This
should not be interpreted to mean that gonadotropin surges do not reflect
differences in GnRH release induced by estradiol. There are marked regional
brain differences in LHRH responsiveness to estradiol (189), the OVLT being
insensitive and the ME sensitive to manipulation of estrogen levels (176, 189).
Other data (341) also imply that estrogens increase the release of GnRH.
Estradiol benzoate decreases the amount of serum LHRH and increases the
amount of this hormone in the MBH of brain-damaged and control animals (176).
Since the MBH is the final common pathway for LHRH transportto the pituitary
this may reflect estrogen-stimulated increases in releasing-hormone concentra-
tions in the portal vasculature. Additional evidence suggests that rhythmic
LHRH release can account for LH surges: pulsatile LHRH infusion produces
pulsatile LH release in OVX rats not treated with estrogens, whereas constant
LHRH infusion leads to a steady increase in plasma LH devoid of pulsatile
characteristics (349).
Pituitary responsiveness to LHRH is greatly augmented by ovarian hor-
mones (85,203). There is a 50-fold increase in rat pituitary sensitivity to LHRH
during proestrus, measured by the amount of LH release. Ovariectomy on the.
day preceding proestrus (diestrus III) abolishes this effect (203). Similar results
have been obtained in vitro; preincubation of a primary culture of rat adeno-
hypophyseal cells with estradiol substantially decreased the concentration of
LHRH required for stimulation of LH release. This effect was detectable after
10 h and maximal after 24 h of incubation. Luteinizing hormone-releasing hor-
mone also induced responses in culture in the complete absence of estradiol
and the maximal LH response to LHRH was not increased by estrogen
treatment (93).
Given in a sequence of two injections spaced 1 h apart, LHRH greatly
increased LH secretion at proestrus; by comparison LH output was only slightly
augmented at other stages of the estrous cycle (75). Evidently in the presence
of estrogen conditioning LHRH sensitized the pituitary to subsequent LHRH
treatment (3, 75). This massive pituitary sensitization to LHRH permits the
modest proestrous increase in GnRH concentration in portal plasma to pro-
duce the LH surge (341).
Single injections of EB produce multiple LH surges at approximately 24-h
intervals in OVX rats in several different photoperiods (202). Only a single
surge is observed during a normal estrous cycle, in part because.progesterone
inhibits estradiol-stimulated LH release (119). Under physiological conditions
increased progesterone secretion that accompanies the LH surge likely pre-
vents subsequent surges until a new cohort of follicles has developed sufficient
estrogenic capabili .ty (11.9). The patterning and amoun .ts of estrad .iol secreted
during the normal cycle also may impose li .mits on the number of surges.

506 BENJAMIN RUSAK AND IRVING ZUCKER Volum 59
Most investigators report an absence of circadian LH release in OVX rats

deprived of estrogens (e.g., ZOZ), but diurnal periodicity of LH release has also
been reported in the absence of estradiol(226). The repeated bleeding of indi-
vidual animals complicates evaluation of the latter finding. If confirmed,
rhythmic release of GnRH would be established as a sufficient stimulus for LH
surges (see discussion of this issue for hamsters). Figure 1 presents a model
of the rat estrous cycle that emphasizes contributions of the circadian system
and incorporates salient information from the preceding sections.
b) Hamsters. Some features of the hamster control system are similar to
those in rats. A substantial LH surge occurs in intact females only on the after-
noon of proestrus (43). Estrogen treatment produces repeated LH surges
separated by 24-h intervals in OVX females in LD 14:lO (276). As with rats
these surges can ‘be blocked with phenobarbital injections (276) that coincide
with a critical period that begins about 10 h after light onset each day (374).
Both spontaneous ovulation and estradiol-induced LH surges are eliminated
by knife cuts that interrupt fibers from the POA to the MBH (278). A paucity
of studies of hamsters with small lesions restricted to individual hypothalamic
nuclei leaves open the possibility that the SCN and not the POA mediate many
of these effects. A preliminary report with incomplete details of histological
analysis suggests that lesions of the SCN induce acyclicity and persistent
vaginal estrus (373); it is not possible, however, to conclude that damage to
other structures is not important for production of this syndrome.
After lesions and knife cuts of the anterior hypothalamus, serum LH levels
are significantly depressed from control values at all clock times tested (278).
Interference with the synthesis and transport of LHRH presumably contributes
to the absence of cyclicity.
Intact, OVX, and adrenalectomized-OVX hamsters housed in LD lo:14
generate daily LH surges of large amplitude in the absence of estrogenic
stimulation (46a, 351). The cyclic LH-release apparatus is clearly functional and
can be activated in the absence of steroid feedback; these data provide excellent
circumstantial evidence for the circadian organization of the LH surge, which
has also been established in more direct tests (372).
By contrast, OVX hamsters maintained in LD 14:lO either show only small
daily LH surges (374a) or fail to generate surges, unless stimulated by exogenous
estrogens (276,351). It remains to be explained why females housed in LD 14:10
require such stimulation to express daily LH surges of substantial amplitude
while females in LD lo:14 do not. Seegal and Goldman (351) suggest that
estradiol and photoperiod stimulate daily surges by an as yet unspecified
common mechanism; Several mediators have been suggested, including a change
in phase relations among constituent circadian oscillators (116), increased
pituitary sensitivity to GnRH, or increased synthesis and release of LHRH.
The hamster is photoperiodic and ceases to show estrous cycles after 6-10
wk of exposure to short days (351). After 20-30 wk of continuous short-day
exposure females resume their estrous cycles (spontaneous ovarian recrudes-

July av’g NEURAL REGULATION OF CIRCADIAN RHYTHMS 507
information
FIG. 1. Features of the system for the circadian timing of the rat estrous cycle. 1) Light-dark
information is relayed via the retinohypothalamic tract to the SCN where it entrains circadian
oscillators. 2) Neurons of the SCN generate circadian rhythms. Projections from the SCN to the
median eminence (383) and probably to the arcuate nucleus (262) impose circadian rhythmicity
on cellular activities of these neural structures. A neurogenic stimulus, generated by the SCN
on a circadian basis, determines the time of release of LHRH (and other releasing hormones)
from the median eminence into the primary capillary plexus of the hypophyseal portal system.
Because it drives many circadian oscillations, the SCN also mediates the diurnal rhy thm of estradiol
secretion (177, 268). 3) Estradiol (E2) acts via negative-feedback mechanisms on the medial basal
hypothalamus-median eminence complex to inhibit LH release. 4) Subsequently E, increases the
release of ME LHRH into the portal vasculature (341) and sensitizes the pituitary to LHRH
(85, 203). The temporal coordination of a) increased LHRH release, b) increased pituitary sensi-
tivity to LHRH, and c) LHRH sensitization of pituitary to subsequent LHRH release (3, 75)
depends on periodic influences from the SCN. Together these events produce the LH surge.
5) Ovarian progesterone fist enhances the estrogen-induced surge but then inhibits the LH release
apparatus*( 119) and restricts the LH surge to 1 day of the cycle. Insufficient estrogenic conditioning
of neural and hypophyseal tissue also prevents transduction of the daily SCN signal into daily LH
surges. AC, anterior commissure; AH, anterior hypothalamic area; AP, anterior pituitary; ARC,
arcuate nucleus; LHRH, luteinizing hormone-releasing hormone; ME, median eminence; OC, optic
chiasm; OVLT, organum vasculosum lamina terminalis; POA, preoptic area; RHT, retinohypotha-
lamic tract; SCN, suprachiasmatic nuclei. [This model is an elaboration and revision of an earlier
version proposed for the hamster estrous cycle (116). ]

cence). Long-term OVX females maintained in the same short-day photoperiod

cease to display daily LH surges at the time intact females resume cyclicity
(351). This indicates that the expression of LH surges, at least in short photo-
periods, is completely independent of ovarian steroid feedback and militates
against Short’s suggestion (354) that hormonal feedback is the sole timing
mechanism of the estrous cycle.
The pineal ‘may modify pituitary sensitivity to LHRH in short days and
thereby promote expression of LH surges. In hamsters responsiveness to
LHRH generally is less dependent on estrogenic priming; unlike OVX rats,
untreated OVX hamsters in LD 14:lO or those injected with estradiol or
progesterone show equally large increases in plasma LH after LHRH treat-
ment; only combined treatment with estradiol and progesterone decreases LH
release in response to LHRH (276). Increased release of melatonin, altered
patterns of melatonin release (387), or similar changes in some other pineal
product may contribute to the LH surges of hamsters maintained in short days.
The structural similarity between LHRH and pineal antigonadal substance (151)
is consistent with these speculations.
An intact cyclic mechanism for LHRH release in short-day females, com-
bined with increased synthesis of LHRH (cf. 293), could account for the daily
LH surges.
c) Guinea pigs. Guinea pigs in which the anterior border of a knife cut is
posterior to the SCN continu .e to undergo estro us cycles with periods somewhat
longer than normal (73). The occurrence of estrous cycles in preparations ex-
cluding the SCN distinguishes guinea pigs from rats and hamsters. The guinea
pigs MBH-hypophyseal system is capable of organizing estrous cyclicity. Steroid
feedback may be necessary and sufficient to the timing of ovulation, in accord
with Short’s suggestion (354).
In a quest for species uniformity one can speculate that the knife cuts may
not have extended completely to the base of the brain, and perhaps neural
information passes in a rostrocaudal direction. This conclusion is plausible in
light of Poulain’s finding that the lesions of the guinea pig SCN eliminate vaginal
estrous cycles and prevent ovulation (299) ,buta similar pattern of results is ob-
tained with rhesus monkeys (see below) where the completeness of the knife
cuts is not open to question.
Deafferentation of the guinea pig hypothalamus anterior to the SCN elimi-
nates estrous cycles (73), although it must be cautioned that the animals were
observed for only 35 days postoperatively (normally a maximum of two cycles).
These more rostra1 cuts probably interfere with the synthesis of LHRH; LHRH-
containing perikarya are found in the guinea pig SCN, MPOA, anterior hypo-
thalamus (28), and MBH (356). This pattern of hormone localization is quite
different from that in the rat and mouse (356). The majority of LHRH-containing
axons in the guinea pig ME have cell bodies within the MBH and 3 wk after
surgical isolation of the MBH there are only slight decreases in ME LHRH
(cf. rats in 176, 353). Teresawa observed normal ovulatory cycles in guinea
pigs with MBH isolation that excludes influences from SCN and POA (cited in

July ;29?‘9 NEURAL REGULATION OF CIRCADIAN RHYTHMS 509
356). In this species, unlike the rat and hamster, ovulation and relatively
normal estrous cycles occur in the absence of neural influences from more
rostra1 hypothalamic structures.
The participation of circadian rhythms in the organization of the guinea pig
cycle has never been established; moreover, the time of ovulation and the onset
of behavioral estrus clearly are not entrained to the LD cycle in ways com-
parable to those reported for rats and hamsters. In guinea pigs exposed to
natural photoperiods the onset of heat occurs at every phase of the solar day
(420). Given these considerations, the persistence of estrous cycles after elimina-
tion or disruption of normal circadian organization attendant on various neural
interventions is neither surprising nor necessarily problematic for the model
developed on the basis of rat studies.
d) Monkeys. Complete deafferentation of the MBH, interrupting neural
connections from the SCN and POA to the median eminence, has no significant
effect on mean plasma gonadotropin levels nor on circhoral patterns of LH
release of OVX lMacaca muZattu (187). Estrogen-induced LH surges also sur-
vive these manipulations. Knobil and co-workers (187, 188) conclude that a
preoptic neural signal is not required for the production of the LH surge. These
data appear to be contradicted by a study (277) in which spontaneous ovulation
and estrogen-induced LH surges were blocked by lesions of the ventralmost
POA, anterior hypothalamic area (AHA), and SCN. Norman et al. (277) suggest
that destruction of only the SCN-MBH pathways, leaving other projections
intact, may inhibit subsequent LH release and could account for the absence
of ovulation or positive-feedback effects of estrogens. A more recent study (188)
has failed to replicate some of these findings. Evidence in favor of SCN partici-
pation in ovulation comes from two other sources. Three of four monkeys with
anterior disconnections rostra1 to the SCN but only one of three with cuts caudal
to the SCN ovulated spontaneously; six of these seven animals generated LH
surges in response to estrogen treatment (195). Monkeys with hypothalamic
islands that included the SCN continued to cycle, whereas one of the animals
with a disconnection caudal to the SCN stopped cycling and failed to respond
to estrogen injections with an LH surge (113). Perhaps in the absence of
exogenous steroids more or different neural circuitry is necessary for spon-
taneous expression of the LH surge than when such steroids are present.
The dispensability of structures rostra1 to the MBH for estrogen-mediated
LH surges was established definitively in a recent study (150). After complete
ablation of the septum, anterior commissure, POA, SCN, all of the OVLT,
most of the supraoptic nuclei, and variable portions of the dorsal hypothalamic
nuclei, EB-treated OVX monkeys generated LH and FSH discharges similar
to those observed in control animals. In monkeys LHRH immunoreactive
perikarya are found in a continuum from the septal-preoptic region to the pre-
mammillary nuclei caudally; reactive axons in the ME have their cell bodies
in the MBH. There is an apparent lack of organization of LHRH elements in the
hypothalamus; they are scattered over a wide area with no distinct, well-
organized fiber tracts (357). Even the major LHRH projections are diffuse and

difficult to follow. The presence of a tuberoinfundibular pathway for LHRH

indicates that the isolated MBH-hypophyseal complex is itself capable of medi-
ating LH surges.
Knobil (187) concludes that in monkeys “the central components of the
control system which initiates the pre-ovulatory gonadotropin surge are resi-
dent in the MBH-hypophysial apparatus.” Clearly the positive-feedback effects
of estradiol require at most the MBH-hypophyseal system. Whether anterior
structures participate in the nomnal regulation of ovulation remains an open
question.
In rhesus monkeys as in guinea pigs the LH surge does not appear to be
linked to the LD cycle (187). Ovulation may be controlled by the timing of
estrogen secretion and its positive-feedback effect on LH. Spies et al. (365)
found no evidence of significant diurnal rhythmicity in serum estradiol or LH
of female monkeys bled at five times of day during the follicular phase of the
cycle although both estradiol and progesterone showed significant diurnal varia-
tions in the luteal phase.
As in guinea pigs there is no evidence for circadian organization of the
menstrual cycle in monkeys. The hypothesis that the SCN are involved in
organization of mammalian estrous cycles should be restricted to those species
in which a circadian component is critical for generation of the LH surge. In
species such as the guinea pig and monkey, where circadian influences are
minimally significant for regulation of estrous cycles (see 259 for further dis-
cussion), the controlling mechanism for the LH surge may reside in the rise
of estradiol during the follicular phase of the cycle. Subcutaneous EB implants
in OVX monkeys exert only negative-feedback effects on LH release and surges
are not observed during the first 36 h. After 42 h of continuous EB stimulation
a single LH surge occurs; implants left in place for weeks produce no additional
surges (187).
Further support for this hypothesis comes from recent experiments (188)
in which LHRH was administered in a pulsatile mode for 6 min each hour to
lesioned monkeys incapable of endogenous LH and FSH release. This regimen
increased gonadotropin values to levels approximating those prior to surgery.
Administration of estradiol while continuing the LHRH treatment produced
first negative- and then positive-feedback effects on LH and FSH discharge.
The LH surge was approximately 50% the magnitude of that observed in
neurologically intact OVX monkeys treated with estradiol. The authors con-
clude that “since endogenous LHRH production is abolished in this experimental
preparation . . . estradiol can exert both its positive and negative feedback
actions . . . on LH and FSH secretion at the level of the pituitary gland” (188).
Although this experiment adds an important dimension to the issues considered
in this section it “does not exclude feedback actions of the steroid at the
neural level in the context of a physiological setting’ (188).
d Sheep. The mechanism controlling cyclic ovulation has properties in
common with those of hamsters and rats and others more similar to primates.
In OVX sheep LH surges occur at a constant interval after estradiol treatment

(167) and not, as in rats, at a specific time of day entrained by the LD cycle (166).
This diminishes the significance of circadian organization of the estrous cycle,
unless secretion of estradiol under physiological conditions is subject to circadian
control. In common with rats and hamsters and unlike rhesus monkeys (187)
a single injection of EB produces multiple LH discharges 16-27 h apart (180).
The hypothalamic control of ovulation cycles and the participation of SCN and
POA in sheep circadian and estrous rhythms remain to be established. Sheep
are particularly interesting in this context because it is not unlikely that circadian
rhythms mediate the seasonal breeding cycle (216, 337); sheep may represent
an intermediate form of organization in which the influence of day length on
seasonal reproduction is mediated by hypothalamic circadian oscillators but
timing of ovulation and behavioral receptivity within a breeding season is not
dependent on circadian organization.
4. Sexual d@brentiation of gonadotropin release
Tonic secretion of gonadotropins is effected by similar mechanisms in male

and female mammals. The phasic or cyclic release of these hormones, of which
LH has been extensively studied, is highly developed in females and absent
in ‘males. One or more LH surges are induced in gonadectomized female rats,
hamsters, and sheep by appropriate treatment with estradiol or estradiol plus
progesterone. In castrated males of these species similar hormone treatments
never produce LH surges. To account for this sex difference it was proposed
that androgens secreted by the testes perinatally suppress the hypothalamic
clock essential for cyclic LH release (129).
In the absence of normal sexual differentiation males retain the potential
for cyclic LH release; male rats castrated on the day of birth and implanted
with ovaries undergo estrous cycles and ovulate (144). The mechanisms by
which cyclicity is suppressed in the male are not known, although several likely
alternatives have been considered (108). Androgens could destroy the central
rhythmic substrate for LH release; we consider this unlikely because other
circadian rhythms coupled to the oscillator(s) that generate LH rhythms are
not affected by sexual differentiation (116) The hypothesis that neonatal expo-
sure to androgens interferes with coupling mechanisms between circadian clocks
and effector systems involved inovulation remains viable and attractive (108,
116). Relatively subtle changes in sensitivity of the pituitary to estrogen feed-
back or in the amounts and chemical compositions of releasing factors available
to males also might be contributory factors. Pituitary changes do not seem to
constitute a sufficient explanation since hypophyseal transplants from adult
males into hypophysectomized females support ovulation (144, 145).
Electrochemical stimulation of the POA of sexually differentiated castrated
male rats induces ovulation in transplanted ovaries (108). The potency of the
male hypothalamohypophyseal axis for ovulatory LH surges is thereby estab-
lished under the influence of these artificial stimuli. Under normal circum-

stances the distinguishing sex-related characteristic may be the males’ inability

to generate the appropriate neurogenic stimulus.
Female rats treated with low doses of androgens during a critical neonatal
period develop a delayed anovulatory syndrome; they undergo a few normal
ovulatory cycles before becoming permanently anovulatory (129). Because there
is no precedent for postnatal chemical suppression of an existing circadian clock,
this suggests that the rhythmic substrate for estrous cycles is functional in
androgen-treated rats of either sex. The mechanisms for elimination of cyclic
LH release in masculinized animals are less likely to be found in the clock per se
than in the coupling between the clock and the structures it regulates (108,
116, 334).
Normal male sexual differentiation leaves rhesus monkeys capable of
estrogen-induced LH surges (179). This represents a notable difference between
this primate and several rodent species. Failure of sexual differentiation of the
cyclic LH release mechanism in rhesus monkeys is consistent with noninvolve-
ment of anterior hypothalamic structures and with the dispensability of circadian
organization in control of ovulation.
B. Male Seasonal Cycles
Seasonal reproductive cycles are often synchronized by changes in day

length. Bunning’s pioneering work (70) established the importance of CRs for
day-length measurement [photoperiodic time measurement (PTM)]. Surprisingly
few studies have assessed the role of the nervous system in generation and
entrainment of these longer duration cycles although the involvement of the
pineal gland and its secretions has been well documented (309).
I. Role of the supachiasmatic nuclei
a) Hamsters. Lesions of the hamster SCN prevent testicular regression

in short days or after blinding (orbital enucleation) (333). Lesions of the SCN
also accelerate testicular regrowth in hamsters whose testes previously have
been regressed by absence of adequate photostimulation. Similar effects were
obtained with lesions limited almost exclusively to the SCN and with larger
lesions that also damaged the periventricular hypothalamus (333). These neural
interventions disrupt circadian rhythmicity in hamsters (331); however, disrup-
tion of the testicular cycle is not contingent on interference with overall circadian
organization since hypothalamic lesions that spare circadian rhythms can pre-
vent testicular regression in short days (373).
The testes of two hamsters with lesions that spared the anterior third of the
SCN failed to regress in short days; the posterior portion of the SCN that was
destroyed receives the RHT projection (251) and may be directly involved in
photic regulation of testicular cycles. Damage to the SCN clearly is sufficient
to prevent or reverse photoperiodically mediated regression (333). Involvement

July &??‘g NEURAL REGULATION OF CIRCADIAN RHYTHMS 513
of the SCN in testicular regression induced by low temperatures or by food

scarcity has not been reported.
b) Ferrets. Lesions that severely damage the SCN and anterior hypo-
thalamus accelerate testicular development in immature ferrets exposed to short
day lengths (31). Lesions that only moderately damage the SCN also hasten
testicular growth; the testes of these animals eventually regress and subse-
quently recrudesce. The neural components for the ferret testicular cycle appar-
ently reside outside the area of hypothalamic destruction (31). Although the
extent of SCN damage was not quantified and there was no assessment of
circadian organization, this study raises the possibility that neural structures
that mediate some seasonal cycles may be different from those involved in the
expression of circadian rhythms. This analysis can also be extended to circannual
body weight, food intake, and hibernation rhythms (263).
2. Pineal-suprachiasmatic nuclei relations
Damage to neural structures other than the SCN also can eliminate the
annual testicular cycle of hamsters. Knife cuts that partially or totally isolate
the MBH are effective, as are pinealectomy or superior cervical ganglionectomy
(308, 310). Some of these procedures do not interfere with the SCN nor with
the generation of nonpineal CRs. Interpretation of these results is problematic
because integrity of the pineal, especially its neural innervation, is essential
for expression of the annual testicular cycle. These findings can be accounted
for in several different ways. 1) The SCN lesions eliminate the nocturnal peak
of pineal NAT activity (250, 251, 254); if melatonin is the primary pineal anti-
gonadal agent, the loss of sufficient melatonin production may be the functional
equivalent of a pinealectomy. 2) The phasing of the pineal’s hormone secretion
relative to the LD cycle may be crucial to its antigonadal activity (387). The
loss of light-controlled pineal rhythms after RHT-SCN destruction may prevent
antigonadal effects even in the presence of sufficient melatonin. 3) The SCN
lesions may remove hypothalamic inhibitory influences and result in increased
prolactin secretion (46). Prolactin is capable in part of protecting the hamster
gonad from the regressive effects of short days (29). 4) The SCN may be part
of the target tissue (67, 152a) on which pineal hormones act to induce testicular
regression (but see 46b).
At present one cannot choose among these possibilities. It seems likely that
neural control of pineal CRs play a crucial role in photoperiodic time measure-
ment and that the SCN regulate male reproductive cycles in some mammals
in ways yet to be specified.
V. PROSPECTUS
We will not attempt to enumerate the many specific research questions

that emerge from the studies reviewed here. We choose instead to focus atten-

514 BENJAMIN RUSAK AND IRVING ZUCKER volume 59
tion on coupling to and among neural oscillators because these processes are
central to unders tanding the neural mechanisms controlling circadian rhythmic-
ity. Coupling refers to the production or modification of rhythmicity in one
system by another. This subsumes: 1) mechanisms for entrainment of oscil-
lators by exteroceptive inputs, 2) the (mutual) interaction of oscillators and
of their constituent osci .Ilatory units, and 8) the efferent influence of central
oscillators on neural and endocrine effecters. Such coupling is likely to be
achieved by a variety of neurophysiological mechanisms; study of these mecha-
nisms may yield new principles of neural function and of circadian organization.
Questions awaiting experimental attention include the following:
I) Virtually no information is available about the neurophysiological basis
for the influence of a remote photoreceptor (e.g., the mammalian retina) on
central oscillators, nor is there any evidence for how a photoreceptor-oscillator
complex (e.g., the ApZysia eye) influences other oscillators and effecters. Since
agents other than light (e.g., temperature, hormones) can influence central
oscillators, questions about access of exteroceptive stimuli to oscillators must
be extended to these agents; this analysis should also consider how and where
these inputs are transduced into neural afference to central oscillators.
2) Several lines of evidence indicate that metazoans comprise a multilevel
complex of oscillators both within and outside the nervous system (10,14,15,51,
154, 165, 258, 284, 296, 331). -Although dissociations may occur in some condi--
tions, normally these oscillators are adaptively integrated through some corn-
binati .on of mutual and hierarchical coupling. The neurophysiological basis for
these interactions among oscillators is unknown . Presumably some combination
of the information-transmission mechanisms familiar to neurophysiologists
serves these functions; the elucidation of which mechanisms permit one oscilla-
tor to influence the ‘period icity of another is likely to provide useful insights
into the molecular basis of rhythm generation in the nervous system.
3) Studies of the control of neural and endocrine effecters by central oscil-
lators (perhaps through intermediary damped oscillators) may prove a source
of new principles of neural integration in relation to control of behavior and
physiology. Even the necessarily brief review we have provided (p. 496) of the
generally ignored dynamic aspects of neurophysiological function indicates that
a rich field of study awaits exploration.
We thank Eric Bittmsn, Julian Davidson, Gail Eskes, Bruce Goldman, Phyllis Johnston, and
Werner Loher for their advice and for criticism of portions of the manuscript. We are grateful
to Claire Almada, Nancy Beattie, Colleen Clattenburg, Gary Dupis, Darlene Frost, Carol McVicar,
and Becky Phillips for bibliographic and secretarial help. Preparation of the manuscript was sup-
ported by a grant from the National Research Council of Canada and by Grant I-ID-02982 from the
National Institute of Child Health and Human Development.
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July 1979 NEURAL REGULATItON OF CIRCADIAN RHYTHMS 525
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Physiological Reviews

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Neural Regulation of Circadian Rhythms

Department of Psychology, Dalhousie University, Halifax, Nova Scotia, Cam&; a&

A. Background and Scope

Rhythmicity is a fundamental property of living matter-from single cells

0031-9333/79/000000-00$01.25 Copyright 0 1979 the American Physiological Society 449

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Circadian rhythms are endogenous oscillations with a period of approxi-

The acceptance of circadian rhythmicity as a property of living organisms

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Three major issues currently attract the attention of neuroscientists inter-

During entrainment the period (T) of a biological rhythm takes on the

A. Extraretinal Light Entrainment

Among invertebrates and. nonmammalian vertebrates the existence and

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Influential early work on extraocular photoreception was generated bY

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The specific retinal receptors involved in entrainment are unknown. Rod

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the rhodopsin-containing rods (scotopic system) while sparing the photopic

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Light impinging directly on the hypothalamus of enucleated rats apparently

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B. Retinal Light Entrainment

a) Receptors. Distribution, receptive fields, and discharge properties of

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Identification of entrainment pathways has been based nearly exclusively

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Primacy of the LD cycle as a xeitgeber has long been acknowledged. The

Ambient temperature fluctuations do not entrain CRs in endotherms,

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several constant temperatures showed temperature compensation of period.

2. Social stimuli and handling

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The efficacy of various food-restriction schedules for synchronization of CRs

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III. RHYTHM GENERATION

Neural control of circadian rhythmicity has been studied in species Tom

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Mollusca: several species of Aplysia, especially A. califowzica; Limax

a) RX R15, a large neuron in the parietovisceral ganglion of Aplysia,

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of locomotion. It is instructive to compare the effects of eye removal inA@@z

A lack of uniform nomenclature in descriptions of insect nervous systems

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hoppers (Romalea microptera). If confirmed, this would suggest that another

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link from brain to thorax is essential to the generation of circadian rhythmicity

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radically alter the level of activity so that rhythmicity cannot be meaningfully

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Both secretory structures (sinus glands, X organs) and vision-related struc-

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