Journal of Comparative and Physiological Psychology

1975, Vol. 88, No. 1, 161-175

OSMOSENSITIVE NEURONS IN THE RAT'S PREOPTIC AREA:

MEDIAL-LATERAL COMPARISON1
EGBERT B. MALMO2 AND WILLIAM J. MUNDL
Allan Memorial Institute, McGill University, Montreal, Quebec, Canada
For the first time, brain-recording data were brought to bear directly on
the question of a critical osmosensitive zone in the lateral preoptic area as
- specifically delimited in the rat by Blass and Epstein, and in the rabbit by
Peck and Novin. Our data clearly showed that this critical zone in the lateral
preoptic area of the rat contains cells that are osmosensitive. Simultaneous
recording from cell populations (a) inside the critical zone and (b) in a zone
medial to it showed that the net acceleratory response to challenge for
the former was much greater than it was for the latter. These findings con-
stitute new evidence for the critical importance of the lateral preoptic area
in cellular dehydration thirst.

The osmoreceptor theory originated with glucose. These findings were confirmed by
Verney (1947) in his research on the sensory Brooks, Ushiyama, and Lange (1962).
aspect of the antidiuretic reflex. Verney In order to demonstrate that neurons in
proposed that central receptor cells, sensi- the supraoptic nucleus were central recep-
tive to increased osmolarity of extracellular tors (i.e., not synaptically driven by neu-
fluids (i.e., osmoreceptors), were located in rons outside the nucleus), Suda, Koizumi,
the supraoptic nucleus. and Brooks (1963) and Ishikawa, Koizumi,
Brain-recording experiments designed to and Brooks (1966) studied small "islands"
discover osmosensitive neurons in and of supraoptic nuclear cells that had been
around the supraoptic nucleus commenced separated by cuts from adjacent neural
with von Euler's (1953) recordings of slow tissue. These isolated neurons reacted to
electrical changes in cats (do "osmopoten- intracarotid injections of hypertonic saline,
tials") that shifted with injection of hyper- thus behaving like first-order neurons.
tonic saline or glucose. Following up the Joynt (1966) and Koizumi, Ishikawa, and
pilot work of Green and Morin (1953), Brooks (1964) found that hypertonic saline
Sawyer and Gernandt (1956) showed that accelerated supraoptic unit discharge more
intracarotid injections of hypertonic saline than hypertonic glucose of equal osmotic
and glucose produced characteristic electro- concentration, while concentrated urea so-
encephalogram (EEG) changes in various lutions had almost no effect. These differ-
telencephalic and diencephalic regions of ential effects on neuronal reaction to solu-
the brain in rabbits. tions of equal osmolal concentration
Cross and Green (1959), who were the correspond with Verney's (1947) findings
first to record from units in the hypothal- of graded effectiveness for these solutions
amus, reported that neurons in the supra- in stimulating antidiuresis. Verney, in not-
optic nucleus generally showed accelerated ing these gradations of effectiveness, had
firing rates in response to intracarotid in- correctly inferred that they were due to
jections of hypertonic sodium chloride or differences in cell permeability to these
1 compounds.
This research was supported in part by grants
to R. B. Malmo from the National Research Coun- Novin and Durham (1969) found 34 re-
cil of Canada and from the National Institute of sponsive cells in testing over 100 rat supra-
Mental Health, U.S. Public Health Service. We optic nucleus units with intracarotid hyper-
express our appreciation to Helen P. Malmo for her tonic saline injections. Comparable results
invaluable assistance in this research. were obtained in opossum by Zeballos,
2
Requests for reprints should be sent to Robert
B. Malmo, 1033 Pine Ave. West, Montreal, P. Q. Wang, Koizumi, and Brooks (1967). In fol-
H3A 1A1, Canada. lowing up earlier work by von Euler (1953)
161
162 ROBERT B. MALMO AND WILLIAM J. MUNDL
on dc potentials, Novin and Durham (1969)
found that after isolating the supraoptic
area of rabbits, they still obtained reactions
(usually stronger ones) to injections of
hypertonic saline. Wayner and Kahan
(1969) reported the results of multiple-unit
recording from a variety of brain sites in the
rat. Salt arousal was produced with injec-
tions of 15% NaCl (an exceptionally high
concentration) into the lateral tail vein or
into the carotid artery.
Hayward and his co-workers (Hayward,
1972; Hayward & Jennings, 1973a, 1973b,
1973c, 1973d; Hayward & Vincent, 1970;
Vincent & Hayward, 1970) have performed
an extensive, carefully controlled series of
experiments, recording from units in the
supraoptic nucleus (and nearby points)
during intracarotid injections of hypertonic
(and control) solutions (chiefly NaCl) in
unanesthetized monkeys.
To the best of our knowledge, the fore-
going completes a nearly exhaustive review
of brain-recording studies aimed at finding
evidence bearing on the osmoreceptor con-
cept. Most of this work has focused on the
supraoptic nucleus and its role in the neuro-
endocrine hypothalamo-pituitary mecha-
nism of the antidiuretic reflex. In some of
the experiments from Hayward's labora-
tory, antidromic stimulation in the posterior
pituitary was used to identify cells as being
part of this reflex system.
Opposition to the Osmoreceptor Concept
The foregoing review of brain-recording
studies is, on the whole, supportive of
Verney's osmoreceptor concept. However,
there is opposition to this concept from
Andersson and his colleagues, who believe
that there are sodium-sensitive receptors
near the third ventricle that respond to
changes in concentration of sodium ions in
the cerebrospinal fluid and that participate
in the control of the complex balance of
fluids in the body through linkage with
vasopressin release and drinking behavior
(Andersson, 1971; Eriksson, Fernandez, &
Olsson, 1971).
Hayward and Jennings (1973d) grant
that a sodium detector in the vicinity of
the third ventricle is a possibility. However,
they observed some changes in firing rate
after intracarotid infusions of hypertonic
D-glucose that were too rapid to be due to a
slow change in sodium ions in the cerebro-
spinal fluid of the third ventricle.
There is disagreement also concerning the
central receptors for cellular dehydration
thirst. As mentioned, Andersson and his
colleagues favor sodium-sensitive receptors,
while a number of other workers believe
that there is compelling evidence that the
lateral preoptic area is the site of osmo-
receptors for cellular dehydration thirst. A
recent exchange between Andersson (1973)
and Peck (1973) highlights the 2 different
points of view. Peck's defense of the osmo-
receptor theory of cellular dehydration
thirst (which Blass, 1973; Epstein, 1973a,
1973b; and Fitzsimons, 1972, 1973, also
favor) is based chiefly on 2 companion re-
searches by Blass and Epstein (1971) and by
Peck and Novin (1971).
Blass and Epstein (1971) presented strong
evidence that a delimited part of the lateral
preoptic area is an important osmosensitive
zone for thirst in rats. Their evidence was
based mainly on alterations of drinking be-
havior produced by lesions, or by cellular
dehydration with intracranial injections of
hypertonic solutions. These findings are in
complete agreement with those of Peck and
Novin (1971), who identified, in the rabbit,
a restricted zone below the anterior commis-
sure and about 2.5 mm. lateral, from which
drinking could be elicited by hypertonic
solutions of sodium chloride or sucrose.
On the other hand, Andersson (1973)
raised the question whether the preoptic
lesions were effective because they impaired
the vascular supply to the choroid plexuses of
the lateral and third ventricles, with con-
sequent delay in transfer of sodium ions
from the systemic extracellular fluid to the
cerebrospinal fluid.
Need jor Brain-Recording Studies of the
Critical Lateral Preoptic Area
Missing here is any substantial evidence
based on brain recordings from the preoptic
area. There are only a few relevant findings,
and they are secondary ones (Brooks, Ishi-
kawa, Koizumi, & Lu, 1966; Cross & Green,
 OSMOSENSITIVE NEURONS
1959; Hayward, 1972; Koizumi et al, 1964;
Vincent, Arnauld, & Bioulac, 1972).
Stellar and Corbit (1973) have called
attention to the dearth of information in this
general area as follows:
Whereas we know some of the stimuli im-
portant in hunger, thirst, and sex, we cannot yet
specify the receptors involved with any confi-
dence For this reason, recording from units
in the hypothalamus and preoptic area during
arousal or satiation of hunger, thirst, and sex
has become a priority experiment [p. 336].
To the best of our knowledge, prior to our
experiments reported in the present article,
the critical zone in the preoptic area, as
delimited by Blass and Epstein (1971) and
Peck and Novin (1971), had not been sys-
tematically studied with any brain-record-
ing technique.
The Present Experiments
In approaching the problem of recording
from the lateral preoptic area, we were
guided by the results of unit recording
from the supraoptic nucleus during hyper-
osmotic challenges. Reports generally stated
that the majority of cells responding to the
challenge showed acceleration; but that
some cells showed inhibition to the chal-
lenge. There is a further complication, which
apparently has not been dealt with in the
literature: A cell that responds with accel-
eration for an hour (or even longer) may
merely be in an acceleratory phase, such
that further observation will reveal a re-
versal in direction (i.e., from an acceleratory
to an inhibitory reaction to challenge).3 If
many cells do reverse direction in this way,
it would be impossible to obtain from single-
unit recordings a reliable estimate of the
3
This possibility was suggested to us by J. N.
Hayward. The findings of Vincent, Arnauld, and
Bioulac (1972) in monkeys and similar observa-
tions of Nicolai'dis (1968) in anesthetized rats must
be viewed in the light of this possibility. Vincent
et al. reported that their "specific" osmosensitive
cells, located in the vicinity of the supraoptic
nucleus, were either activated or inhibited by saline
injection and showed the opposite response during
drinking. Nicolai'dis reported-similar results from
intracarotid injectiojae and stimulations of the
tongue.
163
proportion of cells being accelerated and
the proportion being inhibited during intra-
carotid injection.
On the reasonable hypothesis that cellu-
lar dehydration produces a net increase in
firing in a population of osmoreceptors,
there is an obvious advantage in recording
simultaneously from a population of cells
in the critical area, i.e., the lateral preoptic
area that was delimited by Blass and
Epstein (1971) and Peck and Novin (1971).
This is the main reason for our choice of
multiple-unit recording.
In recording from the critical lateral
preoptic zone, one would expect to find con-
sistent net acceleratory neuronal reactions
to NaCl solutions injected into the carotid
artery. Average degree of reaction should
correspond to the NaCl concentration, with
no reaction to isotonic NaCl.
In recording simultaneously from a point
outside the critical lateral preoptic zone,
one would expect to find significantly lower
reactions to the NaCl injections. For this
area control, we chose a point medial to the
critical zone. This expected lower multiple-
unit reaction from this medial (noncritical)
zone could be due either to the presence of
fewer osmosensitive neurons there (which
is most probable) or to a near equality of
acceleratory and inhibitory single-unit reac-
tions to each challenge.
Finally, since to the best of our knowl-
edge we were the first to conduct a detailed
brain-recording study of this critical lateral
preoptic area, it seemed important to ob-
serve: (a) unit responses to hypertonic
NaCl injections in the absence of response
to isotonic NaCl injections; (b) inhibitory
unit reactions to hyperosmotic challenge,
and (c) a variety of firing patterns in osmo-
sensitive neurons. We were particularly in-
terested in trying to find an osmosensitive
"burster cell" of the kind described by Hay-
ward and Jennings (1973a) in the supra-
optic nucleus and the internuclear zone of
monkeys. If a burster cell were present in the
rat, it wouW be easy to identify because of
its distinctive firing pattern.
In short, multiple-unit recording was de-
signed for the quantitative analysis, and
single-unit recording was designed for sup-
164 ROBERT B. MALMO AND WILLIAM J. MUNDL
plementary qualitative descriptions of in-
teresting cells.
METHOD
Subjects
Seventeen male Charles River Long-Evans
hooded rats, obtained from the Canadian Breeding
Farm & Laboratories, Ltd., St. Constant, Quebec,
were used in this study. At the time of operation,
the weights ranged 325-406 gm. (Mdn = 374 gm.)
after an overnight fast (without water depriva-
tion) . The rats had continuous access to water and
to Charles River Chow (rat, mouse, and hamster
formula). Their diet was regularly supplemented
with lettuce, carrots, and vitamins. Illumination in
their living cage room was on reverse day-night
cycle.
Surgical Procedure
For surgery and for multiple-unit recording,
animals were anesthetized with a mixture of
halothane (Fluotec vaporizer) and nitrous oxide
(50% oxygen) through No. PE 100 polyethylene
nose tubes, and were artificially respired by means
of a C. F. Palmer Miniature Ideal Respiration
Pump. Heart rate was monitored continuously. For
unit recording, anesthesia was shifted from halo-
thane to urethane 1.2 gm/kg ip; and rectal tem-
perature was monitored continuously. A heating
device maintained body temperature at above
37° C.
The rat's head was held in the De Groot plane
(cf. Pellegrino & Cushman, 1967) in the Kopf
stereotaxic instrument by means of a modified
Erickson (1966) headholder, which eliminated ear
bars, thereby making it possible to test reactions
to auditory stimulation. Recording electrodes were
aimed in the right hemisphere for medial and la-
teral preoptic areas (reference: p. 23 of Pellegrino
& Cushman atlas) .8 mm. and 2.5 mm. from mid-
line. Exact depth of the final recording site in each
case depended on the animal's reactions to intra-
carotid hypertonic saline injections as the elect-
trodes were lowered stepwise into the preoptic
area.
The right common carotid artery was exposed
by neck incision, blunt incision, and retraction
of muscle tissue. A section of bent British 30-ga.
(.012-in.) stainless steel tubing, sharpened at its
tip and fitted at the other end with a 21-cm. length
of No. PE 10 polyethylene tubing, was inserted
into the artery for cannulation, while a hemostat
was applied caudally. A Becton Dickinson 1-ml.
tuberculin syringe graduated in .01 ml. was con-
nected via a 27-ga. (.014-in.) hypodermic needle
to a 9-cm. length of No. PE 20 polyethylene tub-
ing, which was joined with the No. PE 10 tubing.
Successful cannulation was indicated by with-
drawing blood into the cannula.
Recording Apparatus
Multiple-unit recording technique. We used a
specially designed unity-gain preamplifier (Mundl,
1971) that was made insensitive to electrical arti-
facts by (a) employing bipolar electrodes4 con-
nected to the differential input of the preamplifier
with the 2 connecting wires being separately
shielded, and (6) driving the shields with a voltage
synchronized with the input signal. The bipolar
recording electrodes were constructed of platinum-
iridium wire coated with Diamel (Johnson, Mat-
they, & Mallory Ltd., Toronto, Ontario). Two wires,
one .005 in., the other .0025 in. in diameter and
.5 mm. longer than the former wire, sharpened
at their tips by diagonal scalpel cuts, were twisted
together and soldered to Amphenol No. 27-9 con-
nectors.
The output from the preamplifier was fed into
a high-gain amplifier equipped with low-pass and
high-pass filters, set at 2 kHz. and 350 Hz., respec-
tively.
The output of the high-gain multiple-unit am-
plifier was fed to an integrator (440-msec. time
constant) with a "phase lag" circuit incorporating
an operational amplifier (Philbrick/Nexus Re-
search, 1968) and to a pulse stretcher (Mundl,
1969). The integrator and pulse stretcher outputs
were recorded on an ink-writing oscillograph. The
output of the high-gain amplifier was also moni-
tored with a Hewlett-Packard 1200B dual trace
oscilloscope and with an audio-amplifier and
speaker. A Grass Model C4 camera was used in
selected instances to record the multiple-unit ac-
tivity from an oscilloscope (see Figure 1).
Calibrations were made with a Hewlett-Packard
208A low-frequency test oscillator and an audio-
frequency microvolter (General Radio Corp., No.
546C).
Unit recording technique. Following a modifica-
tion of Hubel's (1957) method, we made electrodes
of .005-in. 25-mm.-length tungsten wire, elec-
trolytically etched to a tip diameter of .5 /an. or
less and insulated with 4 coats of Epoxylite varnish
(Epoxylite Corp., El Monte, California).
A Kopf 1206 Reduction Drive was used with the
Kopf stereotaxic instrument to provide precise
control of vertical movement of the electrode. The
4
In addition to the advantage of eliminating
artifacts in. the acute experiments described in this
article, it was desirable in the acute experiments
to use the same kind of recording system that is
being used in related ongoing chronic experiments,
in which insensitivity to electrical artifacts gen-
erated by the animal's movements is an extremely
important consideration. In line with the experi-
ence of Geddes (1972), Goodman (1968), and
Schlag (1973), we have found that cautious use of
bipolar electrodes is a convenient way of eliminat-
ing undesired potentials, while still maintaining a
very localized recording radius. Buchwald, Hoi-
stein, and Weber (1973) have reported the same
kind of experience with their concentric electrode.
 OSMOSENSITIVE NEURONS 165
Integroted Multiple Unit Activity

Medial
Preoptic

Preoplic
Area

tunit Signal Photographically Reproduced

esponds to integrator trace for 0.75 M injection above)

Lateral
Preoptic
Aria

FIGUBB 1. Comparison of medial and lateral preoptic areas in multiple-unit reactions to intra-
carotid injections of NaCl. (Upper part: Integrated multiple-unit tracings showing effects of intra-
carotid injections of NaCl in different concentrations. Rating-scale values appear above each section
of tracing. Note higher ratings for lateral preoptic area and correspondence between degree of reaction
and the concentration of NaCl solutions. Lower part: Photographic recording from oscilloscope showing
multiple-unit response in lateral preoptic area to intracarotid injection of hypertonic NaCl solution.
Compare with integrator trace of .75 M solution [rating 4] above.)

signal was led to a preamplifier with an input im-
pedance of 100 MO.
The output of the preamplifier went into a high-
gain amplifier of pass band 380 Hz.-9 kHz. Photo-
graphic recordings were obtained with a Grass
Model C4 camera and a Tektronix Model R5103N
dual beam oscilloscope. The second beam of the
oscilloscope was used to record from the pulse
stretcher. The pulse stretcher traces are displayed
in the bottom lines of the photographic recordings
from the oscilloscope.
The output signal of the high-gain amplifier
was further processed by a pulse stretcher (Mundl,
1969) and an amplitude discriminator. The output
pulses of the amplitude discriminator were inte-
gratated with a phase lag circuit with a 440-msec.
time constant (Philbrick/Nexus Research, 1968).
The pulse stretcher, amplitude discriminator, and
integrator outputs were led to the driver amplifiers
of the Beckman RM oscillograph.
Injection Procedure
We used a manually operated 1-ml. Becton
Dickinson tuberculin syringe, graduated in .01 ml.
for intracarotid injections given at a rate of .02
ml/sec. For injections in the main series, .1 ml. of
fluid was injected over a 5-sec. period. Injections
commenced when both electrodes had been low-
ered about 5 mm. below the surface of the dura.
The first injection was a .75 M (4.5%) sodium
chloride solution.
Medial and lateral electrodes were lowered the
same distance at each step. Reading from the
vertical scale on the stereotaxic instrument, the
steps were .5 mm. until a depth of 6.5 mm. was
reached, when they were reduced to .3, .2, .1, and
.05 mm. in accordance with the strength of multi-
ple-unit reactions to the hypertonic saline injec-
tion. In order to preserve normal physiological
conditions, and as a control, an isotonic saline
solution (.1 ml. of Abbott's Normosol-R pH 7.4)
was injected following every 2 hypertonic saline
injections. Injections were spaced at intervals of
at least 90 sec.
When the first reaction to hypertonic saline oc-
curred, the next descent was reduced to .1 mm.
When it appeared that the responses would not be
increased by further descent, the standard series
of injections was commenced. These were as fol-
lows: .75 M (4.5%), .15 M (.9%), .60 M (3.6%),
.15 M, .45 M (2.7%), .15 M, .30 M (1.8%), .30 M,
.45 M, .15 M, .60 M, .15 M, .75 M, .30 M, .45 M,
.60 M, .75 M, .60 M, .45 M, .30 M, .75 M, .60 M,
.45 M, .30 M, .75 M. Solutions were made with
Fisher biological grade granular sodium chloride
and distilled water.
Histological Procedure
At the end of recording, electrolytic lesions
were made to mark the sites of electrode tips, and
the animals were killed on the operating table
with a lethal dose of anesthesia. Intracardiac perr
fusion was performed, using .15 M (isotonic)
saline followed by 10% Formol saline. Brains were
then removed and stored in 10% formalin for at
least 7 days. The brains were blocked at the
angle used by Pellegrino and Cushman (1967).
Serial frozen sections were stained with luxol fast
blue and counterstained with neutral red.
166 ROBERT B. MALMO AND WILLIAM J. MUNDL

.ISM .30 M .45 M ,.60 M

(iiotonic)
Concentration of NaCI Solution
FIQUBE 2. Graphs showing averaged ratings of multiple-unit reactions in 17 rats to intracarotid
injections of NaCI solutions ranging from isotonic through 4 degrees of hypertonicity. (Note the greater
reactivity of the lateral preoptic areas [see Figure 1].)

Treatment of Multiple-Unit Data
To anticipate the results, in general, the differ-
ences between medial and lateral preoptic areas
were so obvious that no measurements were re-
quired (see Figure 1). However, one of us
(R.B.M.) applied a simple rating method, which
is explained in reference to Figure 1. Look at the
lower integrator-recorder trace showing the re-
action by neurons in the lateral preoptio area to a
.75 M NaCI intracarotid injection. Compare this
lower trace with the upper trace that was simultane-
ously recorded from the medial preoptic area. This
large difference is typical of the findings.
A rating of 1, which was given to the upper
trace, was assigned to a just perceptible change
from base line. Note that a rating of 1 was also
assigned to the reaction from lateral preoptic
neurons to a .30 M NaCI injection (lower trace).
The progressively higher ratings—2, 3, and 4, as-
signed to NaCI injections of .45 M, .60 M, and
.75 M, respectively (lower traces)—were based on
simple inspection of the peak level reached in the
poststimulus period.
In order to explain the way in which the
curves in Figure 2 were plotted, we continue to
use Rat M3-17 as the example. For the other 4
repetitions of the .75 M NaCI concentration in the
lateral preoptic area of Rat M3-17, the ratings
were 4, 4, 4, and 3, yielding a median rating of 4.
A median rating for multiple-unit reaction to
the .75 M NaCI injection into the lateral preoptic
area was calculated in the same way for each of
the other 16 rats. These median values were then
averaged. Figure 2 shows this mean value plotted
in the graph for the lateral preoptic area. Values
used to plot the other points in these graphs were
obtained in the same manner.
RESULTS
Figure 1 illustrates the major finding.
Multiple-unit recordings from lateral pre-
optic areas showed significantly greater re-
sponses to the injection of hypertonic saline
solutions than did the simultaneously re-
corded multiple-unit activity from medial
preoptic areas.
Figure 2 presents graphs based on aver-
aged ratings on all 17 rats. Each point on
the curve represents the mean of the 17
median ratings for multiple-unit reactions
obtained with 1 of the 5 NaCI concentra-
tions. Correspondence between strength of
NaCI concentration and degree of multiple-
unit response is evident in both curves.
With the .75 M concentration, the median
ratings for all rats were higher for the lateral
preoptic area, a difference that is highly
significant by a sign test. With the .60 M
concentration, there were 16 comparisons
favoring the lateral area and one tie, again
a highly significant difference. In the 68
medial-lateral comparisons of median rat-
ing values for all 4 hypertonic NaCI con-
 OSMOSENSITIVE NEURONS 167

Medial Placements o Lateral Placements

FIGUBE 3. Representations of loci of 2 recording sites in each of 17 rats, indicated on frontal diagram-
matic sections from the Pellegrino and Cushman (1967) atlas.

centrations and all 17 rats, there were no sites dorsal to them. In moving the elec-
cases of greater reaction from the medial trodes downward, no attempt was made in
area. this investigation to record systematically
from the septal areas, which contain cells
Histological Results that are responsive to changes in hydration
Figure 3 shows the lateral and medial re- states (Bridge & Hatton, 1973).
cording sites for each animal. Strong multi-
ple-unit reactions were obtained from Level Unit Recording
3.0 to Level 1.8 (millimeters anterior to Data from 3 male hooded rats (U-6, 590
bregma). For the sites indicated in Figure 3, gm.; U-8, 515 gin.; and U-9, 512 gm.) are
the lateral-medial comparison was the only presented. Ten units were studied in these
one that appeared to yield a significant dif- rats. Histology showed that these cells were
ference in the degree of multiple-unit reac- all in the lateral preoptic area (see Figures
tion. Depth of recording site, as previously 4 and 5).
explained, was guided by degree of reaction, As previously stated, the aims in this part
so that in nearly all instances it may be of the study were to observe: (a) unit re-
inferred that the sites shown in Figure 3 sponse to hypertonic NaCl injections in the
yielded stronger reactions than the nearby absence of response to isotonic NaCl injec-
168 ROBERT B. MALMO AND WILLIAM J. MUNDL

RatU-6
2.0

Rat U-8
2.0

FIGUEE 4. Representations of loci of recording sites for unit recording in 3 rats, indicated on frontal
diagrammatic sections from the Pellegrino and Cushman (1967) atlas. (See photomicrograph for Rat
TJ-9 in Figure 5.)

tions; (b) inhibitory unit reactions to hyper-
osmotic challenge, and (c) a variety of
firing patterns in osmosensitive neurons
(with particular interest in the question of
whether a cell of the burster variety could
be found).
From Site 1 in Rat U-6 (Figure 6) the
reaction to intracarotid injection of .75 M
NaCl was in the form of a brief discharge.
To .15 M NaCl there was no reaction (not
shown in the Figure). A quite different reac-
tion to the injection was observed from Site
1 in Rat U-8: a prolonged acceleration. At
Site 2 in Rat U-8 the cell was excited by in-
jection of hypertonic saline, and control in-
jections of isotonic saline showed no re-
sponse. This cell did not respond to visual or
auditory stimuli nor to tail pinch.
Inhibitory reactions were observed in
Rat U-9, Site 2 (see Figure 7). In some in-
 OSMOSENSITIVE NEURONS 169

stimuli, and it showed an extremely strong

reaction to tail pinch.
One cell (Site 3 in Rat U-8) seemed un-
responsive to saline injection, light, sound,
or tactile stimulation.
DISCUSSION
Our experiments were designed to bring
brain-recording data to bear on the question
of a critical osmosensitive zone in the lateral
preoptic area as delimited in the rat by
Blass and Epstein (1971), and in the rabbit
by Peck and Novin (1971). To the best of
our knowledge, we are the first to single out
this delimited lateral preoptic area for con-
centrated study by means of brain record-
ing and to show clearly that this critical
zone in the lateral preoptic area of the rat
contains cells that are osmosensitive.
Furthermore, by means of simultaneous
recording from cell populations (a) inside
the zone designated as critical by Blass and
Epstein and by Peck and Novin, and (b)
in a zone medial to it, we have shown that
the net acceleratory response to challenge
for the former was much greater than it was
FIGURE 5. Photomicrograph showing electrode for the latter. This observation fits with the
track in Rat U-9. (Cathodal electrolytic current
of 90 /*a. for 5 sec. was used to make a lesion mark- lesion data of Blass and Epstein and pro-
ing Site 4. The cell at Site 4 reacted with brief ac- vides further evidence for a critical osmo-
celeration when the .75 M NaCl solution was first sensitive zone as delimited by Blass and
injected. There was no response to light, no re- Epstein and by Peck and Novin.
sponse to the clicker, and only a slight response
to striking the metal dish with a metal rod. ThereKey Importance of Multiple-Unit
were responses to tactile stimuli and a marked re-Recording
sponse to tail pinch, which seemed to facilitate re-
action of this cell to intracarotid injections of hy-As mentioned earlier, osmosensitive neu-
pertonic saline.) rons elsewhere in the brain (e.g., in and
nearby the supraoptic nucleus) have been
stances this slowing, with relatively long observed to show an inhibitory reaction to
latency, appeared to be preceded by slight hyperosmotic challenge; and we observed
acceleration (see top trace in Figure 7). single cells in the lateral preoptic area that
There was no response to a visual stimulus also showed inhibitory reactions to injec-
(flashlight), but the cell was weakly ex- tion of hypertonic saline. If one could
cited by sound (toy cricket clicker, whistle) assume that there are inhibitory and ac-
and by tail pinch. At Site 2 in U-6 we celeratory types of osmosensitive cells, then
recorded a cell that was inhibited by injec- by means of recording from many osmosen-
tion, with a latency of about 2.5 sec. sitive cells one could estimate the approxi-
Figure 8 shows that we succeeded in find- mate percentages of each type of cell in
ing a burster cell, which is of interest in rela- the total population of cells in the delimited
tion to burster cells described by Hayward area under study. However, as previously
and Jennings (1973a) in the monkey. This indicated, it seems possible that osmosensi-
cell, as all others, failed to react to flash- tive cells may shift back and forth between
light, but reacted strongly to the auditory excitatory and inhibitory phases. This
 Lateral Preoptic Area
Rat U-6, Site No. I
O
100)1 V
w
jlOOjjV
H
I 50

W
f .75M NoCI, 5 sec
g
$
g
O
>
Rat U-8, Site No. I 2;
O

lOOjlV

t .75 M NaCI. 5 sec t

continued

s
FIGURE 6. Top trace: Burst of neuronal activity (from Rat U-6) near midpoint of 5-sec. injection period. (See cluster of traces on pulse stretcher
line below the burst. Several units are evident.) Bottom trace: Single unit from Rat U-8 again responding near midpoint of injection interval but
showing prolonged reaction.
 Lateral Preoptic Area

Rat U-9, Site 2

Photographic Recording (Signal and Pulse Stretcher)

1
1 ' ' ' \', ' ' ' i l i l '' ; i i i ; i ; ; 1 1 T ! t.'i [ r
t***+***#**^^ ]>0U'

Oscillographic Recording (Pulse Stretcher) O

QQ

!i

.75M NoCI CO

a
t—I

Injection repeated
I

t .75 M NaCI
FIGUKE 7. Second cell recorded from Rat U-9. (Main effect of intracarotid hypertonic saline injection was to inhibit this cell. Note similar reac-
tions on two occasions, shown in photographic and ink [pulse stretcher] recordings, with suggestion of an initial slight acceleration in top trace.)
 to

Lateral Preoptic Area

Rot U-9, Site3 §

td
Photographic Recording (Signal and Pulse Stretcher)

100/iV
a

g
o
Oscillographic Recording (Pulse Stretcher and Integrator)
d
3

d
2
b
FIGURE 8 Burster cell from Rat U-9 excited by intracarotid hypertonic saline injection, with relatively prompt return to base line. (Response also
shown in ink tracings of pulse stretcher and of integrator [bottom line]. This cell is of particular interest in relation to osmosensitive burster cells
described by Hayward and Jennings (1973a) in the unanesthetized monkey.)
 OSMOSENSITIVE NEURONS
means that it would be impossible to rely
on single-unit recording for estimating the
direction of the net response to hyper-
osmotic challenge in a population of osmo-
sensitive cells. With this possibility in mind,
the technique required is one that samples
the reactions of a population of cells simul-
taneously (i.e., multiple-unit recording).
Single-Unit Recording as Source of
Additional Information
Whether the extremely low multiple-unit
reactions from the medial preoptic area were
due to nearly equal inhibitory and excita-
tory reactions occurring simultaneously is
a question that cannot be answered on the
basis of the present data. It seems more
probable that the low reactivity in the
medial area is due to a much reduced popu-
lation of osmosensitive cells there. In any
event, from the combined behavioral and
brain-recording evidence, it seems reason-
able to suggest that it is the net excitatory
neuronal reaction from an area that deter-
mines the importance of that area for the
mechanism of cellular dehydration thirst.
Single-unit recordings should be used in
future work to search for osmosensitive cells
in the lateral preoptic area (and elsewhere)
and to follow them over periods of hours in
order to look for phase changes. Single-unit
recordings were useful in the present study
in revealing a burster cell, with its dis-
tinctive firing pattern that resembles the
patterns of osmosensitive cells in the supra-
optic and internuclear zone of the unanes-
thetized monkey (Hayward & Jennings,
1973a).
Osmoreceptor Theory
Results of the present experiments pro-
vide new evidence supporting the osmo-
receptor concept.
In order to strengthen our confidence in
the proposition that the lateral preoptic zone
actually contains osmoreceptors, further
brain-recording experiments are required.
Recordings should be taken from the critical
area after it has been surgically isolated
from surrounding tissue. As mentioned
earlier, this has been done for the supraoptic
173
nucleus. In fact, Ishikawa et al. (1966)
found that after surgical isolation, some
supraoptic nucleus neurons showed even
greater response to hyperosmotic challenge.
Similar results from lateral preoptic neurons
following surgical isolation would indicate
that they were central receptors rather than
synaptically driven neurons. Establishing a
locus of osmoreceptors by these procedures
would fix a point of origin for the neural
system underlying cellular dehydration
thirst. Our dual recording technique could
then be used to study relations between the
lateral preoptic area and other brain areas,
such as the lateral hypothalamic area.
The lateral hypothalamic area has an
important involvement in thirst (Mogenson,
1973), but not at the receptor level accord-
ing to the data of Blass and Epstein (1971)
and Peck and Novin (1971). If the lateral
hypothalamus is synaptically driven by
osmoreceptors via the medial forebrain
bundle, which it shares with the lateral pre-
optic area, the lateral hypothalamic neurons
would be osmosensitive; surgical isolation
from surrounding tissue should abolish their
osmosensitivity.
In conclusion, our brain-recording data
have strengthened osmoreceptor theory and
the position of those who believe that there
are osmoreceptors in the lateral preoptic
area. Also, our findings demonstrate the im-
portance of combining multiple-unit and
single-unit recording techniques in the fur-
ther pursuit of problems related to cellular
dehydration thirst.
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(Received September 11, 1973)