Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25

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Autonomic Neuroscience: Basic and Clinical

journal homepage: www.elsevier.com/locate/autneu

Role of dorsolateral periaqueductal grey in the coordinated regulation of

cardiovascular and respiratory function
Roger A.L. Dampney a,⁎, Teri M. Furlong a, Jouji Horiuchi b, Kamon Iigaya a
a
School of Medical Sciences (Physiology) and Bosch Institute for Biomedical Research, University of Sydney, NSW 2006, Australia
b
Department of Biomedical Engineering, Toyo University, Saitama, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The midbrain periaqueductal grey (PAG) contains four longitudinal columns, referred to as the dorsomedial
Received 15 September 2012 (dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG) subdivisions, which collectively have
Received in revised form 18 December 2012 a pivotal role in integrating behavioural and physiological responses to external stressors as well as other func-
Accepted 26 December 2012
tions. This review is focussed on the dlPAG, which is believed to be an important component of the central mech-
anisms that generate the defensive response to acute psychological stressors, such as the presence of a predator
Keywords:
Defensive behaviour
or other immediate threat. The anatomical connections of the dlPAG are highly specific and distinctly different
Stress from those of the other PAG subregions. The chemical properties of the dlPAG are also distinctly different from
Sympathetic activity the other PAG subregions (e.g. there is a very high density of neurons that synthesize nitric oxide in the dlPAG
Respiratory activity but very few such neurons in the other PAG subregions). Recent functional studies have demonstrated that neu-
Periaqueductal grey rons in the dlPAG exert a powerful control over both sympathetic and respiratory activity, and that the pattern of
Superior colliculus the evoked respiratory changes is also distinctly different from those evoked from other PAG subregions. These
studies also showed that the sympathetic and respiratory changes evoked from the dlPAG are highly correlated,
suggesting the possibility that a common population of “command neurons” within this region may generate the
sympathetic and respiratory changes that accompany defensive behavioural responses to acute psychological
stressors. Finally, although the anatomical connections and functional properties of the dlPAG are distinctly dif-
ferent from the other PAG subregions, they have many similarities with adjacent parts of the superior colliculus,
suggesting that the dlPAG and deep layers of the superior colliculus may be part of a common defence system in
the midbrain.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction deep origin in the muscles or viscera, or haemorrhage (Carrive, 1993;

Animals have evolved various types of strategies in defending them-
selves against different types of threatening or stressful stimuli. As
summarised by Bandler et al. (2000) and Keay and Bandler (2001), the
strategies can be classified as either active or passive coping strategies.
Active defensive behaviour is characterised by increased somatomotor
activity, and cardiovascular and respiratory changes that have the effect
of increasing the blood flow and supply of oxygen to active skeletal mus-
cles. Such a response can be triggered by a stimulus that can be escaped
from, such as the presence of a predator or cutaneous pain. In contrast,
passive defensive behaviour is characterised by decreased somatomotor
activity (e.g. quiescence) accompanied by appropriate cardiovascular
changes, including reduced arterial blood pressure and heart rate. Such
responses are triggered by stimuli that are inescapable, such as pain of
⁎ Corresponding author at: School of Medical Sciences (Physiology) and Bosch Institute
for Biomedical Research, F13, The University of Sydney, NSW 2006, Australia. Tel.: +61 2
93514603.
E-mail address: roger.dampney@sydney.edu.au (R.A.L. Dampney).
Bandler et al., 2000; Keay and Bandler, 2001).
There is now a very extensive literature that has clearly documented
the key role of the midbrain periaqueductal grey (PAG) in generating
both active and passive responses to different types of threatening or
stressful stimuli (for reviews see Carrive, 1993; Bandler and Shipley,
1994; Bandler et al., 2000; Keay and Bandler, 2001; Vianna and
Brandao, 2003). The PAG is known to contain four longitudinal columns,
referred to as the dorsomedial (dmPAG), dorsolateral (dlPAG), lateral
(lPAG) and ventrolateral (vlPAG) subdivisions, which differ with respect
to their functional properties, anatomical connections and chemical
properties (Carrive, 1993; Bandler and Shipley, 1994; Bandler et al.,
2000; Keay and Bandler, 2001; Vianna and Brandao, 2003). In regard
to function, it is believed that both the lPAG and dlPAG are crucial com-
ponents of the central circuits that generate active defensive behaviour-
al responses, while the vlPAG is critical for the expression of passive
behavioural responses (Carrive, 1993; Bandler and Shipley, 1994;
Bandler et al., 2000; Keay and Bandler, 2001). Although both the lPAG
and dlPAG can generate active defensive behavioural responses, ana-
tomical studies indicate that the neurons in these two subregions are
activated by distinctly different inputs, and also differ markedly with

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18 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25
respect to their chemical properties (Bandler and Shipley, 1994; Bandler
et al., 2000; Keay and Bandler, 2001; Vianna and Brandao, 2003). Fur-
thermore, recent studies indicate that the dlPAG also has functional
properties that are distinctly different to those of the adjacent lPAG as
well as the dmPAG. In this review we shall describe how the anatomical
connections and chemical and functional properties of the dlPAG differ
markedly from those of other PAG subregions, and will then discuss
the hypothesis originally put forward by Bandler et al. (2000) that the
dlPAG has a unique role in generating the cardiovascular and respiratory
changes associated with defensive responses evoked by unconditioned
threatening psychological stimuli. First, however, we shall briefly de-
scribe the pattern of cardiovascular and respiratory changes evoked by
such stimuli.
2. Cardiovascular and respiratory changes evoked by acute
psychological stimuli
In response to acute alerting or threatening visual, auditory or olfac-
tory stimuli in their external environment rats exhibit a behaviour
consisting of alternate periods of freezing and flight, which has the effect
of making their position less predictable to a predator and thus increas-
ing the probability of escape (Vianna and Brandao, 2003). During the
freezing response that is evoked by noise in rats, there is little change
in arterial pressure, but the cardiac output decreases, indicating that
total peripheral resistance is increased (Miki and Yoshimoto, 2010;
Yoshimoto et al., 2010). The increase in total peripheral resistance is
likely to be due to visceral vasoconstriction, since renal sympathetic
nerve activity is increased while lumbar sympathetic nerve activity is
unchanged (Miki and Yoshimoto, 2010; Yoshimoto et al., 2010). Alerting
stimuli such as a sudden noise or light also evoke an immediate and very
marked increase in respiratory rate (Kabir et al., 2010). Such transient
stimuli evoke either no change or else a decrease in heart rate (Kabir
et al., 2010), whereas a more sustained alerting stimulus (e.g. a noise
maintained for several minutes) can evoke a bradycardia (Yoshimoto
et al., 2010).
The arterial baroreceptor reflex plays a key role in generating the
cardiovascular changes evoked by acute psychological stimuli, because
after baroreceptor denervation the increase in renal sympathetic nerve
activity accompanying the freezing response is attenuated, and the de-
crease in heart rate is abolished (Yoshimoto et al., 2010). Miki and
Yoshimoto (2010) have proposed that during the freezing response a
combination of central command and re-setting of the baroreceptor re-
flex results in visceral vasoconstriction accompanied by bradycardia,
with little change in arterial pressure and skeletal muscle vascular resis-
tance. They point out that the physiological advantage of these effects is
that the visceral vasoconstriction may minimize the time lag for redistri-
bution of cardiac output to the active skeletal muscles during the transi-
tion from freezing to flight behaviour. Further, the bradycardia may
conserve energy in the cardiac muscle, and thus increase the rate at
which cardiac output may increase during this transition phase.
3. Anatomical connections of the dlPAG
The behavioural response of freezing and flight can be reproduced
by direct activation of neurons within the PAG of rats, and the thresh-
old for such effects is lowest in the dlPAG (Bittencourt et al., 2004).
Consistent with this, the dlPAG receives inputs arising from visual,
auditory and olfactory stimuli, that are known to trigger such a defen-
sive behavioural response (Fig. 1, Table 1). In particular, there are di-
rect projections from the primary auditory cortex and from area 18 (a
secondary visual cortical area in the rat) to the dlPAG but not other
PAG subregions (Benzinger and Massopust, 1983; Newman et al.,
1989) (Fig. 1B). There is also a projection from area 18 to the superior
colliculus adjacent to and above the dlPAG, as can be seen in Fig. 1B
(Benzinger and Massopust, 1983). Furthermore, there is a projection
from the superior colliculus to the PAG, which is directed exclusively
to the dlPAG (Rhoades et al., 1989). In addition, there are inputs to the
dlPAG, but not other PAG subregions, from two nuclei known to have
an important role in the control of eye movements, the nucleus
praepositus hypoglossi and the periparabigeminal nucleus (Klop et
al., 2005, 2006) (Fig. 1A). Both of these nuclei also project to the supe-
rior colliculus (Klop et al., 2005, 2006) (Fig. 1A). Klop et al. (2006)
have suggested that these inputs allow neurons in the dlPAG to re-
spond to visual signals generated by the presence of objects in the ex-
ternal visual field rather than changes in visual signals caused only by
eye movements. Consistent with this hypothesis, dlPAG cells are
inhibited just before and during rapid eye movements (saccades)
(Kase et al., 1986).
One of the most dense inputs to the dlPAG arises from neurons in
the dorsal premammillary nucleus (PMD) in the hypothalamus, par-
ticularly its ventrolateral portion (Motta et al., 2009) (Fig. 1A). Le-
sions of the PMD or blockade of excitatory receptors in the PMD
greatly reduce the defensive behavioural response to either the pres-
ence of a predator, or the odour of a predator (Markham et al., 2004;
Blanchard et al., 2005; Do Monte et al., 2008; Motta et al., 2009). The
ventrolateral part of the PMD receives a major direct input from the
dorsomedial part of the hypothalamic ventromedial nucleus (VMH),
which in turn receives inputs from two parts of the amygdala, the
posteroventral part of the medial nucleus and the posterior part of
the basomedial nucleus (Motta et al., 2009). The posteroventral part
of the medial amygdala is activated by predator odour (McGregor et
al., 2004), while the posterior part of the basomedial nucleus is be-
lieved to be activated more generally by signals indicating the pres-
ence of a predator (Motta et al., 2009).
The ventrolateral part of the PMD also receives a major input from
the anterior hypothalamic area (Risold et al., 1994), which together
with the PMD and VMH are part of the medial hypothalamic defen-
sive behaviour system (Swanson, 2000). There is also a strong projec-
tion from a rather circumscribed lateral region in the anterior
hypothalamic area directly to the dlPAG (Semenenko and Lumb,
1992), activation of which evokes increases in arterial pressure,
hindlimb blood flow and respiratory rate, similar to the cardiovascu-
lar response associated with active defensive behaviour (Lumb and
Lovick, 1993).
The dlPAG in the rat also receives direct inputs from neurons in
the medial prefrontal cortex (PFC), particularly its caudodorsal por-
tion (caudal prelimbic and anterior cingulate cortices) (Floyd et al.,
2000; Gabbott et al., 2005). These cortical regions also project to the
anterior hypothalamic nucleus and VMH (Keay and Bandler, 2001),
and so may also indirectly influence neurons in the dlPAG via these
hypothalamic regions. It is believed that the medial PFC, including
those subregions that project to the dlPAG, is involved in complex
cognitive reasoning and the interpretation of stressful stimuli
(Bandler et al., 2000). In primates, the cortical regions that are homol-
ogous to the caudodorsal portion of the medial PFC are areas 10, 25
and 32, and anatomical studies have demonstrated that these areas
are the origin of a dense projection to the dlPAG (An et al., 1998;
Bandler et al., 2000). Damage to the medial PFC in humans impairs
their ability to predict the consequences of risk-taking behaviour,
and also reduces the autonomic responses that are normally associ-
ated with the emotional stress of risk-taking behaviour (Bechara et
al., 1997).
In summary, afferents to the dlPAG arise from a number of nuclei that
receive inputs related to visual, auditory and olfactory signals, or from
nuclei that process such signals. It is particularly striking that, with the
exception of the projection from the PMDvl, all of these inputs target
only the dlPAG, and not any of the other PAG subregion. Conversely,
there are many inputs to the PAG from various regions (e.g. central
nucleus of amygdala, medial preoptic area, pontomedullary tegmental
field, and the spinal cord) that project to all PAG subregions except for
the dlPAG (Fig. 1A, Table 1) (Bjorkeland and Boivie, 1984; Wiberg and
Blomqvist, 1984; Rizvi et al., 1991, 1992; Herbert and Saper, 1992; Klop
 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25 19

A Periparabigeminal Dorsal premanmillary

area nucleus

dl
dl
SC
dm
dl PAG
l Aq
vl

Pontomedullary Central nucleus of

tegmental field the amygdala

dl
dl

B from area 18 in cortex C to midline medulla

Fig. 1. A, Photomicrographs showing examples of anterograde labelling from various nuclei as shown. Note that the inputs from the periparabigeminal nucleus and from the dorsal
premammillary nucleus (modified from Klop et al. (2006) and from Motta et al. (2009), respectively, with permission) terminate almost exclusively in the dorsolateral (dl) periaqueductal
grey (PAG), while inputs from the pontomedullary tegmental field and from the central nucleus of the amygdala (modified from Klop et al. (2005) and from Rizvi et al. (1991), respec-
tively, with permission) terminate in the dorsomedial (dm), lateral (l), and ventrolateral (vl) subnuclei, and not in the dlPAG. B, Terminal labelling in the dlPAG and overlying superior
colliculus after an injection of an anterograde tracer in area 18 in the cortex (visual association area) (modified from Benzinger and Massopust (1983), with permission). C, Retrogradely
labelled cells in the PAG after an injection of a retrograde tracer in the midline medulla (modified from Cowie and Holstege (1992), with permission).
Modified from Cowie and Holstege (1992).

Table 1 et al., 2005). Thus, the afferent inputs to the dlPAG are highly specific and
Origins of inputs to different PAG subregions. distinctive.
Region Nucleus dmPAG dlPAG lPAG vlPAG Similarly, the outputs of the dlPAG are also highly specific and dis-
tinctively different to those of the other PAG subregions. As summarised
Forebrain Primary cortical motor areas X X
(limbs, trunk) in Table 2, there are strong projections to the midline medulla, rostral
Primary auditory cortex X ventrolateral medulla (which contains sympathetic premotor neurons
Secondary visual cortex X (Dampney et al., 2003; Guyenet, 2006)), and to the ventral respiratory
Medial prefrontal cortex X group from the dmPAG, lPAG and vlPAG (Beitz et al., 1983; Van
(rostroventral)
Medial prefrontal cortex X
Bockstaele et al., 1991; Cowie and Holstege, 1992; Gaytan and Pasaro,
(caudodorsal) 1998). In contrast, the dlPAG does not project to any of these regions
Central nucleus of amygdala X X X (Beitz et al., 1983; Van Bockstaele et al., 1991; Cowie and Holstege,
Bed nucleus of stria terminalis X X X 1992; Gaytan and Pasaro, 1998) (Table 2, Fig. 1C). Neurons in the
Medial preoptic area X X X
dlPAG do project, however, to the cuneiform nucleus (Redgrave et al.,
Anterior hypothalamic area X
Dorsal premammillary nucleus X X 1988; Bernard et al., 1989). There is no significant projection to the cu-
(ventrolateral) neiform nucleus from other PAG subregions.
Dorsal premammillary nucleus X X
(dorsomedial)
Midbrain Superior colliculus X
Cuneiform nucleus X
Table 2
Pons/ Pontomedullary tegmental field X X X
Targets of outputs from different PAG subregions.
medulla Ventrolateral medulla X X X
Nucleus tractus solitarius X Region Nucleus dmPAG dlPAG lPAG vlPAG
Spinal trigeminal nucleus X
Nucleus retroambiguus X X Midbrain Cuneiform nucleus X
Nucleus praepositus hypoglossi X Pons/medulla Parabrachial complex X X X X
Periparabigeminal nucleus X Midline medulla X X X
Spinal Cervical segments X X X Rostral ventrolateral medulla X X X
cord Thoracic segments X X X Ventral respiratory group X X
Lumbar segments X X X Spinal cord Cervical segments X X X
20 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25
A second major target of the dlPAG is the parabrachial complex,
specifically its superior lateral nucleus (Krout et al., 1998). While
there are also projections to the parabrachial complex from all four
PAG subregions, the dlPAG is the only subregion that projects exclu-
sively to the superior lateral parabrachial subnucleus (Krout et al.,
1998). It is interesting to note that there is also a very strong projection
from the superior lateral parabrachial subnucleus to the dorsomedial
part of the VMH (Bester et al., 1997) which, as mentioned above, is part
of the medial hypothalamic behavioural defence system (Swanson,
2000). The significance of these output projections from the dlPAG with
respect to cardiorespiratory regulation will be considered below.
4. Chemical properties of the dlPAG
Like its anatomical connections, the chemical properties of the
dlPAG are distinctly different from the other PAG subregions. This is
perhaps most obvious in the case of NADPH diaphorase or nitric
oxide synthase (NOS), both of which are markers of neurons that syn-
thesize nitric oxide (Herbert and Saper, 1992; Onstott et al., 1993;
Bandler and Shipley, 1994). As shown in Fig. 2, neurons in the PAG
containing NADPH diaphorase are essentially confined to the dlPAG
subregion. Similarly, the enzyme acetylcholinesterase in the PAG is
also found only in the dlPAG (Illing and Graybiel, 1986). In contrast,
the enzyme cytochrome oxidase is largely absent from the dlPAG
but is present in all other PAG subregions (Conti et al., 1988) (Fig. 2).
Another distinctive feature of the dlPAG is that it contains a much
higher density of GABA neurons and terminals than the adjacent
dmPAG and lPAG (Barbaresi, 2005, 2010) (Fig. 2). There is also a higher
density of both ionotropic and metabotropic glutamate receptors in the
dlPAG compared to the other PAG subregions, although in this case the
difference is less marked than in the case of GABA neurons and termi-
nals (Albin et al., 1990; Azkue et al., 1997). There is also a very low
NADPH diaphorase
GABA
Fig. 2. Examples of different patterns of labelling in the PAG of NADPH diaphorase (marker of neurons that synthesize nitric oxide), cytochrome oxidase, GABA, and dopamine
β-hydroxylase (marker of noradrenergic neurons and terminals).
Modified from Bandler and Shipley (1994), Conti et al. (1988), Barbaresi (2010), and Herbert and Saper (1992), respectively, with permission.
density of terminals immunoreactive for dopamine-β-hydroxylase in
the dlPAG and dmPAG compared to the lPAG and vlPAG (Herbert and
Saper, 1992) (Fig. 2), indicating that the former subregions do not re-
ceive a significant adrenergic innervation.
5. Functional properties of the dlPAG
As described above, the dlPAG receives inputs arising from visual,
auditory, and olfactory receptors, particularly those that may signal an
external threat. Consistent with these anatomical connections, it has
been shown that an auditory stimulus (ultrasound application) which
is known to trigger defensive behaviour in rats (Beckett et al., 1996), re-
sults in increased c-Fos expression (a marker of neuronal activation) in
the dlPAG (Klein et al., 2010). Similarly, cat odour (olfactory input only)
or exposure to a cat (visual, olfactory and auditory inputs) also results in
increased c-Fos expression in the dlPAG (and to a lesser extent the
dmPAG) at rostral and intermediate levels of the PAG, but not to a sig-
nificant extent in the lPAG and vlPAG at these levels (Canteras and
Goto, 1999; Dielenberg et al., 2001; Motta et al., 2009) (Fig. 3A). Fur-
thermore, these stimuli also evoked strong c-Fos expression in the
PMDvl, which as mentioned above provides a strong direct input to
the dlPAG (Motta et al., 2009). In contrast, stimuli which are essentially
physical, such as radiant heat or muscle pain, evoke increased c-Fos
expression mainly in the lPAG or vlPAG with little effect in the dlPAG
(Keay and Bandler, 2001). Finally, stimuli which have both physical
and psychological components (e.g. an air puff stimulus) evoke c-Fos
expression in all of these PAG subregions (Furlong et al., 2009).
Consistent with the results of the c-Fos studies on the effects of cat
odour or exposure to a cat described above, lesions of the dorsal PAG
that include the dlPAG cause a reduction in the behavioural response
and in the tachycardiac response evoked by cat odour or exposure to
a cat, but has no significant effect on the pressor response to these
Cytochrome oxidase
Dopamine -hydroxylase
 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25 21

A B
160
AP
(mmHg)
80
HR 450
(beats/min) 350
220
Integrated
RSNA
(%baseline)
100
PNA 200
burst rate
(bursts/min)
100
250
Respiratory
activity
(%baseline)
100

DLH (750 pmol) 30 sec

C
70
**# ** P < 0.001 vs dmPAG
# P < 0.02 vs lPAG
60
## P < 0.001 vs lPAG
**
##
50
Response
(% baseline)
40

30

20

10

0
dmPAG dlPAG lPAG
n=16 n=14 n=10

Fig. 3. A, Example of c-Fos expression in the PAG of as rat following exposure to a cat (modified from Canteras and Goto, 1999, with permission). B, Changes in arterial pressure (AP),
heart rate (HR), renal sympathetic nerve activity (RSNA), respiratory rate (i.e. phrenic nerve activity (PNA) burst rate), and overall respiratory activity (i.e. integrated PNA) evoked
by microinjection of D,L-homocysteic acid into the dlPAG (modified from Iigaya et al. (2010), with permission). C, Histograms showing the changes in RSNA, PNA burst rate, and
respiratory (Resp) activity evoked by microinjections in the dmPAG, dlPAG, and lPAG. Note that the largest respiratory responses were evoked from the dlPAG, whereas there
were no significant differences in the magnitudes of the evoked increases in RSNA with respect to PAG subregion.
Modified from Iigaya et al. (2010), with permission.

stimuli (Dielenberg et al., 2004). Thus, neurons in the dlPAG appear to
contribute to the expression of some but not all components of the
cardiovascular and behavioural response to these threatening stimuli.
Other behavioural studies have shown that activation of neuronal
cell bodies in different sites in the PAG of the rat, by microinjection of
an excitatory amino acid, evokes defensive behavioural responses
(e.g. freezing and escape) from both the dlPAG and lPAG, but the thresh-
old for such effects is significantly lower in the dlPAG (Bittencourt et al.,
2004). A similar behavioural response can also be evoked from the deep
layers of the superior colliculus overlying the dlPAG (Bittencourt et al.,
2005). In anaesthetized rats, microinjection of the excitatory amino
acid D,L-homocysteic acid (DLH) into the dlPAG evokes autonomic and
respiratory changes that are appropriate for such behaviours, that is
marked increases in renal sympathetic nerve activity (RSNA) and in re-
spiratory activity (Huang et al., 2000; Subramanian et al., 2008; Iigaya et
al., 2010) (Fig. 3B).
Iigaya et al. (2010) also found, by injecting very small amounts of
DLH (750 pmol in 15 nl), that distinctly different patterns of response
were evoked from the dlPAG as compared to the adjacent dmPAG and
lPAG. In particular, while large increases in RSNA were evoked from all
three PAG subregions, the increase in respiratory activity was much
greater when evoked from the dlPAG (Fig. 3C). Moreover, the increase
in respiratory activity evoked from the dlPAG was due mainly to an in-
crease in respiratory rate, whereas that evoked from the lPAG was due
mainly to an increase in respiratory amplitude (as measured by the
magnitudes of the bursts of phrenic nerve activity) (Fig. 3C). Marked
increases in respiratory activity could also be evoked from the deep
layers of the superior colliculus adjacent to the dlPAG. These increases
were due almost entirely to an increase in respiratory rate, very similar
to the effects evoked from the dlPAG (Iigaya et al., 2010).
Another interesting feature of the responses evoked from the dlPAG
is the close correlation between evoked increases in RSNA and evoked
increases in respiratory activity (Iigaya et al., 2010) (Fig. 4A). There
are two possible explanations for this close correlation. One possibility
is that the changes in respiratory activity and RSNA are evoked indepen-
dently from separate populations of neurons within this subregion, but
these separate populations have very similar overlapping distributions
within the dlPAG. The second possibility is that there is a common
22 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25

population of neurons within the dlPAG that are capable of generating normally regulate the release of nitric oxide in the dlPAG, as well as
simultaneously increases in respiratory activity and RSNA. Regardless actions on GABAergic neurons that are not mediated by nitric oxide,
of which of these two alternative possibilities is correct, the fact that however, remain unknown. Similarly, the source(s) of the GABAergic
powerful respiratory and renal sympathetic responses can be simulta- inputs to the dlPAG has not been determined.
neously elicited from the dlPAG is consistent with the view that this re- There have also been some studies that provide information about
gion generates defensive behavioural responses, in which simultaneous the role of the PAG in humans in generating physiological changes asso-
increases in respiratory and sympathetic activity would be physiologi- ciated with acute psychological stress. In particular, a lactate-induced
cally advantageous. panic attack in patients, which is known to be associated with marked
There was no correlation, however, between increases in heart rate increases in sympathetic and respiratory activity (Esler and Kaye,
and increases in respiratory activity (Fig. 4B), reflecting the fact that 2000; Sinha et al., 2000), has been shown to be associated with in-
changes in heart rate evoked from the dlPAG are small and variable creased blood flow to the superior colliculus and immediately subjacent
(Iigaya et al., 2010). As mentioned above, however, a recent study has region, which would include the dlPAG (Reiman et al., 1989). Second, a
shown that mild alerting auditory or visual stimuli in conscious rats study by Mobbs et al. (2007) using functional magnetic resonance im-
evoke a marked increase in respiratory rate but have little effect on aging showed that activity in the dorsal PAG was increased in human
heart rate (Kabir et al., 2010). These observations support the view subjects confronted with a virtual predator (using computer simula-
that the respiratory and cardiac changes evoked by auditory or visual tion), and that this activity increased as the threat became more proxi-
stimuli are regulated by separate pathways within the brain. mal. Although the resolution of these brain imaging studies was not
As described above, the dlPAG has a high concentration of GABA sufficient to identify the precise PAG subregions that are activated,
receptors and enzymes that synthesize nitric oxide, much higher than they are nevertheless consistent with studies in animals described
in other PAG subregions. The high concentration of GABA receptors above that indicate that the dlPAG has a critical role in integrating the
indicates that neurons in the dlPAG are subject to strong inhibitory responses to acute psychological stress. In support of this, recent studies
inputs. Consistent with this, blockade of GABA receptors in the dlPAG have shown that stimulation of the dorsal part of the PAG in humans
by microinjection of bicuculline evokes strong sympathetic and respira- elicits increases in blood pressure, presumably due to an increase in
tory responses (Iigaya et al., 2012), indicating that the dlPAG neurons sympathetic activity (Basnayake et al., 2011, 2012).
generating these responses receive tonic GABAergic inputs. Similarly,
blockade of nitric oxide synthase in the dlPAG evokes a pressor re- 6. Proposed model of dlPAG functional connections
sponse (Hall and Behbehani, 1997), indicating that nitric oxide also
has a tonic inhibitory effect. Another study using whole-cell recording As summarised above, the anatomical connections and functional
has shown that the mechanism for this effect is that nitric oxide sup- properties of the dlPAG support the view that this PAG subregion can
presses neuronal activity in the dlPAG via a potentiation of GABAergic generate both the defensive behavioural response and associated car-
synaptic inputs (Xing et al., 2008). The physiological stimuli that diovascular and respiratory changes triggered by an acute escapable
psychological threat, such as the presence of a predator. As shown
100 in Fig. 5, auditory, visual and olfactory inputs, that signal the presence
A
90 of an external threat, all converge on neurons in the dlPAG but not on
neurons in other PAG subregions. In contrast, inputs arising from so-
80
matic receptors that signal a physical stressor (e.g. pain) project to
70 the lPAG and vlPAG but not to the dlPAG (Fig. 5).
r = 0.914
60 P < 0.001 As shown in Fig. 5, the dlPAG has rather limited output projections.
This raises the question as to the organization of the output pathways
50
(% baseline) from the PAG that subserve the defensive behavioural response and
40 the associated cardiovascular and respiratory effects, including barore-
30 flex resetting which is an important component of these effects (Miki
and Yoshimoto, 2010). In regard to the cardiovascular and respiratory
20
responses that are evoked by stimulation of the dlPAG, it has been
10 shown that these responses depend upon the activity of neurons in
0 the dorsomedial hypothalamus (DMH) (de Menezes et al., 2009;
20 40 60 80 100 120 Horiuchi et al., 2009). There are two alternative general explanations
for this observation. The first possible explanation is that signals con-
(% baseline) veying cardiovascular and respiratory responses from the dlPAG are
conveyed by an ascending pathway that synapses with neurons within
B the DMH that then generate the cardiovascular/respiratory response
20
r = 0.104
(Fig. 5). Consistent with this, the DMH contains neurons that can gener-
15
P = 0.73 ate such responses, including baroreflex re-setting (Fontes et al., 2001;
10 McDowall et al., 2006, 2007; Tanaka and McAllen, 2008). There is only a
(% baseline)
5 minor direct projection from the dlPAG to the DMH (Thompson and
Swanson, 1998), but as described above there are major projections
0
20 40 60 80 100 120 from the dlPAG to the cuneiform nucleus and superior lateral parabrachial
nucleus, both of which project to the hypothalamus, including the DMH
(% baseline) (Fulwiler and Saper, 1984; Bester et al., 1997; Lam et al., 1997). The de-
scending pathways from the DMH that subserve the cardiovascular and
Fig. 4. Graphs showing the relationship between changes in RSNA and respiratory ac- respiratory effects arising from activation of neurons in the DMH are
tivity evoked by microinjection of DLH in 14 sites in the dlPAG (A) and between evoked not fully defined, although it is known that there are direct projections
changes in HR and respiratory activity at these same sites (B). Note that there was a to the rostral ventrolateral medulla and raphe pallidus, which contain
high degree of correlation between changes in RSNA and respiratory activity but not
between changes in HR and respiratory activity. The straight lines represent the lines
premotor neurons regulating the vasomotor and cardiac sympathetic out-
of best fit in each case. flow (Thompson et al., 1996; Fontes et al., 2001; Samuels et al., 2004).
From Iigaya et al. (2010), with permission. There is also a direct projection to the dorsomedial medulla, which
 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25 23

Escapable psychological stressors of dlPAG neurons. First, there is a projection from the cuneiform nucleus
Auditory Visual Olfactory Perceived to the Kölliker-Fuse nucleus in the dorsolateral pons (Korte et al., 1992),
emotional and this nucleus in turn projects to the ventrolateral medulla, including
stressor regions that contain sympathetic premotor and respiratory neurons
amygdala
(Herbert et al., 1990; Korte et al., 1992). There is also a direct projection
from the cuneiform nucleus to the nucleus tractus solitarius (Korte et
VMHdm al., 1992). Second, stimulation of the cuneiform nucleus evokes increases
visual cortex
auditory superior colliculus Caudodorsal in sympathetic activity as well as freezing responses (Verberne, 1995;
cortex oculomotor nuclei PMDvl medial PFC Mitchell et al., 1988) and may also modulate the baroreceptor reflex
(Korte et al., 1992). Furthermore, cutting the connection between the
cuneiform nucleus and the Kölliker-Fuse nucleus reduced the pressor
response to stimulation of the former nucleus (Korte et al., 1992). It is
therefore possible that the cuneiform nucleus does play an important
Escapable DMH role in generating cardiovascular and respiratory responses, as well as
physical
behavioural responses generated from activation of dlPAG neurons.
stressors
somatic receptors 7. Unresolved questions and future directions
CnF
dlPAG
As we have described above, functional studies have shown clearly
spinal cord lPAG that the dlPAG contains neurons that are capable of generating defen-
& brainstem
sive behavioural responses (e.g. freezing or flight) and cardiovascular
and respiratory responses that appear to be appropriate for such behav-
iours. It is not known, however, whether all the components of the
response to a threatening stimulus (i.e. behavioural, autonomic and
respiratory effects) are generated by a single common population of
neurons (i.e. “command neurons”) or instead by separate distinct
populations of neurons. At least with regard to respiratory and sympa-
thetic effects evoked from the dlPAG, there is some evidence consistent
with the view that a common population of neurons may generate
medulla
these effects (see Fig. 4), but even if that hypothesis is correct, there is
no information available at all as to whether the same neurons also regu-
late the behavioural components of the overall response. In any case,
Sympathetic Respiratory given that the dlPAG is a site at which there is a convergence of inputs
activity activity from a variety of external receptors, as well as from the medial PFC, the
most reasonable hypothesis is that the dlPAG has a key role in integrating
Fig. 5. Schematic diagram showing major inputs to the dlPAG and the proposed output inputs from different sources to evoke a rather stereotyped behavioural,
pathways subserving the co-ordinated changes in sympathetic vasomotor and respira- autonomic and respiratory response to unconditioned threats in the ex-
tory activity regulated by the dlPAG. The lines with arrows indicate connections, that
could be either direct (monosynaptic) or indirect (polysynaptic).
ternal environment. How such integration occurs within the dlPAG is a
question that will need to be addressed in future studies.
A second unresolved question is the organization of the output
includes neurons within the nucleus tractus solitarius that modulate the pathways from the dlPAG that ultimately regulate the behavioural,
baroreceptor reflex as well as neurons in the dorsal respiratory group cardiovascular and respiratory changes evoked by activation of the
(Thompson et al., 1996; Fontes et al., 2001). dlPAG. As indicated above, the DMH does appear to have an impor-
An alternative explanation for the observation that cardiorespirato- tant role in producing such effects, but the precise nature of that
ry responses generated from the dlPAG depend upon the activity of role, and also the relative contributions of ascending and descending
neurons in the DMH is that these responses are conveyed by a descend- pathways to the production of the cardiovascular and respiratory ef-
ing pathway to cardiovascular and respiratory neurons in the medulla, fects evoked from the dlPAG have not yet been clarified.
which also receive inputs from the DMH. If there was a facilitatory sum- As others have noted (e.g. Illing and Graybiel, 1986) several lines
mation of the inputs from the dlPAG and those from the DMH, then of evidence from anatomical and functional studies suggest that the
activation of the dlPAG might only evoke activation of the medullary deep layers of the superior colliculus have a close functional relation-
cardiovascular and respiratory neurons when inputs from the DMH ship with the dlPAG but not with other PAG subregions. First, as noted
were active (Fig. 5). In support of this hypothesis, studies by da Silva above, there is a projection from the superior colliculus to the PAG di-
et al. (2003) and de Menezes et al. (2009) have shown that in conscious rected exclusively to the dlPAG (Rhoades et al., 1989), and it has been
rats cardiovascular responses evoked from activation of either the DMH suggested that this input is necessary for neurons in the dlPAG to per-
or dlPAG are reduced when neuronal activity in the other region is ceive a visual threat (Klop et al., 2006). Second, several nuclei that
blocked. project to the dlPAG (area 18 in the cortex, the caudodorsal part of
What are the descending pathways that might subserve cardiovas- the medial PFC, the PMDvl in the hypothalamus, and the nucleus
cular and respiratory responses evoked from the dlPAG? As noted praepositus hypoglossi in the medulla) also project to the deep layers
above, there are no direct descending projections to the medulla from of the superior colliculus (Benzinger and Massopust, 1983; Wyss and
the dlPAG. Secondly, as already noted the dlPAG does project to the Sripanidkulchai, 1984; Canteras and Swanson, 1992; Klop et al.,
superior lateral parabrachial nucleus, but there are no descending pro- 2005). Third, the cuneiform nucleus in the midbrain receives inputs
jections from this nucleus to the lower brainstem (Fulwiler and Saper, from both the dlPAG and superior colliculus (Vianna and Brandao,
1984). On the other hand, there is a direct projection to the cuneiform 2003). Fourth, the integrity of both the superior colliculus and PAG
nucleus (Redgrave et al., 1988; Bernard et al., 1989), and there is evi- is required to integrate a defensive responsive response to a visual
dence that the cuneiform nucleus may have an important role in gener- threat (Blanchard and Blanchard, 1987). Fifth, NADPH-diaphorase
ating cardiovascular and respiratory responses arising from activation and acetylcholinesterase are found in the dlPAG and parts of the
24 R.A.L. Dampney et al. / Autonomic Neuroscience: Basic and Clinical 175 (2013) 17–25
adjacent superior colliculus, but not in other PAG subregions (Illing
and Graybiel, 1986; Carrive and Morgan, 2012). Sixth, the pattern of
the respiratory changes evoked by microinjection of the excitatory
acid DLH into the superior colliculus is very similar to that evoked
from the dlPAG, but quite different to that evoked from the lPAG
or dmPAG (Iigaya et al., 2010). Finally, we have recently found that
disinhibition of neurons in the deep and intermediate layers of the
superior colliculus evokes a co-ordinated and synchronous activation
of sympathetic and respiratory outputs, and that a similar response
can also be evoked from the dlPAG (Iigaya et al., 2012).
Thus, the dlPAG and adjacent parts of the superior colliculus may
both be part of a common defence system in the midbrain. Redgrave
and colleagues (Keay et al., 1988; Dean et al., 1989) have proposed
that neurons in the superior colliculus can generate both immediate
orienting responses as well as defensive responses (including changes
in cardiovascular and respiratory function) that are appropriate for an
external threat, just as is proposed for the dlPAG. The anatomical orga-
nization and functional properties of the mammalian superior colliculus
are very similar to its homologue in reptiles, the optic tectum (Stein and
Gaither, 1981), indicating that this system developed at an early stage
in evolution. Thus, the mechanisms within the superior colliculus and
dlPAG that generate behavioural, cardiovascular and respiratory re-
sponses to an external threat may represent the mammalian homo-
logue of a phylogenetically ancient defence system. Future studies will
be needed to establish the functional relationship between the dlPAG
and the superior colliculus in regard to the co-ordinated control of
cardiovascular and respiratory function.
Acknowledgements
The studies from our laboratory described in this article were
supported by the National Health and Medical Research Council of
Australia and the Australian Research Council.
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