# Afferent Connections to the Rostrolateral Part of the Periaqueductal Gray: A Critical Region Influencing the Motivation Drive to Hunt and Forage

Authors: Sandra Regina Mota-Ortiz; Marcia Harumi Sukikara; Luciano Freitas Felicio; Newton Sabino Canteras
Journal: Neural Plasticity
 (2009)

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## Abstract

Previous studies have shown that a particular site in the periaqueductal gray (PAG), the rostrolateral PAG, influences the motivation drive to forage or hunt. To have a deeper understanding on the putative paths involved in the decision-making process between foraging, hunting, and other behavioral responses, in the present investigation, we carried out a systematic analysis of the neural inputs to the rostrolateral PAG (rlPAG), using Fluorogold as a retrograde tracer. According to the present findings, the rlPAG appears to be importantly driven by medial prefrontal cortical areas involved in controlling attention-related and decision-making processes. Moreover, the rlPAG also receives a wealth of information from different amygdalar, hypothalamic, and brainstem sites related to feeding, drinking, or hunting behavioral responses.  Therefore, this unique combination of afferent connections puts the rlPAG in a privileged position to influence the motivation drive to choose whether hunting and foraging would be the most appropriate adaptive responses.


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## Body

## 1. Introduction

Previous studies from our laboratory, examining the neural basis of
morphine-induced inhibition of maternal behavior, brought up the suggestion of
a rather unsuspected and integrative role of the periaqueductal gray (PAG) in
influencing the selection of adaptive behavioral responses [1, 2].Examining the neural basis underlying maternal
behavior inhibition by low doses of morphine in morphine-experienced dams, we
found that morphine treatment induces a behavioral switch from maternal to
predatory behavior. Hence, morphine-challenged dams, tested in an environment
containing both pups and roaches (which served as prey), clearly preferred
hunting instead of nursing [2]. We have further shown that, under physiological
conditions, there is a natural endogenous opioid tone that may be able
to stimulate hunting in lactating dams [3].The results of behavioral, neuronal immediate early gene activation, and
lesion experiments indicate that a particular site in the PAG, at the level of
the oculomotor nucleus, located in the outer half of the lateral column, and
referred to as the rostrolateral PAG (rlPAG), should be responsible for this
switching from maternal behavior to prey hunting in morphine-treated dams [2].
First, we showed that the rlPAG upregulates Fos expression in lactating rats
acutely challenged with morphine [1], similar to what had been found for
animals performing insect hunting [4]. Next, by testing morphine-treated dams in an environment containing pups and
roaches, we were able to show that lesions of the rlPAG, but not other parts of
the PAG, impaired predatory hunting and restored the maternal response [2]. These findings support the idea that this
opioid sensitive PAG site is critical for influencing the motivation drive to
hunt and forage; and should be a nodal part of a neural circuit involved in the
decision-making process between hunting, foraging, and other behavioral responses.To start unraveling this circuit, in the
present study, we performed a comprehensive investigation on the rlPAG afferent
connections, using the Fluorogold as retrograde tracer. A number of retrograde
tract-tracing studies have investigated the afferent sources of inputs to the
PAG, but they used a much less sensitive retrograde tracer (i.e., the
retrograde transport of the horseradish peroxidase) and were based on large
injection sites encompassing different PAG functional domains [5]. The
retrograde tract-tracing method using Fluorogold as a tracer, and revealed by
immunohistochemical procedures, is one of the most sensitive retrograde tract-tracing
tools available [6], and yields relatively small injection sites, a feature
particularly suitable for investigating the afferent connections of relatively
small sites, such as the rlPAG, in the present case.Overall, the present results support the idea
that the rlPAG combines a unique set of inputs rendering this region
particularly suitable for influencing the decision-making process between
hunting, foraging, and other behavioral responses.


## 2. Materials and Methods

### 2.1. Animals

Subjects
were adult female Wistar rats (n=18) weighing 190–220 g and
approximately 90 days of age at the beginning of the experiments. Food and water
were available ad libitum to
the animals in light-controlled (06:00 AM to 06:00 PM) and
temperature-controlled (23–25°C)
rooms. Conditions of animal housing and all experimental procedures were
conducted under institutional guidelines of the Committee on Animals of the (Colégio
Brasileiro de Experimentação Animal, Brazil) and
the Committee on the Care and Use of Laboratory Animal Resources, National Research Council.


### 2.2. Retrograde Tracing Experiments

Animals were anesthetized with a
mixture of ketamine (Vetaset; Fort Dodge Laboratory, Campinas, Brazil) and
xylazine (Rompum, 1:2 v/v; 1 mL/kg body weight; Bayer; Sao Paulo, Brazil), and
unilateral iontophoretic deposits of a 2% solution of Fluorogold (Fluorochrome
Inc., Colo, USA)
were placed stereotaxically into the rlPAG (2.9 mm rostral to the interaural
line, 0.65 mm from the midline, and 4.5 mm ventral to the surface of the brain).
Deposits were made over 5 minutes through a glass micropipette (tip diameter,
25μm) by applying a +3 μA current, pulsed at 7-second intervals, with a
constant-current source (Midgard Electronics, Wood Dale, Ill, USA, model CS3).
After a survival time of 7–12 days, the
animals were deeply anesthetized with sodium pentobarbital (65 mg/kg, IP) and
perfused transcardially with a solution of 4.0% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4; the brains were removed and left overnight in a
solution of 20% sucrose in 0.1 M phosphate buffer at 4°C. The brains
were then frozen, and five series of 30-μm-thick sections were cut on a sliding
microtome in the transverse (frontal) plane and collected from the caudal
medulla through the rostral tip of the prefrontal cortex. One complete series
was processed for immunohistochemistry with an antiserum directed against
Fluorogold (Chemicon International, Calif, USA) at a dilution of 1:5000. The antigen-antibody
complex was localized with a variation of the avidin-biotin complex system (ABC)
[7], with a commercially available kit (ABC Elite Kit, Vector laboratories, Calif, USA).
The sections were mounted on gelatin-coated slides and then treated with osmium
tetroxide to enhance visibility of the reaction product. Slides were then
dehydrated and cover slipped with DPX. An adjacent series was always stained
with thionin to serve as reference for cytoarchitecture.Sections were examined under a microscope with bright- and dark-field illumination.
Fluorogold deposits
in the injection sites, and the distribution of retrogradely labeled neurons,
were plotted with the aid of a camera lucida onto maps prepared from adjacent
thionin-stained sections. The distribution of retrograde labeling was
transferred onto a reference atlas of the rat brain [8]. The figures were
prepared using Adobe PhotoShop (v.5.5; Adobe Systems, San Jose, Calif, USA) for
photomicrographs and Adobe Illustrator (v.10, Adobe Systems) for drawings.


## 2.1. Animals

Subjects
were adult female Wistar rats (n=18) weighing 190–220 g and
approximately 90 days of age at the beginning of the experiments. Food and water
were available ad libitum to
the animals in light-controlled (06:00 AM to 06:00 PM) and
temperature-controlled (23–25°C)
rooms. Conditions of animal housing and all experimental procedures were
conducted under institutional guidelines of the Committee on Animals of the (Colégio
Brasileiro de Experimentação Animal, Brazil) and
the Committee on the Care and Use of Laboratory Animal Resources, National Research Council.


## 2.2. Retrograde Tracing Experiments

Animals were anesthetized with a
mixture of ketamine (Vetaset; Fort Dodge Laboratory, Campinas, Brazil) and
xylazine (Rompum, 1:2 v/v; 1 mL/kg body weight; Bayer; Sao Paulo, Brazil), and
unilateral iontophoretic deposits of a 2% solution of Fluorogold (Fluorochrome
Inc., Colo, USA)
were placed stereotaxically into the rlPAG (2.9 mm rostral to the interaural
line, 0.65 mm from the midline, and 4.5 mm ventral to the surface of the brain).
Deposits were made over 5 minutes through a glass micropipette (tip diameter,
25μm) by applying a +3 μA current, pulsed at 7-second intervals, with a
constant-current source (Midgard Electronics, Wood Dale, Ill, USA, model CS3).
After a survival time of 7–12 days, the
animals were deeply anesthetized with sodium pentobarbital (65 mg/kg, IP) and
perfused transcardially with a solution of 4.0% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4; the brains were removed and left overnight in a
solution of 20% sucrose in 0.1 M phosphate buffer at 4°C. The brains
were then frozen, and five series of 30-μm-thick sections were cut on a sliding
microtome in the transverse (frontal) plane and collected from the caudal
medulla through the rostral tip of the prefrontal cortex. One complete series
was processed for immunohistochemistry with an antiserum directed against
Fluorogold (Chemicon International, Calif, USA) at a dilution of 1:5000. The antigen-antibody
complex was localized with a variation of the avidin-biotin complex system (ABC)
[7], with a commercially available kit (ABC Elite Kit, Vector laboratories, Calif, USA).
The sections were mounted on gelatin-coated slides and then treated with osmium
tetroxide to enhance visibility of the reaction product. Slides were then
dehydrated and cover slipped with DPX. An adjacent series was always stained
with thionin to serve as reference for cytoarchitecture.Sections were examined under a microscope with bright- and dark-field illumination.
Fluorogold deposits
in the injection sites, and the distribution of retrogradely labeled neurons,
were plotted with the aid of a camera lucida onto maps prepared from adjacent
thionin-stained sections. The distribution of retrograde labeling was
transferred onto a reference atlas of the rat brain [8]. The figures were
prepared using Adobe PhotoShop (v.5.5; Adobe Systems, San Jose, Calif, USA) for
photomicrographs and Adobe Illustrator (v.10, Adobe Systems) for drawings.


## 3. Results

The distribution of neurons projecting to the rlPAG region was examined by using
Fluorogold. In five experiments, the deposit of the tracer appeared to be
confined almost entirely to the rlPAG (located in the outer half of the lateral
column at the levels of the oculomotor nucleus). The appearance of a
representative Fluorogold injection site in the rlPAG is illustrated in Figure1, and the distribution of retrogradely labeled neurons from this experiment is
illustrated schematically in Figure 2. The results of this experiment are
described in detail, because the injection site was virtually confined to the
rlPAG. Furthermore, in this experiment, the pattern of retrograde labeling was
representative of that one seen in each of the other experiments with deposits
centered in the rlPAG. The following is a summary of regions that appear to send
fibers to the rlPAG.Figure 1
Brightfield photomicrograph
illustrating the appearance of Fluorogold (FG) injection site in experiment PAGlFG15.
Scale bar = 200μmInputs to the rlPAG. The distribution
of retrogradely labeled neurons (black dots) in experiment PAGlFG15 plotted
onto a series of standard drawings of the rat brain arranged from the rostral (a)
to caudal (o) levels. The dark gray area indicates the FG injection site in
this experiment. For abbreviations, see Supplementary Material available
online at doi:10.1155/2009/612698.


### 3.1. Telencephalon

Retrogradely labeled neurons were found in the
isocortex, amygdala and in the septal region. No retrogradely labeled cells
were found in the hippoccampal formation.In the isocortex, a large number of retrogradely labeled neurons was observed in
the prelimbic, infralimbic, anterior cingulate, and secondary motor areas
(Figures2(a), 2(b), 2(c)
and 3(a), 3(b)), in addition to substantial labeling
in the gustatory and visceral areas (Figures 2(b), 2(g), 2(h), and 2(i)). A few
retrogradely labeled neurons were found in the posterior part of agranular insular
area (Figure 3(c)) and primary motor, perirhinal, and ectorhinal areas. All of
the cortical labeled cells were pyramidal neurons of the layer V.Dark field photomicrographs showing (a) the distribution of retrogradely
labeled cells within the ipsilateral secondary motor area and the dorsal part
of the anterior cingulated area; (b) the prelimbic and infralimbic areas; (c)
and the visceral area. Scale bars = 200μm.
(a)(b)(c)In the lateral septal nucleus, a relatively sparse number of marked cells were observed
in the rostral part of the nucleus, distributed mainly through the dorsal
region of the ventrolateral zone (Figures2(b), 2(c)). In the septal region, we
have also observed a substantial number of retrogradely marked neurons in the
posterior division of the bed nuclei of the stria terminalis, particularly in
the interfascicular nucleus, and also, to a lesser degree, in the transverse
nucleus (Figures 2(e), 2(f)). In the amygdala, a large number of retrogradely
labeled cells were found to be restricted to the medial part of the central amygdalar nucleus (Figures
2(g), 2(h), 2(i),
and 4(a)).Dark field photomicrographs showing (a) the distribution of retrogradely
labeled cells within the ipsilateral medial part of the central nucleus of the
amygdala; (b) the median preoptic nucleus; (c) the
anterior part of the anterior hypothalamic nucleus and the retinoceptive region
of the lateral hypothalamic area; (d)
and the anterior part of the ventromedial hypothalamic nucleus and the
retrochiasmatic area. Scale bars = 200μm.
(a)(b)(c)(d)


### 3.2. Diencephalon

Thalamus
At the thalamus, a substantial retrograde labeling was found in the
ventral part of the zona incerta (Figures2(h), 2(i), 2(j)). No marked cells
were observed in the dorsal thalamus.Hypothalamus
In thepreoptic region, a substantial retrograde labeling was found in the median preoptic
nucleus (Figures2(c), 2(d), and 4(b)), in addition to a
sparse number of marked cells in the anteroventral preoptic nucleus (Figures 2(c)
and 2(d)).
At the anterior hypothalamic levels, the anterior part of the anterior
hypothalamic nucleus presented a dense cluster of retrogradely labeled cells,
which were distributed in the region that seems to overlap, at least partially,
with a territory known to contain a large number of neurons expressing
enkephalin [9] (Figures 2(e), 2(f), 2(g), and 4(c)).  Moreover, at these levels, a substantial
retrograde labeling was also found in the lateral hypothalamic area immediately
dorsal to the optic tract and the supraoptic nucleus, which seems to correspond
to a region densely targeted by the lateral component of retinohypothalamic
tract [10] (Figures 2(f), 2(g), and 4(c)).
At tuberal levels, a dense number of
retrogradely labeled cells was found in the anterior part of the ventromedial
nucleus, in addition to a moderate number of marked cells in the
retrochiasmatic area, the ventrolateral and central parts of the ventromedial
nucleus, the tuberal nucleus, and rostral parts of the posterior hypothalamic
nucleus (Figures2(g), 2(h), 2(i), 2(j), and 4(d)). Furthermore, at these levels, we have found a
large number of retrogradely labeled cells in the lateral hypothalamic area,
distributed in the dorsal, suprafornical, justadorsomedial, and
justaventromedial areas (Figures 2(g), 2(h), 2(i), and 2(j)).
At the mammilary levels, a large number of labeled neurons were found in the dorsal premammilary nucleus, mostly
distributed in the dorsal part of the nucleus (Figure2(k)). Finally, at these
levels, we have found moderate retrograde labeling in the parasubthalamic
nucleus, in addition to sparse labeling in the subfornical region of the
lateral hypothalamic area (Figures 2(j) and 2(k)).Brainstem
At mesodiencephalic levels, a large number of
marked cells were found in the precommissural nucleus (Figure2(l)). In the midbrain, at the injection site level,
substantial retrograde labeling was found in the lateral part of the
intermediate layer of the superior colliculus (Figure 2(m)). Additionally, at
this level, a moderate number of retrogradely labeled neurons were also found
in the PAG, which appeared to be distributed within the dorsomedial part
(Figure 2(m)). Proceeding caudally, at the intermediate
rostrocaudal levels of the dorsal raphe nucleus, a moderate number of marked
cells was found in the ventrolateral part of the PAG (Figure 2(n)).
At rostral pontine levels, a few
labeled cells were found in the laterodorsal tegmental nucleus, as well as in
the central lateral, dorsal lateral, and ventral lateral parts of the
parabrachial nucleus (Figure2(o)).
It should be noted that, in the
experiments with Fluorogold deposits centered in the rlPAG, retrograde labeling
was mostly ipsilateral. However, some of the main sources of projections to
this area, including the prefrontal cortex, retrochiasmatic area, anterior hypothalamic
nucleus, ventromedial hypothalamic nucleus, lateral hypothalamic area, zona
incerta, precommissural nucleus, and the PAG, also displayed conspicuous
retrograde labeling contralateral to the injection site.


## 3.1. Telencephalon

Retrogradely labeled neurons were found in the
isocortex, amygdala and in the septal region. No retrogradely labeled cells
were found in the hippoccampal formation.In the isocortex, a large number of retrogradely labeled neurons was observed in
the prelimbic, infralimbic, anterior cingulate, and secondary motor areas
(Figures2(a), 2(b), 2(c)
and 3(a), 3(b)), in addition to substantial labeling
in the gustatory and visceral areas (Figures 2(b), 2(g), 2(h), and 2(i)). A few
retrogradely labeled neurons were found in the posterior part of agranular insular
area (Figure 3(c)) and primary motor, perirhinal, and ectorhinal areas. All of
the cortical labeled cells were pyramidal neurons of the layer V.Dark field photomicrographs showing (a) the distribution of retrogradely
labeled cells within the ipsilateral secondary motor area and the dorsal part
of the anterior cingulated area; (b) the prelimbic and infralimbic areas; (c)
and the visceral area. Scale bars = 200μm.
(a)(b)(c)In the lateral septal nucleus, a relatively sparse number of marked cells were observed
in the rostral part of the nucleus, distributed mainly through the dorsal
region of the ventrolateral zone (Figures2(b), 2(c)). In the septal region, we
have also observed a substantial number of retrogradely marked neurons in the
posterior division of the bed nuclei of the stria terminalis, particularly in
the interfascicular nucleus, and also, to a lesser degree, in the transverse
nucleus (Figures 2(e), 2(f)). In the amygdala, a large number of retrogradely
labeled cells were found to be restricted to the medial part of the central amygdalar nucleus (Figures
2(g), 2(h), 2(i),
and 4(a)).Dark field photomicrographs showing (a) the distribution of retrogradely
labeled cells within the ipsilateral medial part of the central nucleus of the
amygdala; (b) the median preoptic nucleus; (c) the
anterior part of the anterior hypothalamic nucleus and the retinoceptive region
of the lateral hypothalamic area; (d)
and the anterior part of the ventromedial hypothalamic nucleus and the
retrochiasmatic area. Scale bars = 200μm.
(a)(b)(c)(d)


## 3.2. Diencephalon

Thalamus
At the thalamus, a substantial retrograde labeling was found in the
ventral part of the zona incerta (Figures2(h), 2(i), 2(j)). No marked cells
were observed in the dorsal thalamus.Hypothalamus
In thepreoptic region, a substantial retrograde labeling was found in the median preoptic
nucleus (Figures2(c), 2(d), and 4(b)), in addition to a
sparse number of marked cells in the anteroventral preoptic nucleus (Figures 2(c)
and 2(d)).
At the anterior hypothalamic levels, the anterior part of the anterior
hypothalamic nucleus presented a dense cluster of retrogradely labeled cells,
which were distributed in the region that seems to overlap, at least partially,
with a territory known to contain a large number of neurons expressing
enkephalin [9] (Figures 2(e), 2(f), 2(g), and 4(c)).  Moreover, at these levels, a substantial
retrograde labeling was also found in the lateral hypothalamic area immediately
dorsal to the optic tract and the supraoptic nucleus, which seems to correspond
to a region densely targeted by the lateral component of retinohypothalamic
tract [10] (Figures 2(f), 2(g), and 4(c)).
At tuberal levels, a dense number of
retrogradely labeled cells was found in the anterior part of the ventromedial
nucleus, in addition to a moderate number of marked cells in the
retrochiasmatic area, the ventrolateral and central parts of the ventromedial
nucleus, the tuberal nucleus, and rostral parts of the posterior hypothalamic
nucleus (Figures2(g), 2(h), 2(i), 2(j), and 4(d)). Furthermore, at these levels, we have found a
large number of retrogradely labeled cells in the lateral hypothalamic area,
distributed in the dorsal, suprafornical, justadorsomedial, and
justaventromedial areas (Figures 2(g), 2(h), 2(i), and 2(j)).
At the mammilary levels, a large number of labeled neurons were found in the dorsal premammilary nucleus, mostly
distributed in the dorsal part of the nucleus (Figure2(k)). Finally, at these
levels, we have found moderate retrograde labeling in the parasubthalamic
nucleus, in addition to sparse labeling in the subfornical region of the
lateral hypothalamic area (Figures 2(j) and 2(k)).Brainstem
At mesodiencephalic levels, a large number of
marked cells were found in the precommissural nucleus (Figure2(l)). In the midbrain, at the injection site level,
substantial retrograde labeling was found in the lateral part of the
intermediate layer of the superior colliculus (Figure 2(m)). Additionally, at
this level, a moderate number of retrogradely labeled neurons were also found
in the PAG, which appeared to be distributed within the dorsomedial part
(Figure 2(m)). Proceeding caudally, at the intermediate
rostrocaudal levels of the dorsal raphe nucleus, a moderate number of marked
cells was found in the ventrolateral part of the PAG (Figure 2(n)).
At rostral pontine levels, a few
labeled cells were found in the laterodorsal tegmental nucleus, as well as in
the central lateral, dorsal lateral, and ventral lateral parts of the
parabrachial nucleus (Figure2(o)).
It should be noted that, in the
experiments with Fluorogold deposits centered in the rlPAG, retrograde labeling
was mostly ipsilateral. However, some of the main sources of projections to
this area, including the prefrontal cortex, retrochiasmatic area, anterior hypothalamic
nucleus, ventromedial hypothalamic nucleus, lateral hypothalamic area, zona
incerta, precommissural nucleus, and the PAG, also displayed conspicuous
retrograde labeling contralateral to the injection site.


## 4. Discussion

The results of the present retrograde axonal
tract-tracing study suggest that the rlPAG receive inputs from several widely
distributed areas in the forebrain and, to a lesser extent, from the brainstem,
as well. Prefrontal cortical areas represent the major telencephalic source of
inputs to the rlPAG. In addition, clear telencephalic inputs appear to arise
from the gustatory, visceral, and perirhinal cortical areas, as well as from
the medial part of the central amygdalar nucleus and the interfascicular
nucleus of the bed nuclei of the stria terminalis. In the diencephalon, massive
inputs to the rlPAG arise from several hypothalamic sites, including the median
preoptic nucleus, anterior hypothalamic nucleus, retrochiasmatic area, anterior,
and ventrolateral parts of the ventromedial nucleus, dorsal premammillary
nucleus (PMd), and several districts of the lateral hypothalamic area. In
contrast, the rlPAG appears to receive inputs from considerably fewer brainstem
sites, where significant retrograde labeling was found in other parts of the
PAG (i.e., the dorsomedial and ventrolateral parts) and in the intermediate
layers of the lateral part of the superior colliculus.In agreement with previous anterograde
tract-tracing studies, medial prefrontal cortical areas, including the infralimbic,
prelimbic, anterior cingulated, and secondary motor areas, appear to represent
one of the most important afferent sources of projections to the rlPAG [11–20]. In line with previous anterograde findings, we
have also found that the rlPAG receives projections from visceral, gustatory,
and perirhinal cortical areas [12–14, 17]. In the septal region, a
substantial number of retrograde labeled cells were found in the
interfascicular nucleus of the BST, a finding likewise supported by previous
PHAL studies [21]. The
present results also revealed that the medial part of the central nucleus of
the amygdala is an important source of telencephalic inputs to the rlPAG. This
finding has also been confirmed by means of PHAL anterograde tract-tracing
method, which showed that the medial part of the central nucleus of the
amygdala provides a substantial terminal field to the lateral PAG [22].The present findings also revealed that the rlPAG is
targeted by several hypothalamic districts. At preopotic levels, the rlPAG
seems to receive substantial inputs from the median preoptic nucleus, in
addition to somewhat sparse inputs from the anteroventral preoptic nucleus.
Both of these projections have been confirmed by previous PHAL studies [23]. At
anterior hypothalamic levels, a prominent group of retrogradely labeled cells
was found in the anterior part of the anterior hypothalamic nucleus, in a
region containing a characteristic cluster of enkephalinergic cells [9]. We
were able to confirm this projection by means of PHAL deposits placed in the
anterior part of the anterior hypothalamic nucleus, which yielded a distinct
terminal field in the rlPAG [S. R. Mota-Ortiz and N. S. Canteras, personal
observation]. In addition, at these levels, we have found that the rlPAG
receives inputs from the retrochiasmatic area and a lateral hypothalamic region
immediately adjacent to the optic tract and the supraoptic nucleus, which
receives direct projections from the lateral part of the retinohypothalamic
tract [10] and corresponds to the so-called retinoceptive region of the lateral
hypothalamic area. Both the projections from the retrochiasmatic area and from
the retinoceptive region of the lateral hypothalamic area to the rlPAG have
been previously confirmed through PHAL experiments [24, N. S. Canteras, personal
observation]. At tuberal levels, in agreement with the results of our
retrograde transport experiments, previous evidence based on PHAL anterograde
tract-tracing indicates that the rlPAG receives dense projections from the
anterior part of the ventromedial nucleus and the posterior hypothalamic
nucleus, in addition to somewhat weaker inputs from the ventrolateral part of
the ventromedial nucleus and the adjacent tuberal nucleus [25, 26]. In addition,
at these levels, we found that the rlPAG receives substantial inputs from the
dorsal, suprafornical, justadorsomedial, and justaventromedial areas of the
lateral hypothalamus, as well as immediately adjacent parts of the zona
incerta. These projections have been confirmed in PHAL studies [27, M. Goto,
personal observation]. Notably, this region of the lateral hypothalamus
overlaps with the region of cells expressing melanin-concentrating hormone (MCH) and hypocretin/orexin [28].
Moreover, in agreement with previous PHAL studies [29], we have also found, in
the lateral hypothalamic area, that the parasubthalamic nucleus represents
another source of inputs to the rlPAG. Finally, at mammilary levels, we found a
distinct projection mostly from the dorsal part of the PMd. The PMd represents
one of the main hypothalamic sources of inputs to the PAG [5], and previous
PHAL studies have shown that the dorsal and ventral parts of the nucleus provide
a differential pattern of projection to the PAG, and confirmed that the dorsal
part of the PMd provides a strikingly dense projection to the rlPAG [30].The present results also revealed
that a small number of brainstem sites appear to innervate the rlPAG. Evident
retrograde labeling was found in the precommissural nucleus, the dorsomedial
and ventrolateral parts of the PAG, and the lateral part of the intermediate
layer of the superior colliculus. All of these projections have been confirmed
through PHAL anterograde tract-tracing method [31, S. R. Mota-Ortiz and N. S.
Canteras, personal observation] In addition, we have also found that the rlPAG
receives relatively sparse projections from the laterodorsal tegmental nucleus
and the lateral part of the parabrachial nucleus. Unfortunately, to our
knowledge, these projections remain yet to be demonstrated with anterograde
tracer studies.


### 4.1. Differential Inputs to Other PAG Parts: Comparison with the Afferent Connections to the Dorsolateral PAG (dlPAG)

As mentioned above, previous retrograde
tract-tracing studies have investigated the afferent sources of inputs to the
PAG, but they were based on large injection sites, providing just an overall
picture of the afferent inputs to the PAG without differentiating among the
particular PAG domains [5]. By determining the specific set of afferent inputs
to the rlPAG, we have provided important information to distinguish this PAG
site from adjacent different functional domains, such as the immediately
adjacent dorsolateral PAG (dlPAG). In contrast to the rlPAG, the dlPAG upregulates
Fos expression in response to both actual and contextual predatory threats [32–34], and seems to be responsible for organizing the expression of
unconditioned and conditioned antipredatory responses [33]. As for the rlPAG,
the dlPAG also receives substantial inputs from the anterior cingulate,
prelimbic, and infralimbic areas [11, 12, 19, 20], but in sharp contrast to
what has been found for the rlPAG, the visceral area appears to project only
sparsely to the dlPAG [13]. Additionally, in contrast to the rlPAG, the dlPAG
does not seem to receive substantial inputs from the amygdala, and seems to be
densely innervated by medial hypothalamic sites involved in processing
predatory cues, including the posterior part of the anterior hypothalamic
nucleus, the dorsomedial part of the ventromedial nucleus and the dorsal
premammillary nucleus [25, 35, 36]. Notably, the projection from the dorsal
premammillary nucleus to the dlPAG arises chiefly from the ventrolateral part
of the nucleus, which is the hypothalamic site most responsive to the predator
or its cues [30]. In the zona incerta, differently from what we have just found
for the rlPAG, the dlPAG seems to receive dense projections from the rostral
zona incerta, also referred to as the incertohypothalamic area [37]. The superior colliculus also has a
differential pattern of projection to the PAG, where the lateral part of the
intermediate layer projects to the rlPAG, as presently shown, and the medial
part of the intermediate and deep layers target the dorsal PAG [38]. Notably, the medial part of the intermediate and deep
layers of the superior colliculus respond to visual-threatening stimuli, such as
suddenly expanding shadows in the upper visual field (which look like an
approaching predator), and, via a projection to the dlPAG, may exert a marked
influence on the control of defensive responses [38].The evidence just discussed supports the idea that different functional PAG domains
are likely to have particular sets of afferent inputs, and next, we will
provide a discussion on how the diverse inputs to the rlPAG may be related to
its function on hunting and foraging behavior.


### 4.2. Functional Considerations

As commented in the Introduction, the rlPAG is an opioid sensitive site, which is
critical for influencing the motivation drive to hunt and forage, and is likely
to be part of a neural circuit involved in the decision-making process between hunting, foraging, and
other behavioral responses. The present findings help to reveal this complex
network, and reinforce the rlPAG's nodal role in integrating a wealth of
different kinds of information likely to influence foraging or hunting
activity.One of the main findings of the present investigation is that the rlPAG appears to be
particularly driven by inputs from medial prefrontal cortical areas. In the
present context, it is noteworthy that, in the prefrontal cortex, the anterior cingulate, the prelimbic, and the
infralimbic cortices have been associated with diverse emotional, cognitive, and
mnemonic functions that underlie attentional and decision-making processes [39, 40].  In addition to the prefrontal
cortex, the rlPAG also seems to be influenced by the septohippocampal system
via a projection from the enkephalinergic
region of the anterior hypothalamic nucleus [36]. Of particular relevance in
the present account, the septohippocampal system has been shown to have a role
in prioritizing the temporal order of motivated responses, which, in the
present case, seems to utilize an enkephalinergic
pathway. Here, it is noteworthy to recall that the opioidergic influence
on the rlPAG has been shown to control the selection of adaptive behavioral
responses, switching from maternal to hunting behavior [1].As commented in the
Introduction, the rlPAG upregulated Fos expression in animals performing
insect hunting, and in the present study, we have shown that the rlPAG
integrates inputs from a number of neural sites related to the circuitry
underlying predatory hunting. Thus, we have presently found that the medial
part of the central amygdalar nucleus provides a significant projection to the rlPAG.
The medial part of the central amygdalar nucleus is part of a distinct
amygdalar circuit comprising; the anterior
part of the cortical nucleus, the anterior part of the basomedial nucleus and the
posterior part of the basolateral nucleus, which has been shown to be mobilized
during insect predation and seems to be particularly involved in processing
visceral, gustatory and olfactory information [41–44]. The central
nucleus—the main output
way station of this amygdalar circuit—has been shown to
be involved in controlling feeding behavior [45, 46]. In particular, the
nucleus appears to integrate food hedonic values [47, 48] and to influence
searching and consumption of palatable food through a pathway involving
opioidergic neurotransmission [48, 49]. In the context of feeding behavior, it
is also particularly relevant to point out that, according to the present
findings, the rlPAG is also in a position to integrate visceral and gustatory
information from the visceral and gustatory cortical areas, and to a lesser
degree, from the parabrachial area [50].The rlPAG also seems to be innervated by lateral hypothalamic regions
activated during predatory hunting, namely, the parasubthalamic nucleus and the
region containing cells expressing MCH and
hypocretin/orexin [44]. The parasubthalamic nucleus is also targeted by the
medial part of the central amygdalar nucleus, and provides
inputs to hindbrain control regions involved in modulating digestive and
metabolic responses occurring in both cephalic and consummatory phases of
feeding behavior [29]. The lateral hypothalamic region containing cells expressing MCH and hypocretin/orexin appears
to be involved in controlling arousal and exploratory activity related to
feeding behavior [27, 51, 52]. In fact, this lateral hypothalamic region may
also represent an important interface for the hypothalamic periventricular
sites involved in the control of homeostatic feeding [53]. Moreover, according
to the present findings, the rlPAG also receives direct inputs from the
retrochiasmatic region, which has also been included in the hypothalamic
circuit controlling feeding [54].We have presently shown that the rlPAG is also targeted by the lateral
part of the intermediate layer of the superior colliculus, which we have also
previously shown to be mobilized during insect hunting [55]. In fact, cells in
this collicular region seem to respond to moving objects in the temporal visual
field, and we have found that animals bearing lesions in this collicular region,
besides failing to orient themselves toward the moving prey, were also clearly
less motivated to pursue the roaches [I. C. Furigo and S. R. Mota-Ortiz, personal
observation]  perhaps reflecting the functional role of the
superior colliculus—rlPAG pathway.The rlPAG is also significantly targeted by hypothalamic districts that
do not seem to be directly involved in controlling feeding or hunting behavior,
namely, the median preoptic nucleus, the retinoceptive region of the lateral
hypothalamic, area and the dorsal premammillary nucleus. The median preoptic
nucleus is classically known to respond to plasma osmolarity and influence
drinking behavior [23], and this pathway to the rlPAG may be thought of as
having some role in the search of water supplies. Conversely, the projection
from the retinoceptive region of the lateral hypothalamic area may bring the
information regarding the environmental luminescence, which may be thought of
as having a direct impact on foraging activity [N. S. Canteras, personal
observation]. Finally, the dorsal part of the dorsal premammillary nucleus
provides an important projection to the rlPAG. Unfortunately, to the best of
our knowledge, the potential roles of this path remain yet to be determined.Here, it is important to
consider that functional studies using electrical or chemical stimulation have
suggested a role for the lateral PAG in defensive responses. In the rat,
chemical stimulation with kainic acid (KA) in the region of the rlPAG produced
strong backward defense [56], a kind of response that would be expected when the
animal is exposed to a predator. However, this finding is hard to conciliate
with the fact that rlPAG does not seem to be activated in response to predator
exposure [32], as would be expected. In reality, the effects of KA injection in
this region should be taken very cautiously since the immediately adjacent
dlPAG presents a much heavier binding to KA [57] when compared to the rlPAG.
Actually, the dlPAG appears as the PAG site presenting the largest activation
to predator exposure [32], and, as previously discussed, its hodological pattern
is fully compatible with its role in antipredatory defense.In addition, the concept of a distinct functional column for the entire lateral
PAG should also be revised. In fact, the role here assigned to the rlPAG does
not seem to apply to the caudal half of the lateral PAG, since lesions
encroaching upon this latter PAG region do not seem to affect the motivational
drive to hunt [2, M. H. Sukikara and S. R. Mota-Ortiz, personal observation].
Conversely, there is a prominent Fos upregulation in the caudal, but not in the
rostral, lateral PAG in response to predator exposure [32, 33].Overall, the present findings
support the idea that the rlPAG has a central role in a complex network
controlling the decision-making process
between hunting, foraging, and other behavioral responses. On one hand, the
rlPAG appears to be importantly driven by medial prefrontal cortical areas
involved in controlling attentional and decision-making processes. On
the other hand, the rlPAG receives a wealth of information from different
neural sites related to feeding, drinking, or hunting behavioral
responses.  Therefore, this unique
combination of afferent connections puts the rlPAG in a privileged position to
influence the motivation drive to choose whether hunting and foraging are the most appropriate adaptive
responses.


## 4.1. Differential Inputs to Other PAG Parts: Comparison with the Afferent Connections to the Dorsolateral PAG (dlPAG)

As mentioned above, previous retrograde
tract-tracing studies have investigated the afferent sources of inputs to the
PAG, but they were based on large injection sites, providing just an overall
picture of the afferent inputs to the PAG without differentiating among the
particular PAG domains [5]. By determining the specific set of afferent inputs
to the rlPAG, we have provided important information to distinguish this PAG
site from adjacent different functional domains, such as the immediately
adjacent dorsolateral PAG (dlPAG). In contrast to the rlPAG, the dlPAG upregulates
Fos expression in response to both actual and contextual predatory threats [32–34], and seems to be responsible for organizing the expression of
unconditioned and conditioned antipredatory responses [33]. As for the rlPAG,
the dlPAG also receives substantial inputs from the anterior cingulate,
prelimbic, and infralimbic areas [11, 12, 19, 20], but in sharp contrast to
what has been found for the rlPAG, the visceral area appears to project only
sparsely to the dlPAG [13]. Additionally, in contrast to the rlPAG, the dlPAG
does not seem to receive substantial inputs from the amygdala, and seems to be
densely innervated by medial hypothalamic sites involved in processing
predatory cues, including the posterior part of the anterior hypothalamic
nucleus, the dorsomedial part of the ventromedial nucleus and the dorsal
premammillary nucleus [25, 35, 36]. Notably, the projection from the dorsal
premammillary nucleus to the dlPAG arises chiefly from the ventrolateral part
of the nucleus, which is the hypothalamic site most responsive to the predator
or its cues [30]. In the zona incerta, differently from what we have just found
for the rlPAG, the dlPAG seems to receive dense projections from the rostral
zona incerta, also referred to as the incertohypothalamic area [37]. The superior colliculus also has a
differential pattern of projection to the PAG, where the lateral part of the
intermediate layer projects to the rlPAG, as presently shown, and the medial
part of the intermediate and deep layers target the dorsal PAG [38]. Notably, the medial part of the intermediate and deep
layers of the superior colliculus respond to visual-threatening stimuli, such as
suddenly expanding shadows in the upper visual field (which look like an
approaching predator), and, via a projection to the dlPAG, may exert a marked
influence on the control of defensive responses [38].The evidence just discussed supports the idea that different functional PAG domains
are likely to have particular sets of afferent inputs, and next, we will
provide a discussion on how the diverse inputs to the rlPAG may be related to
its function on hunting and foraging behavior.


## 4.2. Functional Considerations

As commented in the Introduction, the rlPAG is an opioid sensitive site, which is
critical for influencing the motivation drive to hunt and forage, and is likely
to be part of a neural circuit involved in the decision-making process between hunting, foraging, and
other behavioral responses. The present findings help to reveal this complex
network, and reinforce the rlPAG's nodal role in integrating a wealth of
different kinds of information likely to influence foraging or hunting
activity.One of the main findings of the present investigation is that the rlPAG appears to be
particularly driven by inputs from medial prefrontal cortical areas. In the
present context, it is noteworthy that, in the prefrontal cortex, the anterior cingulate, the prelimbic, and the
infralimbic cortices have been associated with diverse emotional, cognitive, and
mnemonic functions that underlie attentional and decision-making processes [39, 40].  In addition to the prefrontal
cortex, the rlPAG also seems to be influenced by the septohippocampal system
via a projection from the enkephalinergic
region of the anterior hypothalamic nucleus [36]. Of particular relevance in
the present account, the septohippocampal system has been shown to have a role
in prioritizing the temporal order of motivated responses, which, in the
present case, seems to utilize an enkephalinergic
pathway. Here, it is noteworthy to recall that the opioidergic influence
on the rlPAG has been shown to control the selection of adaptive behavioral
responses, switching from maternal to hunting behavior [1].As commented in the
Introduction, the rlPAG upregulated Fos expression in animals performing
insect hunting, and in the present study, we have shown that the rlPAG
integrates inputs from a number of neural sites related to the circuitry
underlying predatory hunting. Thus, we have presently found that the medial
part of the central amygdalar nucleus provides a significant projection to the rlPAG.
The medial part of the central amygdalar nucleus is part of a distinct
amygdalar circuit comprising; the anterior
part of the cortical nucleus, the anterior part of the basomedial nucleus and the
posterior part of the basolateral nucleus, which has been shown to be mobilized
during insect predation and seems to be particularly involved in processing
visceral, gustatory and olfactory information [41–44]. The central
nucleus—the main output
way station of this amygdalar circuit—has been shown to
be involved in controlling feeding behavior [45, 46]. In particular, the
nucleus appears to integrate food hedonic values [47, 48] and to influence
searching and consumption of palatable food through a pathway involving
opioidergic neurotransmission [48, 49]. In the context of feeding behavior, it
is also particularly relevant to point out that, according to the present
findings, the rlPAG is also in a position to integrate visceral and gustatory
information from the visceral and gustatory cortical areas, and to a lesser
degree, from the parabrachial area [50].The rlPAG also seems to be innervated by lateral hypothalamic regions
activated during predatory hunting, namely, the parasubthalamic nucleus and the
region containing cells expressing MCH and
hypocretin/orexin [44]. The parasubthalamic nucleus is also targeted by the
medial part of the central amygdalar nucleus, and provides
inputs to hindbrain control regions involved in modulating digestive and
metabolic responses occurring in both cephalic and consummatory phases of
feeding behavior [29]. The lateral hypothalamic region containing cells expressing MCH and hypocretin/orexin appears
to be involved in controlling arousal and exploratory activity related to
feeding behavior [27, 51, 52]. In fact, this lateral hypothalamic region may
also represent an important interface for the hypothalamic periventricular
sites involved in the control of homeostatic feeding [53]. Moreover, according
to the present findings, the rlPAG also receives direct inputs from the
retrochiasmatic region, which has also been included in the hypothalamic
circuit controlling feeding [54].We have presently shown that the rlPAG is also targeted by the lateral
part of the intermediate layer of the superior colliculus, which we have also
previously shown to be mobilized during insect hunting [55]. In fact, cells in
this collicular region seem to respond to moving objects in the temporal visual
field, and we have found that animals bearing lesions in this collicular region,
besides failing to orient themselves toward the moving prey, were also clearly
less motivated to pursue the roaches [I. C. Furigo and S. R. Mota-Ortiz, personal
observation]  perhaps reflecting the functional role of the
superior colliculus—rlPAG pathway.The rlPAG is also significantly targeted by hypothalamic districts that
do not seem to be directly involved in controlling feeding or hunting behavior,
namely, the median preoptic nucleus, the retinoceptive region of the lateral
hypothalamic, area and the dorsal premammillary nucleus. The median preoptic
nucleus is classically known to respond to plasma osmolarity and influence
drinking behavior [23], and this pathway to the rlPAG may be thought of as
having some role in the search of water supplies. Conversely, the projection
from the retinoceptive region of the lateral hypothalamic area may bring the
information regarding the environmental luminescence, which may be thought of
as having a direct impact on foraging activity [N. S. Canteras, personal
observation]. Finally, the dorsal part of the dorsal premammillary nucleus
provides an important projection to the rlPAG. Unfortunately, to the best of
our knowledge, the potential roles of this path remain yet to be determined.Here, it is important to
consider that functional studies using electrical or chemical stimulation have
suggested a role for the lateral PAG in defensive responses. In the rat,
chemical stimulation with kainic acid (KA) in the region of the rlPAG produced
strong backward defense [56], a kind of response that would be expected when the
animal is exposed to a predator. However, this finding is hard to conciliate
with the fact that rlPAG does not seem to be activated in response to predator
exposure [32], as would be expected. In reality, the effects of KA injection in
this region should be taken very cautiously since the immediately adjacent
dlPAG presents a much heavier binding to KA [57] when compared to the rlPAG.
Actually, the dlPAG appears as the PAG site presenting the largest activation
to predator exposure [32], and, as previously discussed, its hodological pattern
is fully compatible with its role in antipredatory defense.In addition, the concept of a distinct functional column for the entire lateral
PAG should also be revised. In fact, the role here assigned to the rlPAG does
not seem to apply to the caudal half of the lateral PAG, since lesions
encroaching upon this latter PAG region do not seem to affect the motivational
drive to hunt [2, M. H. Sukikara and S. R. Mota-Ortiz, personal observation].
Conversely, there is a prominent Fos upregulation in the caudal, but not in the
rostral, lateral PAG in response to predator exposure [32, 33].Overall, the present findings
support the idea that the rlPAG has a central role in a complex network
controlling the decision-making process
between hunting, foraging, and other behavioral responses. On one hand, the
rlPAG appears to be importantly driven by medial prefrontal cortical areas
involved in controlling attentional and decision-making processes. On
the other hand, the rlPAG receives a wealth of information from different
neural sites related to feeding, drinking, or hunting behavioral
responses.  Therefore, this unique
combination of afferent connections puts the rlPAG in a privileged position to
influence the motivation drive to choose whether hunting and foraging are the most appropriate adaptive
responses.



---

*Source: 612698-2009-03-19.xml*