www.elsevier.com/locate/brainres

Brain Research 1059

Research Report

Orexin (hypocretin) innervation of the paraventricular nucleus of

the thalamus

Gilbert J. Kirouac*, Matthew P. Parsons, Sa Li

Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada A1B 3V6

Accepted 18 August 2005

Available online 5 October 2005

Abstract

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic nucleus with projections to limbic forebrain areas such as the

nucleus accumbens and amygdala. The orexin (hypocretin) peptides are synthesized in hypothalamic neurons that project throughout the

CNS. The present experiments were done to describe the extent of orexin fiber innervation of the PVT in comparison to other midline and

intralaminar thalamic nuclei and to establish the location and proportion of orexin neurons innervating the PVT. All aspects of the

anteroposterior PVT were found to be densely innervated by orexin fibers with numerous enlargements that also stained for synaptophysin, a

marker for synaptic vesicle protein associated with pre-synaptic sites. Small discrete injections of cholera toxin B into the PVT of rats resulted

in the retrograde labeling of a relatively small number of orexin neurons in the medial and lateral hypothalamus. The results also showed a

lack of topographical organization among orexin neurons projecting to the PVT. Previous studies indicate that orexin neurons and neurons in

the PVT appear to be most active during periods of arousal. Therefore, orexin neurons and their projections to the PVT may be part of a

limbic forebrain arousal system.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Peptides: anatomy and physiology

Keywords: Arousal; Stress; Hypothalamus; Nucleus accumbens; Amygdala

1. Introduction

The paraventricular nucleus of the thalamus (PVT) is part

of a group of midline and intralaminar thalamic nuclei

which are hypothesized to play a role in arousal [23,60].

Consistent with this hypothesis, the midline and intra-

laminar nuclei receive convergent input from a large number

of brainstem cell groups associated with arousal, motor,

somatosensory, and visceral systems [11,13,32–34,45,46].

While receiving common inputs, each nucleus of the

midline and intralaminar group innervates specific cortical

areas as well as restricted regions of the basal ganglia and

amygdala [23,60]. The PVT is notable from the midline and

intralaminar nuclei because only the PVT receives signifi-

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.08.035

* Corresponding author. Fax: +1 709 777 7010.

E-mail address: gkirouac@mun.ca (G.J. Kirouac).

cant projections from the nucleus of the solitary tract, lateral

hypothalamic area, suprachiasmatic, arcuate, and dorsome-

dial nuclei [11,13,44,46]. In turn, PVT neurons project to

exclusive sectors of the nucleus accumbens, amygdala, bed

nucleus of the stria terminalis, and prefrontal cortex

[7,39,60], areas of the forebrain associated with viscero-

limbic functions [10,51,62]. While the function of the PVT

remains poorly understood, experiments looking at the

expression of Fos protein as a measure of neuronal

activation have consistently shown that neurons in the

PVT are activated during periods of arousal or by stress

protocols [5,8,41,42,47,50]. The PVT has also been

implicated in the regulation of food intake and hypothala-

mic–pituitary–adrenal activity during chronic stress [5,29].

The orexin peptides orexin-A and orexin-B (also called

hypocretin-1 and hypocretin-2) are co-localized within

neurons of the lateral hypothalamus [16,55] and innervate

(2005) 179 – 188

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188180

widespread regions of the brain including the midline and

intralaminar thalamic nuclei [14,15,40,52]. A large body of

evidence indicates that orexin neurons are active during the

awake active phase [17,21,31,37,64–66] and may maintain

wakefulness by acting on other arousal systems of the brain

[22,27,56,57]. Central administration of orexins has arousal

effects whereas genetic mutations of different aspects of the

orexin system produce disruption in the maintenance of

arousal and narcolepsy in a number of species [reviewed in

[4,35,57]]. Based on the evidence that the PVT is involved

in arousal and receives a substantial orexin innervation, it is

reasonable to suggest that the PVT may play a part in the

arousal functions of orexins. The purpose of the present

study was to provide a complete description of orexin fibers

in the rostrocaudal extent of PVT as well as the adjacent

midline and intralaminar nuclei. Experiments were also

done to determine if synaptophysin, a synaptic vesicle

protein associated with pre-synaptic terminals [38,63], is

co-localized with orexin fiber staining in the PVT to

provide anatomical evidence for the synaptic release of

orexin in the PVT. In addition, we combined retrograde

tracing with immunofluorescence for orexin to determine

the number and the location of orexin neurons that

innervate the PVT.

2. Methods

The results presented in this study represent experiments

done in a total of 14 male Sprague–Dawley rats (200–250

g) that were obtained from the Memorial University of

Newfoundland vivarium and housed on a 12/12 light/dark

cycle with food and water available ad libitum. All efforts

were made to minimize animal suffering and the number of

animals used. All experiments were carried out in accord-

ance with the Canadian Council on Animal Care and were

approved by the Memorial University of Newfoundland.

2.1. Orexin immunoreactivity in the PVT

Four rats were deeply anesthetized with equithesin (0.4

ml/100 g) and perfused transcardially with 150 ml

heparinized saline followed by 400–500 ml of ice-cold

4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).

The brains were removed immediately, post-fixed in the

same fixative for 2–4 h, and cryoprotected in graded

sucrose concentrations (10% and 20% w/v) over 2 days at

4 -C. Sections of the PVT were cut on a cryostat at 50 Amand placed in wells containing 0.1 M PBS. Briefly, every

fourth section of the PVT was pre-incubated in solution

containing 5% normal donkey serum, 0.3% triton-X 100,

and 0.1% sodium azide, followed by overnight incubation

in a rabbit anti-orexin A primary antibody prepared in the

blocking solution (1:3000; Chemicon, catalogue #AB3704,

lot #24110715). Sections were then rinsed in PBS and

incubated for 2–3 h at room temperature in biotinylated

donkey anti-goat IgG (1:500; Jackson Immunoresearch).

Sections were rinsed again then exposed to an avidin–

biotin complex, prepared according to kit directions (Elite

ABC Kit, Vector Laboratories, Burlingame, CA) for 60

min at room temperature. After a few more rinses, the

tissue was reacted with diaminobenzidine (DAB; Vector)

with nickel intensification for 5–10 min to produce black

orexin fiber labeling. The DAB reaction was terminated

by thorough rinsing in PBS before sections were mounted

onto gelatin-coated slides and coverslipped. Sections of

the thalamus were examined and photographed under low

and high magnification using an Olympus BX51 micro-

scope equipped with real time digital camera (SPOT RT

Slicer, Diagnostic Instruments Inc, Sterling Heights, MI).

Calibration bars were inserted using SPOT software

(Version 3.2, Diagnostic Instruments) and images were

transferred to Adobe Photoshop 5.5 to optimize light and

contrast levels.

2.2. Co-localization of orexin and synaptophysin

Every fourth section of the PVT in six rats perfused and

prepared as described above was incubated in a primary

antibody cocktail of rabbit anti-orexin A (1: 3000; Chem-

icon or 1:2000) or goat anti-orexin A (Santa Cruz, catalogue

#sc-8070, lot #I2302) and mouse anti-synaptophysin (1:500;

Chemicon, catalogue #MAB329, lot #24110798) for 2–3

days at 4 -C. Sections were rinsed and then transferred to a

Cy2 conjugated donkey anti-goat or anti-rabbit depending

on the source of the primary antibody and Cy5 conjugated

donkey anti-mouse cocktail secondary antibody (1:500) for

3 h at room temperature. Sections were then examined using

the Olympus FV300 confocal microscope equipped with

blue argon (488 nm) and red helium neon (633 nm) lasers.

Images sizes of 2048 � 2048 pixels were collected using a

100� oil immersion lens with a zoom setting of 3.0 and z-

steps of 0.4 Am. Since synaptophysin antibody penetrates

fixed tissue with difficulty [9,26], only the surface of the

tissue sections was examined for co-localization of orexin-A

and synaptophysin. Optical sections were examined indi-

vidually to ensure that appearance of double labeling was

confined to a 0.4 Am thickness of tissue and that co-

localization represented staining on the same fiber terminal.

Sequential scanning was used to visualize both Cy2 and

Cy5 fluorescent signals while minimizing channel bleed-

through to a negligible level. In an effort to increase

contrast, color settings were adjusted using the Fluoview 3.0

software (Olympus) so that Cy2 labeling was assigned to the

red channel while Cy5 labeling was assigned to the green

channel. Image files were imported into Adobe Photoshop

5.5 to further optimize light and contrast levels.

2.3. Retrograde tracing experiments

Four rats were anesthetized with equithesin (0.3 ml/

100 g, i.p.) and given supplementary doses (0.15 ml/100

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188 181

g) if necessary. Subjects were placed in a Kopf stereo-

taxic frame and a hand drill was used to expose the brain

surface above the PVT. Iontophoretic injections of 0.5%

cholera toxin B (CTb; List Biological Laboratory, Camp-

bell, CA) were performed by applying a 3–5 AA positive

current (200 ms pulses at 2 Hz for 15 min) through a

chlorinated silver wire placed in a glass pipette (7–10 Amtip diameter). The coordinates used for injecting CTb into

the PVT region were 1.0 mm posterior, 0.0 mm lateral to

bregma, and 5.2 mm ventral to the brain surface. The

scalp incision was sutured and rats were returned to their

home cages for recovery. Following a 7–10 day post-

operative survival, rats were deeply anesthetized with

equithesin (0.4 ml/100 g) and perfused transcardially as

described above. The brains were removed, post-fixed,

and cryoprotected as before. Alternate sections of the

PVT and lateral hypothalamus were taken at 50 Am and

used for the retrograde double-labeling experiments.

A double-labeling immunofluorescent protocol was used

to assess the distribution of orexin neurons in the lateral

hypothalamus that project to the PVT. An antibody against

orexin-A was chosen as a representative marker for all

orexin neurons due to the finding that orexin-A and orexin-

B peptides are co-localized within the same neurons of the

lateral hypothalamus [67]. Sections of the PVT and lateral

hypothalamus were pre-incubated in blocking solution for 1

h at room temperature. Sections were incubated (2–3 days

at room temperature) in a primary antibody cocktail

prepared as before containing goat anti-CTb (1:40,000; List

Biological, catalogue #703, lot #7032H) and rabbit anti-

orexin A (1:3000; Chemicon). After rinsing in PBS, sections

were transferred to a secondary antibody cocktail of Cy2

conjugated donkey anti-rabbit and Cy3 conjugated donkey

anti-goat (1:500; Jackson Immunoresearch Laboratories,

West Grove, PA). Sections were rinsed again, mounted,

and a coverslip was placed on the slide.

Double-labeled neurons were analyzed with an Olympus

fluorescent microscope (BX51) equipped with appropriate

filter cubes for Cy2 (U-MNB2, Olympus), Cy3 (U-MNG2,

Olympus), or combination narrow green and blue filter (U-

M51006ZZ, Olympus) combined with a blue/green excita-

tion balancer (U-EXBABG, Olympus) to optimize the

contrast under the single filter cube. Orexin cells were

manually counted under the Cy2 filter to generate an

approximate number of total orexin cells in the rat brain.

Neurons that were double-labeled for CTb and orexin were

counted bilaterally in every second section and expressed as

a percentage of total orexin-immunopositive cells. Cell

count values were corrected according to the following

formula [1] to avoid counting the same orexin neuron twice:

N = [T/(T + D)]n � 2, where N is the corrected neuron

number, n is the number of orexin neurons counted

(multiplied by 2 because alternate sections were reacted

and counted), T is the section thickness (50 Am), and D is

the average diameter of an orexin neuron (25 Am; [52]).

Sections of the lateral hypothalamus were captured and

edited as before. Coronal sections through the PVT (adapted

from [49]) were scanned into Photoshop and tracer injection

sites for each subject were drawn into the corresponding

area using the orexin immunoreactivity to define the

anatomical boundaries of the PVT (see results of Experi-

ment 1). Representative sections at different rostrocaudal

levels of the lateral hypothalamus were photographed,

traced, and schematic drawings were made in Photoshop

to show the spatial location of CTb, orexin, and double-

labeled neurons.

Control experiments were done for all experiments by

removing the primary antibody (CTb, orexin-A, synapto-

physin) or secondary antibodies which resulted in the

absence of the labeling observed when the immunochemical

reactions were done with the antibodies present.

3. Results

Dense orexin-like immunoreactive fibers were observed

throughout the PVT (Fig. 1). Examination of the PVT at

high magnification revealed unevenly spaced fiber swellings

of various sizes which are characteristic en passant enlarge-

ments (Fig. 1F). Thick branched and un-branched fibers

with a beaded appearance ran in all directions with fibers

often terminating in a cluster of beads. Orexin fiber density

was relatively consistent throughout the PVT with the

heaviest innervation found in the posterior PVT. Despite

changes in PVT shape along its rostrocaudal axis, orexin

innervation patterns accurately mirrored the limits of this

midline thalamic nucleus as previously defined [23]. This

allows the use of orexin staining to distinguish PVT from

other thalamic nuclei (Figs. 1A–E). Orexin immunoreac-

tivity was weak in paratenial nucleus located lateral to the

anterior PVT (Figs. 1A and B) and moderate in the

intermediodorsal nucleus located ventral to the posterior

PVT (Figs. 1D and E). Orexin immunoreactivity was

relatively weaker in the anterior (Fig. 1C) compared to the

posterior aspect of the centromedial nucleus (Fig. 1D).

Orexin staining was low in the paracentral nucleus (Figs. 1C

and D), and slightly above background levels in the

mediodorsal nucleus (Figs. 1C–E). The relative density of

orexin-A fiber immunoreactivity in the thalamus as based

on a qualitative analysis is presented in Table 1.

Experiments were also done to determine if synaptophy-

sin, a synaptic vesicle protein associated with pre-synaptic

terminals [38,63], is co-localized with orexin staining

associated with fiber enlargements in the PVT. Small and

large fibers stained for orexin were found in all regions of

the PVT along with immunofluorescence for synaptophysin

which was distributed at random in the same regions where

orexin fibers were found. Examination of 0.4 Am optical

sections of sequential scans of the PVT revealed consid-

erable overlap with the two makers on enlargements of

orexin-labeled fibers (Fig. 2). The overlap between orexin

and synaptophysin was consistently found on the enlarge-

Fig. 1. The photomicrographs in panels A to E show the distribution of orexin-like immunoreactive fibers in the paraventricular nucleus of the thalamus (PVT).

The shape of the PVT varies slightly throughout its anteroposterior extent but is well defined at each level by heavy orexin immunoreactivity. In general, the

PVT is easily distinguished from adjacent thalamic nuclei by dense orexin innervation. Numbers indicate approximate distance from bregma. Panel F shows

fibers and swellings in the PVT immunostained for orexin and captured under 100� oil immersion lens. AM, anteromedial nucleus of the thalamus; CM,

centromedial nucleus of the thalamus; Hb, habenula; IMD, intermediodorsal nucleus of the thalamus; MD, mediodorsal nucleus of the thalamus; PC,

paracentral nucleus of the thalamus; PT, paratenial nucleus of the thalamus; PVA, anterior paraventricular nucleus of the thalamus; PVP, posterior

paraventricular nucleus of the thalamus; PVT, paraventricular nucleus of the thalamus; sm, stria medullaris.

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188182

ments and not on segments of fibers between enlargements

(Figs. 2C and F).

Results from the tract tracing experiments were obtained

from four animals with CTb injections largely restricted

within the PVT (Fig. 3). Two injections were largely

restricted to the anterior to middle portion of the PVT

(Figs. 3A and B) while the other two injections were found

in the middle to posterior PVT (Figs. 3C and D). Injections

involved a portion of the PVT and there was very limited

tracer diffusion into the adjacent intermediodorsal, medi-

odorsal, and paratenial nuclei. The dense core of the

injection was restricted to the PVT as defined by orexin

fiber immunoreactivity. The hypothalamus from the four

subjects receiving PVT injections was examined for

evidence of orexin and CTb-labeled cells. We estimated

the total number of orexin cells in the rat hypothalamus to

be 4741 T 296 (n = 4). Orexin-labeled cells were restricted

to the lateral and perifornical regions of the posterior

hypothalamus (Fig. 4). Of these, a small proportion (2.45 T0.4%) were double-labeled for orexin and CTb. Fig. 4

schematically depicts the distribution of CTb, orexin, and

double-labeled cells in a representative case (Fig. 3B). The

highest concentration of CTb-labeled cells was found in the

medial hypothalamus and extended laterally into the orexin

cell population (Fig. 4). Double-labeled neurons were found

bilaterally in the anteroposterior extent of the orexin neuron

Table 1

Density of orexin-A fiber immunoreactivity in the dorsal thalamus

Anterior paraventricular nucleus + + +

Middle paraventricular nucleus + + +

Posterior paraventricular nucleus + + + +

Anterior centromedial nucleus +

Posterior centromedial nucleus + + +

Intermediodorsal nucleus + +

Paratenial nucleus +

Mediodorsal nucleus +

Paracentral nucleus +

Anteromedial nucleus �The relative density of fiber staining was classified as absent ( � ),

sparse ( + ), moderate ( + + ), dense ( + + + ), and very dense ( + + + + ).

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188 183

distribution with no preferential labeling of medially or

laterally located orexin neurons. Fig. 5 shows examples of

CTb, orexin, and double-labeled neurons in the perifornical

region.

4. Discussion

Results from the present experiments show that the

orexin peptides are found in fibers located in all regions of

the PVT while a relatively weak to moderate fiber density is

found in the adjacent midline and intralaminar thalamic

Fig. 2. Confocal imaging showing immunostaining for orexin (A and D) and syna

images are shown (C and F). Arrows represent sites of co-localization of orexin

nuclei. Orexin fibers largely avoided the mediodorsal

nucleus and the habenular complex as well as the sensory

relay thalamic nuclei. These observations provide the most

detailed description of orexin innervation of midline and

intralaminar nuclei presented in the orexin literature. We

also show that synaptophysin, a maker for pre-synaptic

terminals [38,63], is co-localized on the many orexin-

positive enlargements and may represent sites of orexin

release [58]. Consistent with these results, previous studies

have reported a strong signal for orexin receptor mRNA

[36] as well as excitatory effects of orexins on neurons of

the PVT [28]. While the co-localization of orexin and

synaptophysin is indicative of sites of orexin release,

confirmation of these observation at the ultrastructural level

is necessary to provide definitive evidence for pre-synaptic

release of orexin in the PVT.

The present paper further shows that orexin fibers in the

PVT originate from a relatively small number of orexin

neurons in the lateral and perifornical regions of the

hypothalamus. Orexin neurons projecting to the PVT were

not found to be preferentially located in a specific region

of the orexin population but dispersed within the entire

orexin cell containing area. It is also interesting to note

that the observation of orexin neurons sending bilateral

projections to the PVT clearly argues against any form of

topography in the projection of orexin neurons to this

ptophysin (B and E) in the paraventricular nucleus of the thalamus. Merged

and synaptophysin on fiber enlargements.

Fig. 3. Schematic drawings of coronal sections through the dorsal thalamus illustrating the location of the retrograde tracer cholera toxin b (CTb) injection in

the paraventricular nucleus. Four injection sites (A, B, C, and D) are drawn at three different stereotaxic levels representing the anterior (�1.4 mm from

bregma), middle (�2.5 mm), and posterior (�3.3 mm) levels. The dense core and diffuse CTb spread are represented by black and gray shading, respectively.

AM, anteromedial nucleus of the thalamus; CM, centromedial nucleus of the thalamus; Hb, habenula; IMD, intermediodorsal nucleus of the thalamus; MD,

mediodorsal nucleus of the thalamus; PC, paracentral nucleus of the thalamus; PT, paratenial nucleus of the thalamus; PVA, anterior paraventricular nucleus of

the thalamus; PVP, posterior paraventricular nucleus of the thalamus; PVT, paraventricular nucleus of the thalamus; sm, stria medullaris.

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188184

midline brain region. There is little information to date

about the precise location of orexin neurons with respect to

their projection targets in the brain. While it was reported

that orexin neurons projecting to the locus coeruleus

appeared to be preferentially localized in the dorsal half

of the orexin cell group, orexin neurons projecting to

forebrain regions were found to be heterogeneously

distributed in the lateral hypothalamus [18]. A lack of a

clear topography in the orexin neurons that innervate the

PVT and other orexin target areas would be consistent with

the notion that at least some orexin neurons work together

to generate coordinated actions in functionally related

terminal fields [18].

We estimated the total number of orexin neurons in the

male Sprague–Dawley rat hypothalamus to be approx-

imately 4741 T 296. Although this estimate is strikingly

similar to a recent report using modern stereological

techniques [3], earlier studies have reported the total number

of orexin neurons to be much lower [12,25,40,52]. Differ-

ences could be attributed to a variety of factors including the

source of the antisera, immunochemical protocols, rat strain,

and counting methods. The greater number of orexin

neurons indicated in the present study as well as a previous

study [3] may have resulted from the cell counts being done

on a larger sample of relatively thick sections through the

hypothalamus.

The number of orexin neurons retrogradely labeled

following injection of CTb in restricted regions of the

PVT may appear low considering the total number of orexin

neurons found in the hypothalamus. However, these

Fig. 4. Schematic drawings of coronal sections through the lateral

hypothalamic region illustrating cells immunopositive for orexin (green

circles) and cholera toxin b (CTb; red triangles) following an injection of

CTb into the paraventricular nucleus of the thalamus (case represented by

Fig. 3B). A small percentage of neurons were found to be double-labeled

for both orexin and CTb (black stars). Numbers indicate approximate

distance from bregma. 3V, third ventricle; AH, anterior hypothalamic area;

DMH, dorsomedial hypothalamic nucleus; f, fornix; opt, optic tract;

PeFLH, perifornical and lateral hypothalamic area; PH, posterior hypo-

thalamic area; VMH, ventromedial hypothalamic nucleus.

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188 185

numbers should be considered to represent a large under-

estimation of the total population because CTb injections

involved only a restricted portion of the entire PVT. The

PVT is a narrow nucleus that extends the entire length of the

thalamus and this limits the extent that injections of tracer

can be made in the PVT without involving adjacent nuclei.

Iontophoretic injections of CTb in the PVT in the present

study produced a small dense core of CTb immunoreactivity

and a relatively more diffuse halo surrounding the core.

While CTb is a very sensitive retrograde tracer, it is unlikely

that the more diffuse CTb staining of the injection halo

could produce significant retrograde transport and labeling

(personal observations). As shown in Fig. 3, the dense

portions of the CTb injections covered only a restricted

portion of the PVT. Therefore, we predict that dense

deposits of CTb that involved the entire PVTwould produce

larger numbers of orexin neurons retrogradely labeled with

CTb. However, attempts at producing injections of CTb that

involved the entire PVT would result in large injections that

would involve most of the dorsal midline thalamus. It is also

possible that dense orexin innervation of the PVT may result

from extensive branching of axons originating from a

relatively small number of orexin neurons.

A large number of neurons labeled following CTb

injections in the PVT were found to be located outside the

orexin group of cells. Many of the labeled cells were found

in the dorsomedial nucleus with the remainder scattered

around the third ventricle as previously reported [45,46].

While the neurochemical identity of the non-orexin CTb

neurons was not investigated in the present study, a recent

investigation reported that half of the neurons in the

dorsomedial nucleus retrogradely labeled from a PVT

injection of CTb were cholecystokinin-positive [43]. Since

fibers in the PVT are immunoreactive to several neuro-

peptides [20], it is likely that other types of peptidergic

neurons in the hypothalamus project to the PVT.

As shown in the present study, the PVT receives a strong

and distinct innervation from orexin neurons in the

hypothalamus. In addition, the PVT is innervated by

serotonergic neurons of the dorsal raphe nucleus, histami-

nergic neurons of tuberomammillary nucleus, and noradre-

nergic neurons of the locus coeruleus [45,46,48]. These

groups of monoaminergic neurons as well as the orexinergic

neurons are most active during periods of arousal and are

believed to play important roles in maintaining wakefulness

and arousal states [22,27,56]. The PVT is also unique in that

it receives projections from nucleus of the solitary tract and

several regions of the hypothalamus [13,34,45,46,54]; brain

regions associated with visceral, autonomic, and endocrine

functions. Furthermore, the PVT is innervated by the

parabrachial nucleus and the periaqueductal gray, two areas

of the brainstem well known as nociceptive relays [32–34].

This has lead to the suggestion that the PVT may have

arousal functions related to visceral and nociceptive stimuli

[32–34,60].

Studies using Fos expression as a measure of neuronal

activation have consistently shown that neurons in the PVT

are activated during arousal [41,42,50] and stress protocols

[5,8,47]. Similar to the activity of PVT neurons, an increase

in the activity of orexin neurons that occurs during the

awake active phase has been reported in different species

[17,21,31,37,64–66]. Evidence also suggests that high

levels of arousal such as stress could also be a strong

activator of orexin neurons [17]. Therefore, information

Fig. 5. Photomicrographs showing lateral hypothalamic neurons immunostained for orexin-A (B and E) and cholera toxin B (CTb; A and D) following an

injection of CTb in the paraventricular nucleus of the thalamus. Images of Cy2-labeled orexin Cy3-labeled CTb taken under separate filters were merged to

show evidence of double-labeling (arrows, C and F).

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188186

about the arousal state of an organism could be in part

relayed from the hypothalamus to the PVT by the release of

orexins. In turn, the PVT could integrate information from a

number of arousal centers and relay this information to the

nucleus accumbens, amygdala, and prefrontal cortex to

place these forebrain structures in a state of arousal or

readiness necessary for behavioral responding. For example,

the nucleus accumbens is connected with behavioral motor

control systems and has been implicated in defensive

behaviors and feeding [30,51,53]. The medial prefrontal

cortex and the amygdala have extensive connections with

brain regions associated with the regulation of the auto-

nomic and endocrine systems and are involved in the

physiological responses to arousal and stress [2,24,59,61].

Experimental evidence suggests that the PVT regulates the

hypothalamic–pituitary–adrenal axis response to chronic

stress, food intake, and energy balance [5,6]. However, the

type of influence that orexins may have on arousal or stress

induced activation of the PVT and its forebrain targets is

unknown. In addition to arousal, orexins have also been

linked to food intake, body temperature regulation, and

metabolism [4,19,35]. The role that the PVT plays in

orexins’ diverse behavioral and physiological function

remains to be determined.

Acknowledgments

This research was supported by the Canadian Institutes

for Health Research (CIHR). GJK is a recipient of a New

Investigator Award from the CIHR/Regional Partnership

Program and MPP is a recipient of a Natural Sciences

and Engineering Research Council (NSERC) graduate

scholarship.

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188 187

References

[1] M. Abercrombie, Estimation of nuclear populations from microtome

sections, Anat. Rec. 94 (1946) 239–247.

[2] I.G. Akmaev, L.B. Kalimullina, L.A. Sharipova, The central nucleus

of the amygdaloid body of the brain: cytoarchitectonics, neuronal

organization, connections, Neurosci. Behav. Physiol. 34 (2004)

603–610.

[3] J.S. Allard, Y. Tizabi, J.P. Shaffery, C.O. Trouth, K. Manaye,

Stereological analysis of the hypothalamic hypocretin/orexin neurons

in an animal model of depression, Neuropeptides 38 (2004) 311–315.

[4] C.T. Beuckmann, M. Yanagisawa, Orexins: from neuropeptides to

energy homeostasis and sleep/wake regulation, J. Mol. Med. 80 (2002)

329–342.

[5] S. Bhatnagar, M. Dallman, Neuroanatomical basis for facilitation of

hypothalamic–pituitary–adrenal responses to a novel stressor after

chronic stress, Neuroscience 84 (1998) 1025–1039.

[6] S. Bhatnagar, M.F. Dallman, The paraventricular nucleus of the

thalamus alters rhythms in core temperature and energy balance in a

state-dependent manner, Brain Res. 851 (1999) 66–75.

[7] M. Bubser, A.Y. Deutch, Thalamic paraventricular nucleus neurons

collateralize to innervate the prefrontal cortex and nucleus accumbens,

Brain Res. 787 (1998) 304–310.

[8] M. Bubser, A.Y. Deutch, Stress induces Fos expression in neurons of

the thalamic paraventricular nucleus that innervate limbic forebrain

sites, Synapse 32 (1999) 13–22.

[9] M.E. Calhoun, M. Jucker, L.J. Martin, G. Thinakaran, D.L. Price, P.R.

Mouton, Comparative evaluation of synaptophysin-based methods for

quantification of synapses, J. Neurocytol. 25 (1996) 821–828.

[10] R.N. Cardinal, J.A. Parkinson, J. Hall, B.J. Everitt, Emotion and

motivation: the role of the amygdala, ventral striatum, and prefrontal

cortex, Neurosci. Biobehav. Rev. 26 (2002) 321–352.

[11] S. Chen, H.S. Su, Afferent connections of the thalamic paraventricular

and parataenial nuclei in the rat—A retrograde tracing study with

iontophoretic application of Fluoro-Gold, Brain Res. 522 (1990) 1–6.

[12] J. Ciriello, J.C. McMurray, T. Babic, C.V. de Oliveira, Collateral

axonal projections from hypothalamic hypocretin neurons to cardio-

vascular sites in nucleus ambiguous and nucleus tractus solitarius,

Brain Res 991 (2003) 133–141.

[13] J. Cornwall, O.T. Phillipson, Afferent projections to the dorsal

thalamus of the rat as shown by retrograde lectin transport: I. The

mediodorsal nucleus, Neuroscience 24 (1988) 1035–1049.

[14] D.J. Cutler, R. Morris, V. Sheridhar, T.A. Wattam, S. Holmes, S.

Patel, J.R. Arch, S. Wilson, R.E. Buckingham, M.L. Evans, R.A.

Leslie, G. Williams, Differential distribution of orexin-A and orexin-

B immunoreactivity in the rat brain and spinal cord, Peptides 20

(1999) 1455–1470.

[15] Y. Date, Y. Ueta, H. Yamashita, H. Yamaguchi, S. Matsukura, K.

Kangawa, T. Sakurai, M. Yanagisawa, M. Nakazato, Orexins,

orexigenic hypothalamic peptides, interact with autonomic, neuro-

endocrine and neuroregulatory systems, Proc. Natl. Acad. Sci. U. S. A.

96 (1999) 748–753.

[16] L. de Lecea, T.S. Kilduff, C. Peyron, X. Gao, P.E. Foye, P.E.

Danielson, C. Fukuhara, E.L. Battenberg, V.T. Gautvik, F.S. Bartlett

II, W.N. Frankel, A.N. van den Pol, F.E. Bloom, K.M. Gautvik, J.G.

Sutcliffe, The hypocretins: hypothalamus-specific peptides with

neuroexcitatory activity, Proc. Natl. Acad. Sci. U. S. A. 95 (1998)

322–327.

[17] R.A. Espana, R.J. Valentino, C.W. Berridge, Fos immunoreactivity

in hypocretin-synthesizing and hypocretin-1 receptor-expressing

neurons: effects of diurnal and nocturnal spontaneous waking,

stress and hypocretin-1 administration, Neuroscience 121 (2003)

201–217.

[18] R.A. Espana, K.M. Reis, R.J. Valentino, C.W. Berridge, Organiza-

tion of hypocretin/orexin efferents to locus coeruleus and basal

forebrain arousal-related structures, J. Comp. Neurol. 481 (2005)

160–178.

[19] A.V. Ferguson, W.K. Samson, The orexin/hypocretin system: a critical

regulator of neuroendocrine and autonomic function, Front. Neuro-

endocrinol. 24 (2003) 141–150.

[20] L.J. Freedman, M.D. Cassell, Relationship of thalamic basal forebrain

projection neurons to the peptidergic innervation of the midline

thalamus, J. Comp. Neurol. 348 (1994) 321–342.

[21] N. Fujiki, Y. Yoshida, B. Ripley, K. Honda, E. Mignot, S. Nishino,

Changes in CSF hypocretin-1 (orexin A) levels in rats across 24

hours and in response to food deprivation, NeuroReport 12 (2001)

993–997.

[22] C. Gottesmann, Brain inhibitory mechanisms involved in basic and

higher integrated sleep processes, Brain Res. Brain Res. Rev. 45

(2004) 230–249.

[23] H.J. Groenewegen, H.W. Berendse, The specificity of the ’non-

specific’ midline and intralaminar thalamic nuclei, Trends Neurosci.

17 (1994) 52–57.

[24] H.J. Groenewegen, H.B. Uylings, The prefrontal cortex and the

integration of sensory, limbic and autonomic information, Prog. Brain

Res. 126 (2000) 3–28.

[25] T.A. Harrison, C.T. Chen, N.J. Dun, J.K. Chang, Hypothalamic orexin

A-immunoreactive neurons project to the rat dorsal medulla, Neurosci.

Lett. 273 (1999) 17–20.

[26] J.J. Hiscock, S. Murphy, J.O. Willoughby, Confocal microscopic

estimation of GABAergic nerve terminals in the central nervous

system, J. Neurosci. Methods 95 (2000) 1–11.

[27] M. Hungs, E. Mignot, Hypocretin/orexin, sleep and narcolepsy,

Bioessays 23 (2001) 397–408.

[28] M. Ishibashi, S. Takano, H. Yanagida, M. Takatsuna, K. Nakajima, Y.

Oomura, M.J. Wayner, K. Sasaki, Effects of orexins/hypocretins on

neuronal activity in the paraventricular nucleus of the thalamus in rats

in vitro, Peptides 26 (2005) 471–481.

[29] A. Jaferi, N. Nowak, S. Bhatnagar, Negative feedback functions in

chronically stressed rats: role of the posterior paraventricular thalamus,

Physiol. Behav. 78 (2003) 365–373.

[30] A.E. Kelley, Ventral striatal control of appetitive motivation: role in

ingestive behavior and reward-related learning, Neurosci. Biobehav.

Rev. 27 (2004) 765–776.

[31] T. Kodama, S. Usui, Y. Honda, M. Kimura, High Fos expression

during the active phase in orexin neurons of a diurnal rodent, Tamias

sibiricus barberi, Peptides 26 (2005) 631–638.

[32] K.E. Krout, A.D. Loewy, Parabrachial nucleus projections to midline

and intralaminar thalamic nuclei of the rat, J. Comp. Neurol. 428

(2000) 475–494.

[33] K.E. Krout, A.D. Loewy, Periaqueductal gray matter projections to

midline and intralaminar thalamic nuclei of the rat, J. Comp. Neurol.

424 (2000) 111–141.

[34] K.E. Krout, R.E. Belzer, A.D. Loewy, Brainstem projections to

midline and intralaminar thalamic nuclei of the rat, J. Comp. Neurol.

448 (2002) 53–101.

[35] J.P. Kukkonen, T. Holmqvist, S. Ammoun, K.E. Akerman, Functions

of the orexinergic/hypocretinergic system, Am. J. Physiol., Cell

Physiol. 283 (2002) C1567–C1591.

[36] J.N. Marcus, C.J. Aschkenasi, C.E. Lee, R.M. Chemelli, C.B.

Saper, M. Yanagisawa, J.K. Elmquist, Differential expression of

orexin receptors 1 and 2 in the rat brain, J. Comp. Neurol. 435

(2001) 6–25.

[37] G.S. Martinez, L. Smale, A.A. Nunez, Diurnal and nocturnal rodents

show rhythms in orexinergic neurons, Brain Res. 955 (2002) 1–7.

[38] W.D. Matthew, L. Tsavaler, L.F. Reichardt, Identification of a synaptic

vesicle-specific membrane protein with a wide distribution in neuronal

and neurosecretory tissue, J. Cell Biol. 91 (1981) 257–269.

[39] M.M. Moga, R.P. Weis, R.Y. Moore, Efferent projections of the

paraventricular thalamic nucleus in the rat, J. Comp. Neurol. 359

(1995) 221–238.

[40] T. Nambu, T. Sakurai, K. Mizukami, Y. Hosoya, M. Yanagisawa, K.

Goto, Distribution of orexin neurons in the adult rat brain, Brain Res.

827 (1999) 243–260.

G.J. Kirouac et al. / Brain Research 1059 (2005) 179–188188

[41] C.M. Novak, A.A. Nunez, Daily rhythms in Fos activity in the rat

ventrolateral preoptic area and midline thalamic nuclei, Am. J.

Physiol. 275 (1998) R1620–R1626.

[42] C.M. Novak, L. Smale, A.A. Nunez, Rhythms in Fos expression in

brain areas related to the sleep–wake cycle in the diurnal Arvicanthis

niloticus, Am. J. Physiol., Regul. Integr. Comp. Physiol. 278 (2000)

R1267–R1274.

[43] K. Otake, Cholecystokinin and substance P immunoreactive projec-

tions to the paraventricular thalamic nucleus in the rat, Neurosci. Res.

51 (2005) 383–394.

[44] K. Otake, Y. Nakamura, Single midline thalamic neurons projecting to

both the ventral striatum and the prefrontal cortex in the rat,

Neuroscience 86 (1998) 635–649.

[45] K. Otake, D.A. Ruggiero, Monoamines and nitric oxide are employed

by afferents engaged in midline thalamic regulation, J. Neurosci. 15

(1995) 1891–1911.

[46] K. Otake, D.A. Ruggiero, Y. Nakamura, Adrenergic innervation of

forebrain neurons that project to the paraventricular thalamic nucleus

in the rat, Brain Res. 697 (1995) 17–26.

[47] K. Otake, K. Kin, Y. Nakamura, Fos expression in afferents to the rat

midline thalamus following immobilization stress, Neurosci. Res. 43

(2002) 269–282.

[48] P. Panula, U. Pirvola, S. Auvinen, M.S. Airaksinen, Histamine-

immunoreactive nerve fibers in the rat brain, Neuroscience 28 (1989)

585–610.

[49] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates,

1997.

[50] Z.C. Peng, G. Grassi-Zucconi, M. Bentivoglio, Fos-related protein

expression in the midline paraventricular nucleus of the rat thalamus:

basal oscillation and relationship with limbic efferents, Exp. Brain

Res. 104 (1995) 21–29.

[51] C.M. Pennartz, H.J. Groenewegen, F.H. Lopes da Silva, The nucleus

accumbens as a complex of functionally distinct neuronal ensembles:

an integration of behavioural, electrophysiological and anatomical

data, Prog. Neurobiol. 42 (1994) 719–761.

[52] C. Peyron, D.K. Tighe, A.N. van den Pol, L. de Lecea, H.C.

Heller, J.G. Sutcliffe, T.S. Kilduff, Neurons containing hypocretin

(orexin) project to multiple neuronal systems, J. Neurosci. 18

(1998) 9996–10015.

[53] S.M. Reynolds, K.C. Berridge, Glutamate motivational ensembles in

nucleus accumbens: rostrocaudal shell gradients of fear and feeding,

Eur. J. Neurosci. 17 (2003) 2187–2200.

[54] D.A. Ruggiero, S. Anwar, J. Kim, S.B. Glickstein, Visceral afferent

pathways to the thalamus and olfactory tubercle: behavioral implica-

tions, Brain Res. 799 (1998) 159–171.

[55] T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R.M. Chemelli, H.

Tanaka, S.C. Williams, J.A. Richardson, G.P. Kozlowski, S. Wilson,

J.R. Arch, R.E. Buckingham, A.C. Haynes, S.A. Carr, R.S. Annan,

D.E. McNulty, W.S. Liu, J.A. Terrett, N.A. Elshourbagy, D.J.

Bergsma, M. Yanagisawa, Orexins and orexin receptors: a family of

hypothalamic neuropeptides and G protein-coupled receptors that

regulate feeding behavior, Cell 92 (1998) 573–585.

[56] C.B. Saper, T.C. Chou, T.E. Scammell, The sleep switch: hypothala-

mic control of sleep and wakefulness, Trends Neurosci. 24 (2001)

726–731.

[57] J.M. Siegel, Hypocretin (orexin): role in normal behavior and

neuropathology, Annu. Rev. Psychol. 55 (2004) 125–148.

[58] M.A. Silver, M.P. Stryker, A method for measuring colocalization of

presynaptic markers with anatomically labeled axons using double

label immunofluorescence and confocal microscopy, J. Neurosci.

Methods 94 (2000) 205–215.

[59] L.W. Swanson, G.D. Petrovich, What is the amygdala? Trends

Neurosci. 21 (1998) 323–331.

[60] Y.D. Van der Werf, M.P. Witter, H.J. Groenewegen, The intralaminar

and midline nuclei of the thalamus. Anatomical and functional

evidence for participation in processes of arousal and awareness,

Brain Res. Brain Res. Rev. 39 (2002) 107–140.

[61] A.J. Verberne, N.C. Owens, Cortical modulation of the cardiovascular

system, Prog. Neurobiol. 54 (1998) 149–168.

[62] D.L. Walker, D.J. Toufexis, M. Davis, Role of the bed nucleus of the

stria terminalis versus the amygdala in fear, stress, and anxiety, Eur. J.

Pharmacol. 463 (2003) 199–216.

[63] B. Wiedenmann, W.W. Franke, Identification and localization of

synaptophysin, an integral membrane glycoprotein of Mr 38,000

characteristic of presynaptic vesicles, Cell 41 (1985) 1017–1028.

[64] Y. Yoshida, N. Fujiki, T. Nakajima, B. Ripley, H. Matsumura, H.

Yoneda, E. Mignot, S. Nishino, Fluctuation of extracellular hypocre-

tin-1 (orexin A) levels in the rat in relation to the light–dark cycle and

sleep–wake activities, Eur. J. Neurosci. 14 (2001) 1075–1081.

[65] J.M. Zeitzer, C.L. Buckmaster, K.J. Parker, C.M. Hauck, D.M. Lyons,

E. Mignot, Circadian and homeostatic regulation of hypocretin in a

primate model: implications for the consolidation of wakefulness,

J. Neurosci. 23 (2003) 3555–3560.

[66] J.M. Zeitzer, C.L. Buckmaster, D.M. Lyons, E. Mignot, Locomotor-

dependent and -independent components to hypocretin-1 (orexin A)

regulation in sleep–wake consolidating monkeys, J. Physiol. 557

(2004) 1045–1053.

[67] J.H. Zhang, S. Sampogna, F.R. Morales, M.H. Chase, Co-localization

of hypocretin-1 and hypocretin-2 in the cat hypothalamus and

brainstem, Peptides 23 (2002) 1479–1483.