Activation of feeding-related neural circuitry after unilateral injections of muscimol into the nucleus accumbens shell

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Brain Research 1048

Research report

Activation of feeding-related neural circuitry after unilateral injections

of muscimol into the nucleus accumbens shell

Thomas R. Stratford*

Laboratory of Integrative Neuroscience, University of Illinois at Chicago, 1007 West Harrison Street, Chicago, IL 60607-7137, USA

Department of Psychology (m/c 285), University of Illinois at Chicago, 1007 West Harrison Street, Chicago, IL 60607-7137, USA

Accepted 3 May 2005

Available online 25 May 2005

Abstract

Chemical inhibition of neurons in the nucleus accumbens shell (AcbSh) elicits intense, behaviorally specific, feeding in satiated rats. We

have demonstrated previously that this treatment activates a number of brain regions, most significantly the lateral hypothalamus (LH). This

activation could be elicited through a direct neural connection with the AcbSh or secondarily through changes in autonomic activity, stress, or

circulating levels of orexigenic or satiety factors. In the present study, we used the immunohistochemical localization of Fos protein to map

neuronal activation after unilateral muscimol injections into the AcbSh to determine whether AcbSh-mediated Fos expression remains

lateralized in the circuit and whether secondary systemic changes in the rat can be excluded as primary factors in the activation of downstream

component nuclei. Rats receiving only saline injections exhibited very little Fos immunoreactivity. In contrast, unilateral injections of

muscimol into the AcbSh consistently increased Fos expression in several brain regions. Three distinct patterns of expression were observed.

Fos synthesis in the LH was increased only on the side of the brain ipsilateral to the muscimol injection. Fos expression remained primarily

ipsilateral to the injection site in the septohypothalamic, paraventricular hypothalamic (PVN), paratenial thalamic, and lateral habenular nuclei,

and medial substantia nigra, but was increased bilaterally in the piriform cortex, supraoptic nucleus, central nucleus of the amygdala, and

nucleus of the solitary tract. Smaller numbers of Fos-immunoreactive cells were seen unilaterally in the bed nucleus of the stria terminalis,

medial ventral pallidum, arcuate nucleus, and ventral tegmental area and bilaterally in the supraoptic and tuberomammillary nuclei. The

labeling in the LH, PVN, and other unilaterally labeled structures provides evidence that these brain regions are components of an AcbSh-

mediated neural circuit and suggests that they may be involved in the expression of AcbSh-mediated feeding behavior.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Ingestive behaviors

Keywords: Food intake; Feeding behavior; Fos; Lateral hypothalamic area; GABA; Rat

1. Introduction

Data acquired over the past several years suggest that the

shell subregion of the nucleus accumbens (AcbSh) may be an

important component of a neural system involved in the control

of feeding behavior. Inhibiting neurons in the ventromedial

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

doi:10.1016/j.brainres.2005.05.002

* Department of Psychology (m/c 285), University of Illinois at Chicago,

1007 West Harrison Street, Chicago, IL 60607-7137, USA. Fax: +1 312

413 4122.

E-mail address: [email protected].

AcbSh with local injections of GABA agonists [41,42,50] or

glutamate antagonists [31,44] elicits a powerful, but behavior-

ally specific, feeding response in satiated rats. This feeding is

characterized by its short latency and intensity, yet the treatment

does not increase water intake, non-ingestive gnawing, or

locomotor activity [44]. The fact that large specific increases in

food intake are elicited by increasing levels of endogenous

GABA or blocking the action of endogenous glutamate in the

AcbSh strongly suggests that these neurochemical systems in

this particular brain region participate in the physiological

control of feeding.

(2005) 241 – 250

T.R. Stratford / Brain Research 1048 (2005) 241–250242

In order to investigate the neural circuitry underlying the

effects of muscimol injected into the AcbSh, previous

studies have examined the ability of these injections to

induce synthesis of the immediate early gene product Fos, a

widely employed marker of neuronal activation [16,37].

These studies have shown that bilateral injections of

muscimol into the AcbSh result in pronounced bilateral

expression of Fos in several brain regions including the

lateral hypothalamic area (LH) and the paraventricular

hypothalamic nucleus (PVN) [42]. These results are

significant given the substantial evidence indicating that

these regions play an important role in the control of food

intake [5,13,40]. The AcbSh projects directly to the

ipsilateral LH [21,27,53] and can additionally influence

this region through projections to the ventral pallidum

[21,33], which, in turn, sends ipsilateral efferents to the

hypothalamus [19,20]. These findings are all compatible

with the possibility that the AcbSh elicits feeding through a

neurally mediated influence on the activity of hypothalamic

neurons. However, the results on Fos expression do need to

be interpreted with caution. For example, studies have

shown that Fos synthesis in the LH or PVN can be induced

by a variety of stressors [7,10,38,39] and by changes in

circulating levels of glucose or a number of hormones

[2,6,8,11,17,30,32,34,36,47]. Thus, while it is possible that

the hypothalamic Fos expression seen after bilateral intra-

AcbSh muscimol injections may reflect alterations in the

activity of neural circuits linking the AcbSh and the

hypothalamus, it is also quite plausible that this Fos

expression might be secondary to AcbSh-mediated changes

in autonomic activity, hormone levels, arousal, stress, or

other systemically or behaviorally mediated factors.

One approach to the problem of determining whether the

AcbSh exerts a direct control over neural activity in the

other brain structures is to examine the effects on Fos

expression produced by unilateral, as compared to bilateral,

injections of muscimol. Under these conditions, one would

expect to see bilateral labeling if Fos synthesis reflects

alterations in the general physiological or behavioral state of

the animal. In contrast, a pattern of unilateral Fos expression

would strongly suggest that neurons in the affected regions

are part of an uninterrupted neural circuit involving the

AcbSh. In the present study, we mapped neuronal activation

after unilateral muscimol injections into the AcbSh to

determine whether AcbSh-mediated Fos expression remains

lateralized in the circuit and whether secondary systemic

changes in the rat can be excluded as primary factors in the

activation of downstream component nuclei.

2. Materials and methods

2.1. Subjects

Male Sprague–Dawley SD rats were obtained from

Harlan (Madison, WI). The animals weighed between 280

g and 320 g at the time of surgery. They were housed

individually in wire-mesh cages and were maintained on a 12

h:12 h light:dark cycle (lights on at 07:00) in a temperature-

controlled environment (¨22 -C) with food (Harlan Teklad

7001) and tap water available ad libitum, except as noted

below. All experiments conformed to the NIH Guide for the

Care and Use of Laboratory Animals and were approved by

the Institutional Animal Care and Use Committee.

2.2. Surgery

The rats were anesthetized with sodium pentobarbital (50

mg/kg), and bilateral 26-gauge stainless steel guide cannu-

lae (Plastics One, Roanoke, VA) were implanted using

standard, flat-skull stereotaxic techniques. The guide can-

nulae were aimed so that they terminated 2.0 mm dorsal to

the AcbSh using coordinates anterior–posterior (AP): +1.4,

lateral–medial: T0.75, dorsal–ventral: �6.1 (mm from

bregma) and were held in place using stainless steel screws

and denture lining material. A stainless steel obturator was

inserted into the lumen of each cannula to help maintain

patency. Each rat was allowed to recover at least 7 days

before the start of testing, during which time the rats were

handled daily.

2.3. Intracerebral injections

In order to acclimate the rats to the microinjection

procedure, the obturators were removed, and a 33-gauge

injection cannula, extending 2.0 mm beyond the ventral

tip of the guide, was inserted into each guide cannula on

three consecutive days. The obturators were then

replaced, and the rats were returned to their home cages.

On the final acclimation day, each rat received bilateral

0.5 Al intracerebral injections of sterile 0.15 M saline at a

rate of 0.32 Al/min.

2.3.1. Feeding behavior

Forty-eight hours after the final acclimation run, seven rats

received simultaneous 0.5 Al injections of muscimol (0, 25,

50, or 100 ng; Sigma, St. Louis, MO) into one AcbSh and the

sterile saline vehicle into the opposite AcbSh. After the

infusions, the injection cannulae were left in place for an

additional 60 s in order to minimize leakage up the cannula

track. The order in which the different doses were adminis-

tered and the side of the brain receiving the muscimol

injection were randomized between rats. Following the

microinjections, the rats were placed in test cages with a

preweighed quantity of the maintenance diet and a graduated

bottle containing tap water available. Food intake (corrected

for spillage) was calculated at 30, 60, and 120 min. Total

water intake was determined at the end of the 120 min test.

2.3.2. Muscimol-induced Fos expression

Rats in Group I (n = 9) received simultaneous 0.5 Alinjections of 100 ng muscimol into one AcbSh and the

T.R. Stratford / Brain Research 1048 (2005) 241–250 243

sterile saline vehicle into the opposite AcbSh. The side of

the brain receiving the muscimol injection was randomized

between rats. In Group II (n = 6), infusion cannulae were

lowered through both guide cannulae, and saline was

infused into one AcbSh, while no injection was made into

the opposite AcbSh. Immediately following the injections,

the rats were returned to their home cages without food or

water present. The rats remained in their home cages for 90

min, at which time they were deeply anesthetized with

sodium pentobarbital and were perfused transcardially with

50 ml of a 0.15 M saline solution followed immediately by

500 ml of a 10% buffered formalin solution. The brains

were removed and postfixed in the formalin solution for

2–3 h, transferred to 20% sucrose in 0.01 M phosphate

buffer at 4 -C for 48–72 h, and then immunohistochemi-

cally processed.

2.4. Immunohistochemistry

The brains were frozen quickly with a chemical freeze

spray (Freeze’It; Curtis Matheson Scientific, Houston, TX)

and, guided by our previous results in which bilateral

muscimol injections induced strong Fos expression primar-

ily in the diencephalon and NTS [42], 34 Am-thick coronal

sections were taken from the level of the AcbSh (AP level:

1.4 mm) to the caudal pole of the interpeduncular nucleus

(AP level: �7.0 mm) and through the NTS (AP level: �14.0

mm). Every second section was placed in a blocking serum

composed of 0.01 M phosphate-buffered saline (PBS, pH

7.2) containing 10% normal goat serum (NGS; Vector

Laboratories, Burlingame, CA) and 0.3% Triton X-100

(Sigma, St. Louis, MO) for 30 min. The sections were rinsed

in PBS and incubated on a rotary shaker table for 44 h at 4

-C in a polyclonal rabbit anti-Fos primary antibody (diluted

1:50,000 with 0.01 M PBS containing 4% NGS; Oncogene

Research Products, Boston, MA). The sections were rinsed

again in PBS and were processed using a Vectastain Elite

ABC kit (Vector Laboratories). The sections were incubated

in the biotinylated goat anti-rabbit secondary antibody

(diluted 1:200 with PBS containing 4% NGS) for 60 min

at room temperature, rinsed in PBS (3 � 10 min), and

incubated in the avidin–biotin complex solution for 60 min.

Following another series of rinses in PBS, the peroxidase

was visualized by incubating the tissue for 5 min in the

nickel-enhanced chromogen solution from a Vector 3,3V-diaminobenzidine tetrahydrochloride peroxidase substrate

kit. The sections were mounted on chrom–alum-coated

slides, air-dried, cleared in xylene, and coverslipped with

Permount. Alternate sections were stained with cresyl violet

to assist in delineating section level and structure boundaries

and to insure placement accuracy of the injection sites.

2.5. Quantification

Fos-like-immunoreactive nuclei were visualized using a

Leitz Diaplan microscope at a total magnification of 250�.

The brains of rats receiving unilateral muscimol injections

were examined thoroughly to identify regions exhibiting a

consistent, substantive increase in Fos-immunoreactivity in

response to the drug. Based on this examination, the number

of Fos immunopositive nuclei was counted in individual

matched coronal sections from Group I and Group II rats at

four different levels of the LH corresponding approximately

to plates 25, 30, 34 and 37 of the Paxinos and Watson rat

brain atlas [35], respectively. Fos was also quantified in nine

additional structures which were observed in preliminary

studies to display a strong response to muscimol. All

sections were examined to determine the general AP level

showing maximal Fos expression for each structure and a

single representative section through that level from each rat

was analyzed. Section levels were matched between groups,

photographed using 35 mm slide film, and digital images

were captured using a 35 mm slide scanner. Because

digitally captured images vary slightly in brightness and

contrast, an image processing program (Adobe Photoshop)

was used to make minor adjustments to these parameters in

order to standardize the images before quantification. Using

Nissl-stained adjacent sections as a guide, structure borders

were outlined digitally on both sides of the brain, and Fos-

immunopositive nuclei within those borders were identified

and counted using Scion Image software (Scion Corpora-

tion, Frederick, MD). Identification parameters for Fos-

immunopositive nuclei were optical density, shape, and size

and were set so that the automated counts closely

approximated careful counts of several representative

sections by the experimenter.

2.6. Statistical analyses

Food and water intakes were analyzed with separate

repeated-measures, one-factor ANOVAs followed by

planned comparisons between the saline and each muscimol

treatment using a Bonferroni adjustment to determine

significance.

Three different comparisons were made for each

structure in which Fos expression was quantified. In

Group I, a paired Student’s t test was used to evaluate

the effect of muscimol on Fos synthesis by comparing the

number of Fos-positive cells in the drug-injected side to

those in the same region of the saline-injected side.

Similarly, in Group II, a paired Student’s t test was used

to evaluate the effect of the saline injection by comparing

the number of Fos-positive cells in the saline-injected side

to those in the non-injected side. Finally, in order to

evaluate the effect intra-AcbSh muscimol injections have

on Fos synthesis in the contralateral side, an unpaired

Student’s t test was used to compare the mean number of

Fos-positive nuclei in the saline-injected side of Group I to

those in the saline-injected side of Group II. Because 13

brain regions were analyzed, the alpha level for rejection

of the null hypothesis was corrected to P < 0.003 using the

Bonferroni method.

T.R. Stratford / Brain Research 1048 (2005) 241–250244

3. Results

3.1. Verification of microinjector placement

In all rats, the microinjector tracks terminated in the

ventromedial AcbSh at placements similar to those illus-

trated in previous studies [41,42]. Fig. 1 incorporates a

schematic illustration of typical microinjector placements.

3.2. Feeding behavior

Unilateral injections of muscimol into the AcbSh

consistently elicited feeding in satiated rats (F(3,18) =

Fig. 1. Schematic illustration of representative injection sites. Group I is comprised

AcbSh on one side of the brain and saline into the opposite AcbSh. Group II is c

side of the brain and no injection into the opposite AcbSh. Data represent the

investigated structure. Labeled neurons were counted in single, matched sections t

(parenthetical anterior–posterior LH coordinates are given as millimeters from br

and NTS. Statistical analysis confirmed what was apparent from examining the

AcbSh exhibited one of three patterns of labeling. Fos synthesis in the LH was

immunoreactivity in the PVN, PT, LHab, SHy, and SN was increased significant

ipsilateral side. In contrast, Fos immunoreactivity in the Pir, SCh, Ce, and NTS

typeface indicates comparisons that differed significantly with asterisks indicating

significantly from counts on the saline-injected side of the brain, and daggers ind

significantly from counts on the saline-injected side of rats in Group II. Abbrevia

vagus, ac: anterior commissure, AH: anterior hypothalamic area, AM: anteromedia

of the stria terminalis, Ce: central amygdaloid nucleus, cp: cerebral peduncle, C

dorsal endopiriform nucleus, DMN: dorsomedial hypothalamic nucleus, f: fornix

intermediodorsal thalamic nucleus, IPN: interpeduncular nucleus, La: lateral am

nucleus, lo: lateral olfactory tract, MD: mediodorsal thalamic nucleus, MHb: m

nucleus, MnPO: median preoptic nucleus, mp: mammillary peduncle, NTS: nucleu

opt: optic tract, PeF: perifornical nucleus, Pir: piriform cortex, pm: principal mam

PT: paratenial thalamic nucleus, PVA: anterior paraventricular thalamic nucleus, P

thalamic nucleus, SHy: septohypothalamic nucleus, sm: stria medullaris, SNC: su

supraoptic nucleus, sol: solitary tract, st: stria terminalis, SuM: supramammillary

vtgx: ventral tegmental decussation, VTM: ventral tuberomammillary nucleus.

36.5, P < 0.0001). Comparisons of the effects of

individual doses with that of the vehicle treatment

showed that food intake was increased significantly (P <

0.03) by each dose of muscimol tested (Fig. 2). Water

intake was not affected by the treatment. Informal

observation of the rats showed that the unilateral injections

of muscimol did not elicit any gross motor asymmetries

during the tests.

3.3. Muscimol-induced Fos synthesis

The mean numbers of Fos-immunopositive cells

observed on each side of the brain in the Group I and

of rats that received simultaneous injections of muscimol (100 ng) into the

omprised of rats that received an injection of saline into the AcbSh on one

mean number of cells (TSEM) expressing Fos immunoreactivity in each

aken through four levels spaced along the anterior–posterior axis of the LH

egma) and through one level of the SHy, PT, PVN, LHb, SN, Pir, SCh, Ce,

sections—that brain regions activated by injections of muscimol into the

increased significantly only on the side ipsilateral to the injections. Fos

ly on both sides of the brain, but there was a much greater increase on the

was found in equivalent numbers of cells on both sides of the brain. Bold

Group I Fos counts on the muscimol-injected side of the brain that differed

icate Fos counts on the saline-injected side of rats in Group I that differed

tions in the figures: 3n: oculomotor nerve, 10: dorsal motor nucleus of the

l thalamic nucleus, AStr: amygdalostriatal transition area, BST: bed nucleus

Pu: caudate–putamen, cu: cuneate fasciculus, Cu: cuneate nucleus, Den:

, Gr: gracile nucleus, ic: internal capsule, IF: interfascicular nucleus, IMD:

ygdaloid nucleus, LH: lateral hypothalamic area, LHb: lateral habenular

edial habenular nucleus, ml: medial lemniscus, MM: medial mammillary

s of the solitary tract, NTSc: nucleus of the solitary tract, commissural part,

millary tract, PMD: dorsal premammillary nucleus, PN: paranigral nucleus,

VN: paraventricular hypothalamic nucleus, PVP: posterior paraventricular

bstantia nigra–pars compacta, SNR: substantia nigra–pars reticulata, SO:

nucleus, VMH: ventromedial hypothalamic nucleus, VP: ventral pallidum,

Fig. 2. Mean (TSEM). Food and water intake after unilateral injection of

various doses of muscimol (25–100 ng/500 nl) or vehicle into the AcbSh.

Unilateral muscimol dose-dependently increased food intake at each dose

tested but had no effect on 120 min water intake (*P < 0.05, .P < 0.001).

T.R. Stratford / Brain Research 1048 (2005) 241–250 245

Group II rats are presented in Fig. 1. Fos synthesis did

not differ significantly between the saline-injected side

and the non-injected side in any of the brain structures

examined in the Group II rats. In contrast, we observed a

Fig. 3. Photomicrographs showing Fos synthesis after injection of 100 ng muscim

different levels of the LH. Muscimol greatly increased the number of neurons ex

significantly change Fos immunoreactivity on the contralateral side. Scale bars: 50

show the major structures visible at each level and the anterior–posterior (AP) lev

large and consistent increase in Fos synthesis in the rats

that received unilateral muscimol injections. It should be

noted that no obvious circling or other asymmetric

locomotor behaviors were noted in any of the subjects.

Statistical analyses showed that three distinct patterns of

Fos expression occurred in these structures. Exclusively

ipsilateral labeling was indicated when the muscimol-

injected side of the Group I rats showed significantly

more Fos immunoreactivity than the saline-injected side,

and the saline-injected side of the Group I rats did not

differ significantly from the saline-injected side of the

Group II rats. This pattern suggests that the drug

increased Fos synthesis ipsilaterally, while synthesis in

the saline-injected side remained at baseline levels.

Primarily, ipsilateral labeling was indicated when the

muscimol-injected side of Group I rats showed signifi-

cantly more Fos synthesis than the saline-injected side,

and the saline-injected side of Group I showed a

significant increase in Fos synthesis compared to the

saline-injected side of Group II. This pattern indicates

that the drug had its largest effect ipsilaterally but that

ol into the right AcbSh (as depicted) and saline into the left AcbSh at four

pressing Fos on the side ipsilateral to the muscimol injection but did not

0 Am. In this figure and Figs. 4–6, the accompanying schematic diagrams

el coordinates relative to bregma (modified from Paxinos and Watson [35]).

Fig. 4. Photomicrographs showing Fos synthesis after injection of 100 ng

muscimol into the right AcbSh and saline into the left AcbSh. Fos synthesis

was increased on both sides of the brain in the (a) PT, (b) PVN, and (c)

LHb, however, a much larger, and highly significant ( P < 0.001), increase

was seen on the side ipsilateral to the injection as compared to the

contralateral side. Scale bars: 500 Am.

T.R. Stratford / Brain Research 1048 (2005) 241–250246

there was also a significant degree of contralateral

activation. Bilateral labeling was indicated when the

saline-injected side of Group I rats did not differ from

Fig. 5. Photomicrographs showing Fos synthesis after injection of 100 ng muscim

SN. Again, although Fos synthesis was increased on both sides of the brain, a mu

ipsilateral to the injection as compared to the contralateral side. Scale bars: 500 A

the muscimol-injected side but did show significantly

more Fos synthesis than the saline-injected side of Group

II. This pattern represents a significant and equivalent

increase in Fos synthesis in both sides of the rats injected

with muscimol.

3.3.1. Structures demonstrating exclusively ipsilateral

activation

The LH was the only structure in which the muscimol-

elicited increase in Fos synthesis was exclusively ipsilateral.

A very large increase in the number of cells exhibiting Fos

immunoreactivity was observed throughout the entire

rostrocaudal extent of the LH in the side ipsilateral to the

drug injection, with little Fos immunoreactivity seen on the

saline-injected side (Figs. 1 and 3).

3.3.2. Structures demonstrating primarily ipsilateral

activation

Five of the heavily labeled brain regions exhibited a

pattern of primarily ipsilateral Fos synthesis (Fig. 1). These

included the paratenial thalamic nucleus (PT; Fig. 4a), PVN

(Fig. 4b), lateral habenula (LHb; Fig. 4c), septohypothala-

mic nucleus (SHy; Fig. 5a), and medial substantia nigra

(SN; Fig. 5b). Smaller, primarily ipsilateral, increases in Fos

synthesis were noted in other brain regions including the

bed nucleus of the stria terminalis, medial ventral pallidum,

arcuate hypothalamic nucleus (Arc), and ventral tegmental

area.

3.3.3. Structures demonstrating bilateral activation

Fos immunoreactivity was significantly increased bilat-

erally in four of the structures examined (Fig. 1): the

piriform cortex (Pir; Fig. 6a), suprachiasmatic nucleus

(SCh), central nucleus of the amygdala (Ce; Fig. 6b), and

the NTS (Fig. 6c). Less consistent bilateral increases in Fos

ol into the right AcbSh and saline into the left AcbSh in the (a) SHy and (b)

ch larger, and highly significant ( P < 0.001), increase was seen on the side

m.

Fig. 6. Photomicrographs showing Fos synthesis after injection of 100 ng muscimol into the right AcbSh and saline into the left AcbSh demonstrating a large

bilateral increase in Fos synthesis in the (a) Pir, (b) Ce, and (c) NTS. Scale bars: (a) 1 mm, (b) 250 Am, (c) 250 Am.

T.R. Stratford / Brain Research 1048 (2005) 241–250 247

immunoreactivity were seen in the supraoptic and tuber-

omammillary nuclei.

4. Discussion

Bilateral injections of muscimol into the AcbSh elicit a

robust, behaviorally specific increase in food intake in

satiated rats [41]. In the present study, unilateral injections

of muscimol into the AcbSh also reliably induced feeding

without eliciting any apparent asymmetric motor impair-

ments (Fig. 2). Furthermore, similar unilateral injections of

muscimol activate populations of neurons throughout the

brain in several distinct patterns based on the laterality of

Fos synthesis in those structures. When Fos is synthesized

in a brain region in response to a unilateral stimulus, three

broad patterns of expression are possible. Fos synthesis may

be increased exclusively on one side of the brain, increased

equally on both sides of the brain, or may be increased on

both sides of the brain, but in significantly greater amounts

on one side. Examples of each pattern were found in the

brains of rats receiving unilateral injections of muscimol

into the AcbSh. A significant increase in Fos synthesis in

both sides of the brain might represent secondary activation

by a systemically acting factor, such as a change in blood

pressure, gastrointestinal state, or the circulating levels of

some hormone or physiologically relevant compound such

as glucose or sodium. Of course, unilateral activation could

result under these conditions if structures on one side of the

brain were preferentially sensitive to such changes. How-

ever, in that case, one would expect the unilateral labeling to

be located consistently on the sensitive side of the brain

regardless of which side received the injection of muscimol.

In the current study, the injection side was varied between

rats, and all structures that displayed unilateral increases in

Fos synthesis showed the largest increase on the side

ipsilateral to the drug injection. This pattern effectively

precludes a primary role for systemic intermediaries in the

downstream activation of those brain regions, suggesting the

existence of a neurally linked network. It should be noted

that rats in the present study did not have access to food, so

increases in Fos synthesis cannot be attributed to the effects

of ingestion. Furthermore, Fos synthesis was evaluated in

rats after their first exposure to muscimol in order to

minimize the possibility that the results might be compli-

cated by expectation or conditioning effects.

The present study confirms and extends our previous

finding that injections of muscimol into the AcbSh increase

Fos synthesis in neurons throughout the entire rostrocaudal

extent of the LH [42]. In the current study of unilateral

injections, LH Fos expression increased exclusively on the

drug-injected side of the brain (Figs. 1 and 3). These

findings strongly suggest that the ability of AcbSh injections

to activate neurons in the LH is mediated through uncrossed

neural pathways linking the two structures rather than

through alterations in behavior or in circulating factors to

T.R. Stratford / Brain Research 1048 (2005) 241–250248

which the LH is known to be sensitive. The potential

importance of these connections in mediating the influence

of the AcbSh on feeding is supported by the observation that

local injections of a NMDA receptor antagonist into the

perifornical region of the LH potently suppress AcbSh-

mediated feeding behavior [42]. Two recent investigations

found that a subset of the LH neurons activated by AcbSh

muscimol contain the orexigenic peptide orexin [3,55].

Interestingly, Zheng and colleagues (2003) reported that

AcbSh muscimol injections induced an increase in Fos

expression in the vicinity of NPY-containing neurons in the

Arc, another nucleus in which we noted a unilateral increase

in Fos synthesis. Arcuate NPY neurons contain orexin

receptors [1], receive projections from LH orexin neurons

[23], and express Fos in response to ICV administration of

orexin [52]. Furthermore, ICVorexin elicits feeding, at least

in part, through actions on an NPY system [25,52]. In

addition, we have recently discovered that ICV injections of

NPY Y1 or Y5 antagonists potently suppress AcbSh-

mediated feeding [43]. Together, these data suggest the

possibility that AcbSh-mediated feeding is effected, at least

in part, through an activation of LH orexin neurons which,

in turn, activate NPY neurons in the Arc. It is important to

note, however, that the neurochemical phenotype of the vast

majority of activated LH cells remains unknown, and other

neurotransmitter systems undoubtedly participate in the

expression of AcbSh-mediated feeding.

Several other brain regions exhibited a pattern of

primarily unilateral Fos synthesis, suggesting that, like the

LH, they too are components of a lateralized AcbSh circuit.

The PT, PVN, LHb (Fig. 4), SHy, and SN (Fig. 5) all

showed large, consistent increases in Fos synthesis ipsi-

lateral to the muscimol injection, although each also showed

a smaller, but significant, increase in the contralateral side as

well (Fig. 1). Of these brain regions, only the PVN has been

shown to play a well-defined role in the control of food

intake [13,40]. Neurons in the PVN are activated by

orexigenic conditions such as hypoglycemia [8,34] and

ICV injections of ghrelin [30] but also by increased levels of

hormones that reduce food intake [6,11,17,47]. PVN

neurons are also activated in response to a variety of

noxious or anxiogenic stimuli [10,38], as are cells in both

the SHy [4,9] and the LHb [10,51]. However, all of these

conditions induce Fos equally on both sides of the brain,

while AcbSh muscimol increases Fos synthesis to a much

larger degree in the ipsilateral PVN. This surprising result

suggests that this response must be mediated largely through

uncrossed neural pathways originating in the AcbSh. The

AcbSh does not appear to project directly to the PVN but

can potentially influence it through the LH, which sends

projections to the medial hypothalamus. Little is known

about the function of the PT or the small, circumscribed

region of the medial SN that expresses Fos after AcbSh

inhibition, however, the latter does receive a monosynaptic

projection from the AcbSh [21,54]. Further investigations

will be necessary to determine whether these regions

mediate the influence of the AcbSh on feeding or other

behaviors.

Four nuclei, the Pir, SCh, Ce, and NTS (Fig. 6) showed an

equivalent increase in Fos expression on both sides of the

brain following unilateral muscimol injections into the

AcbSh (Fig. 1). The design of the current study does not

allow for strong conclusions to be reached about the origin of

these effects. Projections arising in the AcbSh itself are

almost entirely ipsilateral, but structures several synapses

removed from the AcbSh might well give rise to projections

with a contralateral component. For example, the PVN sends

a bilateral projection to the NTS [22,45,49], which may

utilize the orexigenic peptide galanin [28], and, thus, be

capable of influencing the firing rate of NTS neurons on both

sides of the brain. There is also some evidence that certain LH

neurons send bifurcating projections to the NTS [12,24],

suggesting that an LH-NTS projection that bypasses the

medial hypothalamus could also be involved in AcbSh-

mediated NTS activity. Therefore, it is possible that Fos

expression in bilaterally labeled regions might reflect

activation of crossed neuronal pathways. Alternatively, Fos

expression in these structures might be mediated through one

of the behavioral or circulating intermediates to which they

have been shown to be sensitive [6,8,14,18,29,30,38,47]. It

should be noted, however, that only weak Fos expression was

seen in the PVN opposite to the side of the muscimol

injection, even though this structure is sensitive to many of

the same stimuli as are the structures showing bilaterally

symmetrical labeling. While each of these four regions

appears to play some role in the control of food intake

[15,26,46,48], further work will be required to show whether

they are involved in the effects of muscimol injections on

feeding or other behaviors.

In conclusion, the present study demonstrates that

unilateral injections of muscimol into the AcbSh signifi-

cantly increase Fos synthesis in the ipsilateral, but not

contralateral, LH. This result strongly suggests that neurons

in the LH are activated as part of a functional neural circuit

and not in response to an AcbSh-mediated change in

circulating levels of orexigenic or satiety factors. The

unilateral nature of LH activation, combined with our

previous observation that blocking NMDA receptors in

the perifornical LH completely suppresses AcbSh-mediated

feeding, reinforces the idea that the AcbSh controls food

intake through a neural mediation of activity in NMDA

receptor-bearing LH neurons, although evidence suggests

that the circuit may be polysynaptic [42]. These injections

also resulted in a primarily ipsilateral activation of the SHy,

PT, PVN, LHb, and SN, suggesting that these structures

may be downstream components of a lateralized neural

circuit controlled by the AcbSh. Taken together, the data

indicate that systemic intermediaries such as hypoglycemia,

hypotension, or changes in circulating levels of other

orexigenic or satiety factors are not involved in the increase

in Fos synthesis seen in the LH, PVN, and other unilaterally

activated brain regions. Furthermore, based on the primarily

T.R. Stratford / Brain Research 1048 (2005) 241–250 249

unilateral activation of the PVN, none of these conditions

appear to be induced in the rats by intra-AcbSh muscimol

injections and, thus, it also appears unlikely that these

conditions play a significant role in the activation of the

bilaterally labeled structures. However, these data do not

preclude the possibility that such systemic changes are an

upstream component of this system and that the firing rate

of AcbSh neurons themselves are mediated by these factors.

It is possible that multiple systemic orexigens initiate

feeding by inhibiting neurons in the AcbSh which, in turn,

activates neurons in the LH, Arc, and PVN.

Acknowledgments

The author would like to thank Dr. David Wirtshafter for

his many insightful observations concerning the data and for

his critical review of the manuscript. This research was

supported by NIH grant NS33992.

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