Neuropeptide Y in hamster limbic nuclei: Lack of colocalization with substance P

Peptides, Vol. 17, No. 6, pp. 1053-1062. 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved

0196.9781/96 $15.00 + .OO

ELSEVIER PII SO196-9781(96)00130-l

Neuropeptide Y in Hamster Limbic Nuclei: Lack of Colocalization With Substance P

LANCE F. BURROUGHS,* JEANNIE M. FIBER? AND JENNIFER M. SWANN”’

*Department of Biological Sciences and flnstitute of Animal Behavior, Rutgers University, Newark, NJ 07102, USA

Received 22 May 1995

BURROUGHS, L. F., J. M. FIBER AND J. M. SWANN. Neuropeptide Y in hamster limbic nuclei: Lack of colocalization with substance P. PEPTIDES 17(6) 1053-1062,1996.-The medial nucleus of the amygdala (Me), bed nucleus of the stria terminalis (BNST), and medial preoptic area (MPOA) regulate copulation in the male hamster. The present study identified neuropeptide Y-immunoreactive (NPY-IR) neurons in the BNST and Me with the greatest concentration in the posteromedial and posterior- dorsal subdivisions of these nuclei, respectively. NPY-IR fibers are found in all three nuclei with dense plexi of NPY-IR varicosities in the most medial subdivisions. Substance P neurons are also densely concentrated in the posterior BNST and Me; however, no neurons contained both peptides. Thus, NPY and substance P neurons comprise two distinct populations within the BNST and Me of the hamster.

NPY Tachykinin Mating behavior

Medial preoptic nucleus Bed nucleus of the stria terminalis Medial nucleus of the amygdala

ONE of the fundamental goals in the study of neuroscience is to identify the neuronal components involved in the regulation of behavior. Identification of the neuronal cell groups, the pathways that connect them, and the neurotransmitters, modulators, and receptors that regulate a particular function often establishes ba- sic principles of organization that generalize to other systems. The vomeronasal pathway of the male Syrian hamster has emerged as an excellent model in studying the neuronal regulation of copulation. This pathway processes pheromonal cues from the female that are essential for the initiation of copulatory behavior. Receptors in the vomeronasal organ and olfactory mu- cosa project to the accessory and main olfactory bulbs, respectively. Destruction of thse olfactory bulbs abolishes mating in male hamsters ( 18,21,34). Output neurons in the AOB project to the medial nucleus of he amygdala (Me) and bed nucleus of the stria terminalis (BNS’T) (30). The Me projects to the BNST via the stria terminalis and a ventral pathway ( 14,16). The BNST and the Me both project to the MPOA (22,3 1) . The integrity of this system is critical for normal male mating behavior. Destruc- tion of the Me, BNST, or MPOA or the pathways that connect them severely disrupts or completely abolishes mating behavior in the male hamster (1726).

Although the neural circuitry of this pathway has been described in detail, the neurotransmitters/neuromodulators that mediate copulation are not completely known. Recent studies suggest that neuropeptide Y (NPY) , a putative peptide neuromodulator ( 1 ), may play a role in the regulation of copulation. Intraventricular injections of NPY decrease mating behavior in male rats (5). Decrements in sexual performance are associated with a decline in NPY concentration in the hypothalamus of cas-

trated (29), hypertensive (6), or aged (4) rats. NPY has been localized within the hypothalamus, amygdala, and medial preoptic area in a variety of mammalian species (3,13,27,32,33). Since the publication of these studies these nuclei have been further divided into subdivisions which maintain separate connections and functions. The goal of this study was to determine if NPY is found within the neurons and fibers of the subdivisions of the BNST, MPOA, and Me that regulate mating behavior in the hamster.

Neurons in the BNST, MPOA, and Me of the hamster also contain substance P (23), a tachykinin neuromodulator. Sub- stance P may also play a role in the regulation of mating behavior. Injections of this peptide facilitate mating behavior in the male rat (8 ) . Because both NPY and substance P are localized within the same nuclei and both are implicated in the regulation of male copulatory behavior, we determined whether neurons within this pathway contain both neuropeptides using double label immunofluorescence.

ABBREVIATIONS

AC, anterior commissure; ADPN, anterodorsal preoptic nucleus; AHA, anterior hippocampal area; AVPN, anteroventral preoptic nucleus; Bla, basolateral amygdaloid nucleus, anterior subdivision; BLp, basolateral amygdaloid nucleus, posterior subdivision; BM, basomedial amygdaloid nucleus; BNSTal, bed nucleus of the stria terminalis, anterolateral subdivision; BNSTam, bed nucleus of the stria terminalis, anteromedial subdivision; BNSTav, bed nucleus of the stria terminalis, anteroventral subdivision; BNSTpi, bed nucleus of the stria terminalis, posteroin-

’ Kequests for reprints should be addressed to Jennifer Swann, Department of Biological Sciences, 111 Research Drive, Bethlehem, PA 18012,

1053

1054 BURROUGHS, FIBER AND SWANN

termediate subdivision; BNSTpl, bed nucleus of the stria terminalis, posterolateral subdivision; BNSTpm, bed nucleus of the stria terminalis, posteromedial subdivision; ACo, anterior cortical amygdaloid nucleus; PLCo, posterolateral cortical amygdaloid nucleus; Ce, central amygdaloid nucleus; ENDO, endopiriform nucleus; F, fornix; GP, globus pallidus; HDB, nucleus of the horizontal limb of the diagonal band; La, lateral amygdaloid nucleus; LPOA, lateral preoptic area; LS, lateral septal nucleus; LSv, lateral septal nucleus, ventral subdivision; Me, medial amygdaloid nucleus; MeA, medial amygdaloid nucleus, anterior subdivision; MeP, medial amygdaloid nucleus, posterior subdivision; MCPO, magnocellular preoptic nucleus; MI, massa intercalata; MPN, medial preoptic nucleus; MPNmag, medial preoptic nucleus, magnocellular; MPOA, medial preoptic area; OC, optic chiasm; OT, optic tract; OVLT, vascular organ of the lamina terminalis; PVN, paraventricular hypothalamic nucleus; SHY, septohypothalamic nucleus; SM, stria medulla&; ST, stria terminalis; TH, thalamus; TU, olfactory tubercle; VP, ventral pallidum; Pir, piriform cortex.

METHOD

Animals and Procedures

Eight male adult Syrian hamsters (Mesocricetus auratus) obtained from Charles River (Kingston, NY) were used for this study. Animals were group housed on a 14-h light/lo-h dark schedule with food and water available ad lib. Four of the hamsters were anesthetized with sodium pentobarbital (9.7 mg per 100 g body weight, IP) and pretreated with a unilateral injection of (320 mg/2 ~1) colchicine into the lateral ventricle 48 h before sacrifice to increase neuropeptide labeling within neuronal peri- karya. This step was necessary to visualize the peptide in the soma. Only NPY fibers are seen in the areas studied in the absence of colchicine treatment. At the close of the experiment all animals were sacrificed with an injection of a lethal dose ( 19.5 mg per 100 g body weight, IP) of sodium pentobarbital. All eight animals were perfused with 0.1% sodium nitrate, and fixed with either 4% paraformaldehyde or 2% pamformaldehyde and 2.5% benzoquinone in potassium phosphate buffer (0.01 M K2P04, pH 7.4). The brains were removed and postfixed for 6-12 h, and then placed in 17% sucrose solution for 24 h. Each brain was then frozen on a sliding microtome and cut into 40-pm sections. This procedure has been approved by the Institutional Review Board for the Use and Care of Animals at Rutgers University (protocol # 90-058).

Immunocytochemistry and Analysis

Every third section from each brain was immunolabeled using commercially prepared antisera raised in rabbit to NPY and obtained from Incstar Corporation. This antiserum has minimal crossreactivity with related structurally similar peptides: less than 0.1% cross-reactivity with peptide YY and less than 0.01% crossreactivity with APP ( 15). All antisera were diluted in potassium phosphate buffer (0.01 M K2P04, pH 7.4). Tissue was pretreated with 0.5% hydrogen peroxide for 20 min to eliminate endogenous peroxidase. Tissue was subsequently incubated in anti-NPY antisera (1:15,000 with 0.3% T&on-X) for 48-72 h at 4°C. The sections were then sequentially incubated in biotinylated anti- rabbit (raised in goat) from Vector Labs ( 1:400) or Jackson Labs ( 1:2500) for 1 h at room temperature, and Avidin-Biotin-HRP Complex from Vector Labs (1:200) for 1 h. The complex was visualized with diaminobenzidine (DAB) (0.0125%) in a solution containing 0.06% hydrogen peroxide, and 0.015% nickel

chloride. The sections were mounted on gelatin-coated slides, dehydrated, and coverslipped.

Labeled tissue was examined by eye for immunoreactive neurons and fibers. Immunolabeled neurons were identified by a dark brown to black reaction product that filled the cells and their processes and easily distinguished them from the background. Neither cells nor processes were labeled in the absence of the primary (data not shown). Neuronal size was determined using a reticule that contained a scale; the scale was calibrated with a stage micrometer. Neurons and fibers in representative sections were drawn by hand using a drawing tube and 10 times objective on a Nikon Labophot light microscope. Neurons were drawn from the side ipsilateral to the colchicine injection. Fibers were drawn from tissue obtained from non-colchicine-injected animals. The density of labeled cells in each subdivision of the BNST and Me was determined by counting the neurons in the side ipsilateral to the colchicine injection in one section through the subdivision in each of the four brains. The density of fiber labeling was computer generated from an image obtained with a video camera attached to a microscope and analyzed using the NIH Image program. The density of fiber labeling was calculated by subtracting the pixels of the nonspecific labeling from the pixels of the labeled image. The labeled image was 0.16 mm square through each subdivision of the BNST and Me. Nonspe- cific labeling in each section was obtained from a 0.16-mm square of unlabeled area in the same section. One section through each subdivision of each of four brains was analyzed. When drawings and analysis were completed, coverslips were removed and sections were counterstained with cresyl violet to identify conventional cell groups.

Double Labeling and Analysis

To determine the extent of colocalization, additional sections from the four colchicine-treated hamsters were processed for immunofluorescence for both NPY and substance P. The sections were incubated in potassium phosphate buffer (0.0 1 M K,PO,, pH 7.4) containing both anti-NPY raised in rabbit ( 1:1500) from Incstar Corp. and anti-substance P raised in rat from Accurate Scientific (1:500) for 48-72 h at 4°C. The sections were then incubated for 1 h in rhodamine-conjugated anti-rat IGg (1:50) raised in donkey, followed by incubation with fluorescein conjugated anti-rabbit IGg ( 1:50) raised in donkey (Jackson Labs) for 1 h at room temperature. The sections were mounted on gelatin-coated slides and coverslipped with a phosphate-buffered medium containing glycerol and phenylenediamine (25).

The extent of double labeling was determined by eye. One section through the BNSTpm and one section through the MeP were examined in each of four brains under a fluorescence microscope. The number of immunolabeled NPY and substance P cells was determined by counting these neurons in the side ipsilateral to the colchicine injection. Following this analysis coverslips were removed from some slides and the tissue was stained with cresyl violet to identify conventional cell groups. The total number of cells per area was determined by counting the number of cresyl violet-stained cells per a 0.16~mm square area in one section through the BNSTpm and one section through the MeP in each of four animals.

Areas and subdivisions within the MPOA were described using the nomenclature and cytoarchitecture of Maragos and Win- ans (20) that were adapted for the hamster from Simerly and Swanson (3 1). Areas and subdivision of the BNST and Me were described using the nomenclature and cytoarchitecture of Gomez and Newman (11).

NPY IN HAMSTER LIMBIC AREAS 1055

C I

GP

FIG. 1. Distribution of NPY-IR cells drawn from representative sections from colchicine-treated hamsters at four levels through the BNST and preoptic area. Levels are from rostra1 (A) to caudal (D). Open circles represent l-5 neurons and closed circles represent 5- 10 neurons.

RESULTS

Distribution of NPY Cell::

BNST. Neuropeptide Y immunoreactive (NPY-IR) neurons are distributed in all subdivisions of the BNST except the posterior lateral (Fig. 1) . The number of cells is almost evenly divided between the anterior and posterior subdivisions. The posterior medial subdivision contains the greatest concentration of

labeled neurons in the BNST (Table 1). Within the anterior BNST NPY-IR neurons are lo- 12 pm in diameter and oval to fusiform in shape with one, or occasionally two, visible processes. In the anterior medial subdivision the labeled neurons are located just under the vemricle or clustered just above the anterior commissure [Fig. 3 (A)].

The posterior medial IBNST is densely populated with NPY- IR cells [Fig. 1 (B-D)]. These neurons are lo- 14 pm, fusiform to ellipsoid in shape, contain one to three processes, and are ori-

TABLE 1

NUMBER OF NW-IR CELLS IN THE SUBDIVISIONS OF THE BNST AND Me

Area NPY-IR Cells (per 0.16 md)

BNSTam 86.75 ? 7.5 BNSTav 42.75 5 5.8

BNSTal 78.0 + 14.0 BNSTpm 193.5 2 27.6%

BNSTpi 63.5 k 11.8

MeA 9.8 IT 4.9

MeP 13.5 2 6.8t

Values are 2 SE. * Greater thzln all other groups in BNST,

p < 0.0001. t Greater than the MeA, p < 0.0001.

ented with their long axes parallel to the long axis of the subdivision. The posterior intermediate subdivision contains far fewer NPY-IR cells than the posteromedial subdivision [Fig. 1 (B- D)]. These cells are similar in shape and size to those in the medial subdivision [Fig. 3 (C)l. Unlike those of the medial subdivision, these neurons are oriented with their long axes perpen-

FIG. 2. Schematic representation of sections through the anterior (A) and posterior (B) subdivisions of the BNST and the posterior (C) subdivision of the medial nucleus of the amygdala. Squares indicate the position of photographs in Figs. 3 and 6.

1056 BURROUGHS. FIBER AND SWANN

FIG. 3. Photomicrographs of NPY-IR neurons through the BNST (A-C) and medial nucleus of the amygdala (D). Note that in both the anterior (A) and posterior (B. C) subdivisions the medial subdivision (M) contains more fibers and varicosities than the more lateral subdivisions (L. Int). (D) Section through posterior medial nucleus of the amygdala (Me). Note: cells closest to the optic tract (OT) are organized with their long axes parallel to it. Cells further away (lower left hand corner) are more randomly arranged.

dicular to the long axis of the nucleus and the incoming fibers of the stria terminalis. The posterior lateral subdivision contains the lowest concentration of immunoreactive cells. These neurons are lightly stained, fusiform in shape, and oriented with their long axes pointed in a dorsoventral direction.

MPOA. Few NPY-IR neurons are found in the medial preoptic area (Fig. 1) . These cells are just ventral to the medial and intermediate BNST [Fig. l(B)]. Two or three neurons are located within the MPN magnocellular [Fig. 1 (C)l. No NPY-IR cells are found in the medial aspect of the MPN or the rest of the MPOA at any level.

Other areas in the forebrain that contain NPY-IR cells are: lateral septum, medial septum, ventral pallidum, olfactory tubercle, magnocellular preoptic area, lateral preoptic area, and horizontal limb of the diagonal band of Broca.

Amygdala. NPY neurons are scattered throughout the amygdaloid complex. Examination by eye suggests that the highest concentrations are found in the more lateral subdivisions of the amygdala and the olfactory cortex (Fig. 4). The medial nucleus contains a moderate concentration of NPY-IR neurons ranging from round to fusiform in shape. These neurons are almost 10 times more concentrated in the posterior than in the anterior subdivision (Table 1) . Within the anterior subdivision, NPY-IR neurons have one to two processes and no particular orientation. Two different cell types are found in the posterior dorsal subdivision: fusiform neurons that are oriented parallel to the fibers of the stria terminalis and round neurons with processes that are oriented perpendicular to the stria terminalis. Neurons in the posterior ventral subdivision, like those in the anterior subdivisions, are lightly stained and sparsely distributed with no discernible orientation [Fig. 3 (D)]

Other nuclei of the amygdala that contain NPY-IR cells are: lateral, basolateral, central, massa intercalata, anterior cortical, endopiriform, basomedial, posterior lateral. NPY-IR was also found in the primary olfactory cortex.

Distribution of NPY Fibers

BNST, MPOA, and Me are densely innervated with NPY-IR fibers. These fibers appeared as short branched [Fig. 6(A)] or long, beaded, and covered with punctate-immunoreactive material [Fig. 6(B )] . In addition, patches of punctate-immunoreactive material are seen in several areas. These may be fibers cut in cross section or terminal nerve endings-we refer to them as varicosities.

BNST. NPY-IR fibers are found in all subdivisions of the BNST (Fig. 5). Unlike the labeled cells, which are equally distributed between the anterior and posterior regions, the concentration of labeled fibers is greatest in the anterior subdivisions. Within the anterior subdivisions the lateral subdivision is the most densly innervated. Within the posterior subdivisions the medial subdivision is the most densely innervated (Table 2).

The anterior medial subdivision is innervated with long, beaded, unbranched fibers that course diagonally from the lateral ventricle to the medial edge of the anterior limb of the anterior commissure [Fig. 3(A)]. Fibers in the anterior lateral subdivision are branched. These fibers are concentrated on the medial and lateral edges of this subdivision [Fig. 3(A)]. Fibers on the medial edge form two clusters. One cluster lies just under the lateral ventricle; the other lies just dorsal to the anterior commissure. The ventral subdivision is densely innervated with NPY-IR varicosities and short, branched, densely beaded fibers.

NPY IN HAMSTER LI:MBIC AREAS 1057

FIG. 4. Distribution of NPY-IR neurons drawn from representative sections of tissue from colchicine-treated animals through (A) anterior and (B) posterior subdivisions of the medial nucleus of the amygdala. Open circles represent l-5 neurons and closed circles represent 5- 10 neurons.

Long, unbranched NPY-IR fibers are seen in all subdivisions

of the post commissural BNST [Fig. 6 (A-B )] Within the posteromedial BNST (BNSTpm) these fibers begin in the stria terminalis and extend diagonally to enter the medial preoptic area and MPN. Immunoreactive varicosities are lightly distributed throughout the BNSTpm and are highly concentrated in a band along the stria medularis and fornix [Fig. 6(A)]. Posterior intermediate BNST (BNSTpi) contains far fewer immunoreactive fibers than the BNSTpm [Fig. 6(B)]. These fibers are long oc-

C \ I

Tal

casionally beaded and unbranched. The majority of these fibers are oriented parallel to the stria terminalis with an occasional perpendicular fiber. Posterior lateral BNST contains short, branched, densely beaded fibers, and a dense patch of immunoreactive varicosities [Fig. 6 (B )] .

MPOA. In the most rostra1 levels a medium concentration of long, unbranched, beaded, immunoreactive fibers extends ven- trally from the ventral subdivision of the ADPN through the MPOA, into the anterior ventral preoptic nucleus (AVPN). At

B

D

FIG. 5. Distribution of NPY-IR fibers drawn from representative sections from untreated hamsters at four levels through the BNST and preoptic area. Levels are from rostra1 (A) to caudal (D).

10.58 BURROUGHS, FIBER AND SWANN

TABLE 2

DENSITY OF NPY-IR FIBER LABELING IN

THE SUBDIVISIONS OF THE BNST AND Me

NPY-IR Fibers (per 0.16 mm’)

BNSTpm

BNSTpi

BNSTpl

MeA

MeP

22.4 2 3.8*

11.5 + 3.5

4.4 2 2.2

4.9 k 1.8

15.4 k 2.q.

Values are k SE. * Greater than all other groups in BNST,

p < 0.05. t Greater than the MeA, p < 0.02.

more caudal levels these fibers extend from the BNST through the MPOA, terminating along the third ventricle in the MPN. A dense population of short, branched fibers and immunoreactive varicosities was found along the third ventricle throughout the MPOA [Fig. 6(D)] . The MPN mag was only lightly innervated with fibers. These fibers did not enter the MPN mag but appeared to encircle it [Fig. 5(C)].

Amygdala. The amygdala and neighboring cortex are densely innervated with NPY-IR fibers throughout their rostra]-caudal extent. It should be noted that the innervation is greater in the surrounding cortical region than any region within the amygdala [Fig. 7(A-B)]. Within the medial nucleus of the amygdala, the posterior subdivision is more densely innervated than the anterior subdivision (Table 2).

The anterior subdivision of the medial nucleus (MeA) and the anterior cortical nucleus ( ACo) receive the lightest innervation in the amygdala. These areas are lightly innervated with long, unbranched, beaded fibers generally oriented towards the ventral surface. An occasional immunoreactive fiber can be seen extending through the ventral region into the molecular layer. NPY-IR varicosities were moderately concentrated within the molecular layer and contained a few short, densely beaded fibers.

In contrast to the MeA, the posterior medial nucleus (MeP) is densely innervated. These long, unbranched, beaded fibers originate within the stria terminalis and terminate just above the molecular layer within the nucleus. A high concentration of immunoreactive varicosities extends in a narrow band along the optic tract [Fig. 6 (C )]

A medium concentration of short, branched, densely beaded fibers is distributed in the intra-amygdaloid BNST, just lateral to the medial nucleus [Fig. 7(A)]. Primary olfactory cortex is densely innervated with long. unbranched fibers throughout its rostral-caudal extent. The central, basolateral, and lateral nuclei are lightly innervated with fibers of passage.

Other areas containing NPY-IR fibers were the lateral septum, lateral preoptic area, thalamus and the hypothalamic paraventricular nucleus (PVN) . The thalamus and PVN also contain varicosities.

Double Labeling

In the present study, incubation with both antibodies successfully labeled both NPY-IR and substance P immunoreactive (SP- IR) cells within the BNST and MeP. Our results indicate that these peptides are differentially distributed within these areas. SP-IR-neurons are distributed in two clusters, one in the dorsal

FIG. 6. Photomicrograph of NPY-IR fibers in posterior BNST (A, B), posterior medial nucleus of the amygdala (C), and medial preoptic area (D). Arrows indicate: (A) terminal plexus in posterior medial BNST (M); (B) long beaded fibers in posterior intermediate BNST (Int), (C) terminal field along the optic tract (OT), and (D) dense terminal plexus along the third ventricle (3).

NPY IN HAMSTER LIMBIC AREAS

FIG. 7. Distribution of NPY-IR fibers drawn from representative sections of untreated tissue through (A) anterior and (B) posterior subdivisions of the medial nucleus of the amygdala.

and one in the ventral aspects of the BNSTpm whereas NPY-IR cells are more evenly distributed in a band that extends more laterally than those contaning substance P (Figs. 8 and 9). A similar pattern of distribution is found in MeP. SP-IR cells were arranged in clusters along the optic tract, with the greatest concentration within the dorsal subdivision of this nucleus. NPY-IR cells are more sparsely distributed throughout the ventral and dorsal subdivisions of this, nucleus (Figs. 9 and 10).

SP-IR cells extend in a band through the lateral part of the MPOA. There are no NPY-IR cells in the MPOA.

BNSTpm and MeP contain the same number of NPY- and

SP-IR cells, as shown in Table 3. The percentage of the total neurons labeled for either peptide is similar for both peptides in both areas. NPY and substance P each represent only 7-10% of the total cell population. Only three neurons out of all the neurons counted in both areas contained both NPY and SP.

DISCUSSION

The results of the present study indicate that NPY-immunoreactive cells and fibers are found in all subdivisions of BNST

0 =NPY t = Substance P

FIG. 8. Schematic representation and distribution of NPY and substance P in the BNSTpm. Note that although the cells are intermingled the peptides are not colocalized.

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and Me of the male Syrian hamster with high concentrations in the posteromedial and posterior dorsal subdivisions, respectively. The MPOA, in contrast, contains few immunoreactive neurons. All three areas are densely innervated with fibers and varicosities. Fibers within the BNSTpm, BNSTpi and MeP appear to originate within the stria terminalis, suggesting that NPY may be contained within the neurons that connect these areas. In the rat, NPY fibers in the stria appear to terminate in the BNSTam and the dorsal subdivision of the BNSTpm (2,13 ) Destruction of the stria terminalis or the medial nucleus of the amygdala in this species eliminates NPY fibers just under the lateral ventricles. This ap- proach, however, does not eliminate immunoreactivity within the MPOA or more ventral subdivisions of the BNST. Therefore, these plexi must arise from other sources. The medial nucleus of the amygdala also projects to the BNST via a ventral pathway (16). This pathway contained NPY fibers in the present study, suggesting that NPY neurons may project to the BNST via this route. The posterior medial BNST also receives projections from the central nucleus and intra-amygdaloid BNST ( 12)) areas that are dense with NPY neurons. Varicosities within the MPOA could arise from sources within the hypothalamus. In particular, the arcuate nucleus contains a high percentage of NPY neurons (data not shown) and sends a heavy projection to the MPOA (31) Alternatively, NPY varicosities in the MPOA could originate in the medulla. Neurons in the medulla contain NPY ( 10) and project to the MPOA (7). Knife cuts that eliminate projections from the brain stem decrease NPY levels in the MPOA (28). Further studies that combine tract tracers and immunocytochemistry are warranted to accurately identify the source of NPY terminal plexi in the BNST, Me, and MPOA.

In the present study we found a high concentration of NPY-IR neurons within the posterior subdivision of the BNST, a finding not reported in previous studies in this species (27). Preliminary studies in our lab using the same antisera as in this study successfully labeled cells in the anterior BNST but not in the posterior subdivisions. The commercially prepared antisera used in this study labeled neurons in all subdivisions of this nucleus. These findings suggests that differences between these studies may be due to the potency

1060 BURROUGHS. FIBER AND SWANN

FIG. 9. Photomicrographs of NPY labeled with fluorescein (left) and substance P labeled with rhodamine (right) in tissue immunolabeled for both peptides. Pictures (A) and (B) are through the BNSTpm in the area indicated in the box in Fig. 8. Pictures (C) and (D) are through the MeP in the area indicated in the box in Fig. 10. In all four photographs, the big arrows indicate the location of substance P neurons and the smaller arrows indicate NPY neurons.

of the antibodies. Neurons in the anterior BNST that reliably label with different antibodies may contain more NPY than those in the posterior subdivisions, preventing less potent antibodies from labeling neurons in the latter while still de- tecting neurons in the former subdivisions. Alternatively, these areas may contain different forms of NPY. To date only one peptide has been identified with a structure similar enough to cross-react with antibodies to NPY. This peptide, PPY, does not appear to be localized within neuronal cell groups of the limbic system (9,19.24). This does not rule out the possibility that novel, structurally similar peptides pres-

O=NPY + = Substance P

FIG. 10. Schematic representation of the distribution of NPY and substance P in the posterior medial nucleus of the amygdala.

ent within the anterior BNST are cross-reacting with these antibodies.

Both substance P and NPY are present in the neurons and/or fibers of the Me, BNST, and MPOA. We found little evidence of colocalization of these peptides within these areas. Our results indicate that these peptides represent different populations of neurons. This is of interest because these two peptides appear to play different roles in the regulation of mating behavior. Intra- cerebral injections of substance P facilitate mating behavior (S), whereas injections of NPY inhibit the behavior (29 ) . These peptides may influence behavior via different areas within the MPOA. Substance P stimulates mating behavior when injected into the lateral ventricles (8). These injections would most likely influence the BNST and MPNmag, which lie just ventral to the ventricles. Both areas play critical roles in the regulation of mating behavior. Lesions of the BNST or the MPNmag disrupt or eliminate copulation in male hamsters (26). Preliminary studies in our lab failed to find an effect of NPY on mating behavior when injected into the lateral ventricles of intact male hamsters. NPY has been shown to inhibit mating behavior in the male rat when injected into the third ventricle (5). NPY varicosities are densest along the periventricular region of the MPOA, supporting the hypothesis that this region is the endogenous site of action of NPY in the MPOA. Thus, NPY and substance P may play different roles in regulating copulation by acting on functionally distinct areas within the MPOA.

In summary, we have demonstrated NPY immunoreactivity in the neurons and fibers of the BNST, MPOA, and Me of the male golden hamster. Because these areas play a major role in the regulation of mating behavior of the male hamster, our

NPY IN HAMSTER LIMBIC AREAS 1061

TABLE 3

NUMBER OF NPY-IR AND SUBSTANCE P-IR CELLS IN THE BNSTpm AND MeP RELATIVE TO THE TOTAL NUMBER OF CELLSfAREA

NPY Cells % of Total Cells Containing Substance P Cells % of Total Cells Containing Ar<:a (n = 4) NPY (n = 4) Substance P

BNS’Tpm 83.3 ? 7.3 10.1 79.34 2 17.3 9.6

MeP 39.9 + 4.7 7.2 48.19 2 3.9 8.8

Neurons were counted from a 0.16 mm2 area indicated by the box in the schematic drawing of Figs. 8 and 10.

results suggests that NPY may also play a role in this behavior. We have also shown that substance P is not colocalized with NPY within the neurons, indicating that substance P- and

NPY-immunoreactive neurons comprise two separate popu-

lations of neurons within these interconnected areas of the

hamster limbic system.

ACKNOWLEDGEMENTS

This work was supported by grants from the NIGMS-MBRS-3 SO6 PHS GM08223-06S2 and the NICHHD BP0 R29 PHSHD28467 to J.M.S. and a Sigma Xi award to J.M.F. The authors wish to thank Jean Marie Gabriel-Sidhom and Hany Aziz for their technical assis- tance.

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Neuropeptide Y in hamster limbic nuclei: Lack of colocalization with substance P