In the ventromedial nucleus of the rat hypothalamus, GABA-immunolabeled neurons are abundant and are innervated by both enkephalin- and GABA-immunolabeled axon terminals

Brain Research 816 Ž1999. 58–67

Research report

In the ventromedial nucleus of the rat hypothalamus, GABA-immunolabeled neurons are abundant and are innervated by both enkephalin- and GABA-immunolabeled axon terminals Kathryn G. Commons a

a, )

, Lee-Ming Kow a , Teresa A. Milner b , Donald W. Pfaff

a

Laboratory of Neurobiology and BehaÕior, The Rockefeller UniÕersity Box 275, 1230 York AÕenue, New York, NY 10021, USA Department of Neurology and Neuroscience, Cornell UniÕersity Medical College, 411 E 69th St., New York, NY 10021, USA

b

Accepted 13 October 1998

Abstract Immunohistochemical-labeling for the neurochemicals g-aminobutyric acid ŽGABA. and enkephalin are abundant in the ventromedial nucleus of the hypothalamus ŽVMN.. In VMN, both GABA and enkephalin may function to regulate feeding behavior, as well as other hormone-controlled behaviors. Importantly, in several brain areas, enkephalin is often thought to modulate GABAergic neurotransmission. Therefore, we used dual-labeling immunohistochemistry with electron microscopic analysis to study the circuitry of neurons containing GABA- andror enkephalin-labeling within the VMN. Somato-dendritic profiles containing GABA-labeling were three fold more abundant than GABA-labeled axon terminals Ž117 soma or dendrites vs. 34 axons.. In addition, axon terminals containing GABA-labeling sometimes synapsed onto GABA-labeled somata or dendrites Ž25% or 9r34.. In contrast, under these conditions labeling for enkephalin was primarily restricted to axon terminals, which were very abundant throughout VMN. Enkephalin-containing terminals accounted for a large fraction Ž25% 23r92. of the axons in contact with GABA-labeled dendrites, although they also contacted unlabeled dendrites. These observations suggest that a population of VMN neurons are GABAergic. These may be either local circuit ‘interneurons’ or projection neurons. In addition, GABA-labeled VMN neurons may be regulated by either enkephalin or GABA. These morphologic observations provide the basis for disinhibitory mechanisms to function within the VMN. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Feeding; Lordosis; Opiate; Ultrastructure; Estrogen; Glucose; GAD

1. Introduction Accumulating evidence has established that GABAergic neurotransmission within the VMN is involved in regulating feeding behavior w2,15,18,19,22,23,30,31x. GABA agonists stimulate feeding behavior, and likewise GABA antagonists inhibit feeding when administered directly to the VMN w15,18x. In the VMN levels of endogenous GABA and the activity of the GABA synthetic enzyme, glutamate decarboxylase ŽGAD., are regulated by blood glucose levels: low blood glucose leads to an elevation of GABA release w2,4,16x and an increase in GAD activity, while high blood glucose has the opposite effects w16x. Moreover, in several hyperphagic models, GAD activity is enhanced and GABA levels are heightened in the VMN w3,35x.

)

Corresponding author

Although GABA appears to play an important functional role in VMN, several observations have contributed to the impression that only a few VMN neurons themselves use GABA as a neurotransmitter. First, electrophysiological studies have shown that increased activity of neurons of the VMN correlates with excitation in efferent brain areas w1x suggesting VMN neurons primarily use excitatory neurotransmitters. Secondly, GABA-immunolabeling in VMN, although intense, is very diffuse and darkly GABA-labeled cell bodies are not commonly found w32,37x. Thirdly, even though GAD enzymatic activity is high w38,40x, the expression of the GABA synthetic enzymes GAD-65 or GAD-67 is not detectable by in situ hybridization within VMN neurons w24x. Therefore, it is assumed that for the most part GABA-labeling in VMN pertains to GABAergic axon terminals. In several brain areas, GABAergic neurotransmission is modulated by opiate ligands w42x. The opioid peptide enkephalin is abundant throughout VMN and is produced

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 0 8 4 - 1

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within many VMN neurons w17x. In fact, enkephalin mRNA is elevated by estradiol w34x in a manner correlated with behavioral changes w20x. Opioid ligands are also known to modulate feeding behavior w13,14x. Systemic morphine administration causes an initial decrease in food intake and a subsequent marked increase in feeding in rats. These effects may be mediated in part in the VMN, since injection of the opioid antagonist naloxone in the VMN suppresses food intake of food-deprived rats w41x. These observations raise the possibility that there may be interactions between neurons containing GABA and enkephalin within the VMN. The aim of this study was two fold: first we sought to determine if GABA-labeling was restricted to axon terminals in VMN, or there were also GABA-labeled neurons within VMN. Second, we wished to determine if there was a relationship between enkephalin-containing axon terminals and GABA-labeled neuronal profiles. For this study we used dual-labeling immunohistochemistry for GABA and enkephalin with electron microscopic analysis. 2. Methods 2.1. Section preparation Five male Sprague–Dawley rats were anesthetized with sodium pentobarbital and perfused transcardially with Ž1. 10–15 ml of heparin Ž1000 unitsrml. in normal saline Ž0.9%., Ž2. 50 ml of 3.75% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4. ŽPB. and Ž3. 200 ml 2% paraformaldehyde in PB. The brains were removed, cut into a coronal blocks and stored in the last fixative for an additional 30 min. Sections Ž30–40 mm thick. were cut on a Vibrotome and incubated with 1% sodium borohydride in PB, and then with 0.5% bovine serum albumin ŽBSA. in 0.1 M Tris saline pH 7.6 ŽTS. before immunohistochemistry. 2.2. Antisera A rat GABA antiserum supplied by Dr. Andrew C. Towle ŽLab. of Molecular Neurobiology, Burke Rehabilitation Center 785 Mamaroneck Dr. White Plains, NY 10605. was used at a dilution of 1:1000 with immunogold–silver labeling. Previous studies have established the specificity of this antisera as follows: the immunoreactivity is not blocked by unconjugated GABA or 100 mM BSA conjugated glutamate, b-alanine, or taurine but is completely abolished by preincubation with 10 mM BSA conjugated GABA w21,28,29x. In addition, a polyclonal rabbit GABA antiserum obtained from Sigma was used at dilution of 1:35,000 for immunoperoxidase and 1:10,000 for immunogold–silver labeling. Previously we have shown labeling with this antiserum in the hippocampus is specific for GABA containing cell populations w6x. Enkephalin was labeled using a polyclonal antiserum against leu-enkephalin raised in rabbits, purchased from

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Immunotech, at a dilution of 1:40,000 with immunoperoxidase method of visualization. This antiserum has 50-fold greater sensitivity for leu-enkephalin over dynorphin peptides, and immunocytochemical pattern of labeling is distinct from that of dynorphin w5x. 2.3. Imunohistochemistry Tissue was dually labeled using the immunoperoxidase method to label the rabbit antisera for leu-enkephalin, and the immunogold–silver method to label the rat antisera for GABA w6x. Tissue sections were incubated in both primary antibody diluted in 0.1% BSA in TS overnight. For the immunoperoxidase-labeling, the sections were then incubated through Ž1. a 1:400 dilution of biotinylated goat anti-rabbit ŽVector Labs. for 30 min, Ž2. the peroxidase avidin complex ŽVectastain Elite kit, Vector Laboratories Burlingame, CA. at double the recommended dilution Ži.e., 1r2 the concentration. for 30 min and Ž3. 3,3X-diaminobenzindine ŽDAB. and H 2 O 2 for 6 min. Subsequently the tissue was processed for immunogold–silver labeling as follows. Non-specific binding sites were blocked with 0.1% gelatin and 0.8% BSA in PBS for 30 min, incubated with goat anti-rat IgG conjugated to 1 nm gold particles ŽGoldmark Biologicals. for 3 h at room temperature. Sections were rinsed in PBS, then 0.2 M sodium citrate buffer pH 7.4. Gold particles were enhanced by treatment with silver solution ŽInstenSE, Amersham. for 6 to 10 min. In separate experiments, tissue was singly labeled using the Sigma rabbit GABA antisera using the immunoperoxidase method. For light microscopic analysis, some sections were also singly labeled for leu-enkephalin using the immunoperoxidase method. For electron microscopy sections were fixed for 1 h in 2% osmium tetroxide in 0.1 M PB, then dehydrated and flat embedded in EMbed 812. Ultra thin Ž50 nm thick. sections were cut from mid rostro-caudal extent of the ventromedial nucleus and collected on copper grids and counterstained with uranyl acetate and Reynold’s lead citrate and examined on a Phillips CM10 or Joel electron microscope. 2.4. Ultrastructural analysis Thin sections cut from at least two vibrotome sections from each of three animals dually labeled for enkephalin and GABA were examined electron microscopically. Only ultrathin sections from the surface of the tissue were collected and examined since immunolabeling has a limited penetration through the vibrotome section. Within areas of those sections which exhibited detectable labeling, low magnification ‘fields’ were photographed which usually contained several labeled profiles. Only tissue that was dually labeled was used for final quantitative analysis. In addition, two vibrotome sections from one animal singly labeled for GABA using the rabbit antisera from Sigma were examined. This tissue was examined solely to deter-

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Fig. 1. ŽA. Immunoperoxidase-labeling for GABA in the VMN primarily consists of homogenous diffuse labeling, consistent with previous descriptions. Cell bodies vary from extremely lightly stained Žcurved arrows. to apparently unstained Žstraight arrows.. Punctate labeling is seen throughout the section. ŽB. Enkephalin-labeling in the VMN using immunoperoxidase consists of homogeneously distributed puncta Žsmall arrows. and occasionally labeled somata Žlarge arrows.. Bar s 50 mm.

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mine if the pattern of labeling for GABA was consistent between antisera. Photographs were subsequently analyzed using classification and descriptions of cellular elements based on the descriptions of Peters et. al. w33x. Profiles were considered

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GABA-labeled if they contained gold–silver particles at a density approximately 4 times greater then neighboring profiles of similar size. Small profiles such as dendritic spines or small caliber dendrites Ždiameter - 1 mm. were counted as labeled if they contained at least two gold

Fig. 2. This perikaryon contains numerous gold–silver particles for GABA-labeling. Two GABA-labeled axon terminals form symmetric synapse Žcurved arrows. on the cell body Žboxed region, shown at higher magnification in inset A.. This perikaryon also contains dense-core vesicles immunoperoxidaselabeled for enkephalin arrows Žsmaller boxed region, shown in inset B.. Main panel, bar s 0.85 mm, inset A and B, bar s 0.5 mm.

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particles and the surrounding neuropil contained goldlabeling at a density of 0–1 particlerm2 . Cell bodies containing GABA-labeling were sampled and analyzed independently since cell bodies were not encountered frequently within random ‘fields’. Only cells

containing high levels of GABA-labeling were photographed, and subsequently analyzed. Thus the percentage of all VMN cells containing GABA-labeling was not determined. Although gold–silver labeling for GABA sometimes appears nuclear for unknown reasons, cell bod-

Fig. 3. A field illustrating the high density of GABA-labeled dendrites observed. Three GABA-labeled ŽGABA. and several unlabeled ŽUL. dendritic profiles are visible. One GABA-labeled dendrite receives three synaptic contacts from unlabeled axon terminals in the plane of section Žarrows., while no visible synapses are formed by the other two terminals. Glial wrapping Ž). is visible on all three dendrites. Bar s 0.4 mm.

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ies were only considered labeled if gold–silver particles were present in the cytoplasm. Many cell bodies contained approximately 2 times higher density of GABA-labeling then the surrounding neuropil, presumably representing the enrichment of GABA within the somatic metabolic pool. Therefore, cell bodies were considered GABA-labeled only if they contained gold particles estimated to be at a density of at least 5 times greater then the surrounding neuropil. This sampling criterion was used to reduce the frequency of incorrectly identifying cells as GABA-labeled Žfalsepositives.. 3. Results 3.1. Light microscopy Immunohistochemistry with each of the GABA antisera using either the immunoperoxidase method or the immunogold–silver method resulted in equivalent patterns of labeling. The pattern and intensity of labeling was similar between the VMN and other hypothalamic nuclei such as the dorsomedial hypothalamus, and the arcuate nucleus. The neuropil had a granular appearance but also contained diffuse labeling throughout. Cell body labeling varied from very light to apparently unstained ŽFig. 1A.. To serve as a positive control, cortical areas in the same tissue section were analyzed. Cerebral cortex, in contrast to the hypothalamus, had a very distinct pattern of labeling with darkly labeled cell bodies and very little diffuse neuropil staining. These observations were consistent with previous descriptions of GABA-labeling both in the hypothalamus, and in cortical regions w28,29,32,37x. Enkephalin-labeling was less diffuse then GABA-labeling, but was also widespread with a discrete punctate appearance ŽFig. 1B.. Enkephalin-labeled puncta were dispersed evenly throughout VMN. Occasionally perikarya containing enkephalin-labeling were visible. The number of labeled perikarya appeared to vary between animals suggesting it was fixation-dependent. When visible, enkephalin-labeled perikarya were concentrated within the ventrolateral portion of VMN.

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neuropil ŽFig. 2.. Labeled cell bodies were not homogeneous either in size and shape, or amount of rough endoplasmic reticulum and Golgi apparatus; however, most had an invaginated nucleus. GABA-labeled perikarya usually received few Ž- 2. synaptic contacts in the plane of section sampled. In a few of these cases, GABA-labeled somata were contacted by GABA-labeled terminals Ž4 of 15 axon terminals contacting GABA-labeled soma.. In addition, a large fraction of the plasmalemmal surface of GABA-labeled cell bodies was covered with astrocytic processes. 3.4. GABA-labeled dendrites Numerous GABA-labeled dendritic profiles were observed, Ž106 sampled, Fig. 3., and they varied widely in size Ž1–7 mm diameter.. Labeled dendritic profiles typically appeared aspiny in the plane of section sampled. Synapses onto GABA dendrites were more often symmetric Žinhibitory-type. than asymmetric Žor excitatory-type. Ž19 asymmetric, 31 symmetric, 42 appositions.. A fraction of terminals in contact with GABA-labeled dendrites were

3.2. Electron microscopy Since the VMN can be subdivided into ventrolateral and dorsomedial subnuclei which exhibit different neurochemical characteristics, tissue was initially sampled from each subdivision separately. However, the data represent a pooling of the regions since almost all morphological aspects studied appeared the same between subnuclei. The only exception to this statement was that enkephalin-labeled perikarya and dendrites were observed only within the ventrolateral subdivision of VMN. 3.3. GABA-labeled perikarya Eleven perikarya were observed that had GABA-labeling at least 5 times greater density then the surrounding

Fig. 4. An example of a GABA-labeled axon terminal ŽGABA. forming a symmetric synapses on an unlabeled dendrite ŽUL.. The axon terminal contains small clear vesicles as well as larger, dense-core vesicles ŽDCV.. Bar s 0.15 mm.

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Fig. 5. ŽA. A dendrite containing immunogold particles for GABA receives a symmetric synapses from an enkephalin-labeled axon terminals Žblack arrows.. An adjacent unlabeled axon terminal also forms a symmetric synapse onto the GABA-labeled dendrite Žopen arrow.. The upper-left enkephalin-labeled terminal contains several intensely labeled dense core vesicles Žlarge straight arrow., while the lower terminal contains one dense-core vesicle Žlarge straight arrow., in addition to an unlabeled dense core vesicles Žopen arrow.. Bar s 0.2 mm. ŽB. An enkephalin-labeled axon terminal contains peroxidase-labeling intense in two foci probably over dense core vesicles Žstraight arrows.. It forms a symmetric synapse Žcurved black arrow. on a GABA-labeled dendrite which also receives a synapse from an unlabeled axon terminal Žopen curved arrow.. Also in the field is an enkephalin-labeled unmyelinated axon Žarrowhead.. Bar s 0.15 mm.

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GABA-labeled Ž5r92; 1 symmetric synapse, 4 appositions.. On average GABA-labeled dendrites were in contact with one axon terminal Ž92 contactsr106 dendrites., and the remaining portion of each dendritic profile was covered with astrocytic processes.

Ž1. within VMN, enkephalin and GABA may act in part by disinhibiting GABA-containing VMN neurons, and Ž2. that the VMN may provide a source of GABA which could function to modulate behaviors in which VMN participates.

3.5. GABA containing axons

4.1. Methodological considerations

Although less numerous than GABA-labeled dendrites, GABA-labeled axon terminals also were observed ŽFig. 4.. GABA-labeled axons terminals varied in size from 0.3 to 0.7 mm in diameter. In addition to small clear vesicles, GABA-labeled terminals often contained a few dense-core vesicles ŽFig. 4.. Most GABA-labeled axon terminals did not form synapses in the plane of section sampled. When a synapse was seen, it was exclusively of the symmetric type usually onto unlabeled dendrites or soma. Of the GABAlabeled terminals sampled, a few synapsed on dendrites Ž5r34. or soma Ž4r34. that had GABA-labeling. Occasionally myelinated GABA-labeled axons were observed.

A high level of confidence in the pattern of GABAlabeling described can be derived from parallel lines of evidence. First, the pattern of GABA-labeling within VMN was consistent between two different antisera and visualization techniques. Furthermore, the antisera used have been previously shown to have specific labeling patterns in other brain regions w21,28,29x. In addition, the pattern of labeling we observed at the light microscopy level for GABA-labeling was consistent with previously published descriptions for labeling in the hypothalamus w32,37x. The antisera used to visualize enkephalin has been shown to preferentially detect enkephalin over dynorphin peptides w6x. Although the number and intensity of enkephalin-labeling in cell bodies was variable without the use of colchicine, axon terminal labeling with enkephalin was consistent and similar to previous studies w10x.

3.6. Enkephalin-labeled profiles Enkephalin-labeling was primarily found in axon terminals Ž92% or 113r123 labeled profiles.. Enkephalin-labeled axons were very abundant throughout VMN and ranged from 0.3 to 0.85 mm in diameter. Enkephalin-labeled dense-core vesicles coexisted with axon terminals with unlabeled dense-core vesicles as well as small-clear Žsynaptic. vesicles. Additionally, a few enkephalin containing somata Ž n s 5. and dendrites Ž n s 5. were found in the ventrolateral subdivision of VMN. Of the 95 enkephalin-labeled axon terminals that were associated with dendrites, more contacted unlabeled Ž76% or 72r95. than GABA-labeled dendrites Ž24% or 23r95.. However, enkephalin-containing terminals comprised a large fraction of the axon terminals in contact with GABA-labeled dendrites or soma Ž25%, 23 of 92. ŽFig. 5.. Although only some enkephalin-containing terminals formed synapses in the plane of section sampled, these were primarily symmetric synapses Ž85% or 30 of 36 terminals forming synapses.. Occasionally axon terminals Ž n s 8. and a few somata Ž n s 3. contained labeling for both enkephalin and GABA. 4. Discussion The results of this study indicate that the rich, but diffuse GABA-labeling viewed by light microscopy in VMN corresponds to labeling within a subset of the dendrites and soma of VMN neurons as well as some axon terminals. This concentration of GABA within the somato-dendritic compartment is commonly interpreted as evidence that these cells use GABA as a neurotransmitter at their axonal synaptic junctions. In addition, GABAlabeled VMN neurons were innervated by both GABAand enkephalin-containing axons. These data suggest that

4.2. Significance of GABA-labeling The abundance of GABA-labeled neurons in VMN was unexpected because messenger RNAs for the two enzymes known to synthesize GABA, GAD-65 and GAD-67, have eluded detection within the VMN w11,12,24x. Thus, these observations present the unusual case of neurons containing GABA-labeling in the apparent absence of a known GAD. It has been suggested that in addition to GAD-65 and GAD-67, other forms of GAD may be present within brain w7,8,30,31x. Indeed, the level of GAD enzymatic activity in VMN is among the highest of several brain areas w37x. Therefore it remains a possibility that GABA is synthesized within VMN neurons by a novel mechanism. It is important to point out that GABA-labeling methods cannot discriminate the source or functional importance of GABA-labeling. Specifically, GABA-labeled VMN neurons may not synthesize GABA but rather actively take up GABA released from afferents to the VMN. Without the demonstration that VMN neurons use GABA as a neurotransmitter by electrophysiological means, both the source and function of the enriched GABA-labeling observed in this study remain subject to speculation. However, we favor the interpretation that the structural attribute of GABA-labeling has a functional correlate. That is, the presence of GABA-labeling within neuronal dendrites could be interpreted to reflect that these neurons may be GABAergic. Presumably, cells which use a particular neurotransmitter in their axon terminals, can have levels of this transmitter which overflow into the cytoplasm of soma and dendrites. Immunolabeling for several neurotransmitters or their synthetic enzymes is commonly found within dendrites w28,29x.

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Several observations in combination with our results raise the interesting possibility that some GABA-labeled neurons in the VMN could be projection neurons. First, the high proportion of GABA-labeled dendrites in comparison to axon terminals may indicate that some GABA-labeled neurons have axons outside of VMN. Second, it is known that surgical isolation of the medial basal hypothalamus dramatically lowers GAD enzymatic activity w36x. Our observation that dendrites outnumber axons containing GABA in the VMN suggests the loss of GAD activity could result from retrograde degeneration of cell bodies, rather than from removal of GABAergic afferents. Third, in several regions including the septum, hippocampal formation and medulla, we have observed a correlation between very light somatic GABA-labeling and GABAergic projection neurons, and conversely, between cells with more intense somatic GABA-labeling and local circuit neurons ŽTAM, unpublished observation.. 4.3. Enkephalin-labeled neurons A small number of neuronal somata or dendrites containing enkephalin-labeled dense-core vesicles were sampled. Since enkephalin immunoreactivity here was found preferentially within axon terminals, the sampling of enkephalin containing somata probably greatly under-represents the quantity of enkephalin containing neurons in VMN. Indeed, a large fraction of VMN neurons, especially those in the ventrolateral subdivision of VMN express the enkephalin precursor gene, preproenkephalin w17x. Symmetric synapses were more commonly formed by enkephalin containing terminals then asymmetric synapses. This suggests enkephalin containing terminals may commonly exert inhibitory influences over VMN neurons. However, enkephalin containing axon terminals in VMN may arise from mixed sources. First, they may be local collaterals of VMN neurons themselves. In addition, several areas rich in enkephalin containing neurons densely innervate VMN, including the amygdala and the periaqueductal gray w9,17x. 4.4. Potential functional role of GABA and enkephalin in VMN GABA has been implicated in modulating behaviors in which the VMN participates, especially feeding w15,18x. In the female rat, VMN is a key region in regulating the hormone-dependent expression of the female sexual response, lordosis. The distribution of enkephalin and GABA in the female rat is similar to that described here in the male ŽKGC, unpublished observations., which may be relevant to the role of GABA and enkephalin in lordosis w25–27x. In the female rat, feeding and reproductive behavior have a reciprocal relationship. Most manipulations which decrease feeding, facilitate lordosis and vice versa, correlating with the activity of VMN. For example, in-

creased activity of VMN inhibits feeding while promoting reproductive behavior. The exception to this rule is GABA A agonists which stimulate both feeding and reproduction w15,18,27x. The suggestion that VMN neurons use GABA as a neurotransmitter and that these neurons may project outside of VMN would explain several observations relating to feeding behavior. There is a good correlation between the action of VMN neurons and the effects of GABA agonists on feeding in specific brain areas to which VMN projects. Specifically in the lateral hypothalamic area w18x and the central nucleus of the amygdala w28,29x GABA agonists inhibit feeding. In contrast to effects outside the VMN, within VMN GABA functions to stimulates feeding w15,18x. Our results establish that 25% of GABA-labeled axon terminals contact GABA-labeled soma or dendrites in VMN. Therefore, we postulate that the behavioral effects of GABA may be mediated in part by a GABAergic inhibition of VMN GABA-neurons. However, the role that GABAergic neural circuits might play in lordosis behavior is more dependent on the specific neuroanatomical region in question. In two areas to which the VMN projects heavily, the medial preoptic area and the midbrain central gray, GABA inhibits w25x and facilitates lordosis w27x, respectively. In addition, within the VMN specific GABA A agonists facilitate lordosis w25x. Likewise, enkephalin provides a robust innervation of VMN GABA-labeled neurons, suggesting it may also function in controlling GABAergic tone within VMN. Endogenous opioids such as enkephalin could modulate feeding from within VMN, as naloxone in the VMN inhibits food intake in food-deprived rat w41x. Enkephalin may act in part by inhibiting GABA neurons in the VMN. Like GABA agonists, some opioid agonists injected into the VMN increased feeding w39x. Thus, the interplay between these two inhibitory neurotransmitter system within the VMN may be critical in determining the behavioral output of this nucleus. Acknowledgements This work was supported by: DK 07313-18 ŽKGC., NS30824 ŽL-MK., NIDA DA08259, NIMH MH42834, NIH 18974 ŽTAM. and HD05751 ŽD.W.P... References w1x P.M. Beart, L.S. Nicolopoulos, D.C. West, P.M. Headley, An excitatory amino acid projection from ventromedial hypothalamus to periaqueductal gray in the rat: autoradiographic and electrophysiological evidence, Neuroscience Letters 85 Ž1988. 205–211. w2x J.L. Beverly, M.F. Beverly, M.M. Meguid, Alterations in extracellular GABA in the ventral hypothalamus of rats in response to acute glucoprivation, American Journal of Physiology 269 Ž1995. 1–2. w3x J.L. Beverly, R.J. Martin, Increased GABA shunt activity in VMN of three hyperphagic rat models, American Journal of Physiology 256 Ž1989. 1–2.

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