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Endocrinology Vol. 140, No. 2 933-940
Copyright © 1999 by The Endocrine Society

ARTICLES

Interactions between Neuropeptide Y and {gamma}-Aminobutyric Acid in Stimulation of Feeding: A Morphological and Pharmacological Analysis1

Shuye Pu, Mukul R. Jain, Tamas L. Horvath, Sabrina Diano, Pushpa S. Kalra and Satya P. Kalra

Departments of Neuroscience (S.P., S.P.K.) and Physiology (M.R.J., P.S.K.), University of Florida College of Medicine, Gainesville, Florida 32610; and the Department of Obstetrics and Gynecology, Yale University School of Medicine (T.L.H., S.D.), New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Shuye Pu, M.D., Department of Neuroscience, University of Florida College of Medicine, P.O. Box 100244, Gainesville, Florida 32610. E-mail: pu{at}neocortex.health.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y (NPY) produced in neurons in the arcuate nucleus and brain stem and released in the paraventricular nucleus (PVN) and surrounding areas is involved in stimulation of feeding in rats. We recently reported that {gamma}-aminobutyric acid (GABA) is coexpressed in a subpopulation of NPY neurons in the arcuate nucleus. To determine whether GABA is colocalized in NPY terminals in the PVN, the site of NPY action, light and electron microscopic double staining for NPY and GABA using pre- and postembedding immunolabeling was performed on rat brain sections. GABA was detected in NPY-immunopositive axons and axon terminals within both the parvocellular and magnocellular divisions of the PVN. These morphological findings suggested a NPY-GABA interaction in the hypothalamic control of feeding. Therefore, the effects of muscimol (MUS), a GABAA receptor agonist, on NPY-induced food intake were examined in sated rats. When injected intracerebroventricularly, both NPY and MUS elicited dose-dependent feeding responses that were blocked by the administration of 1229U91 (a putative Y1 receptor antagonist) or bicuculline (a GABAA receptor antagonist), respectively. Coadministration of NPY and MUS intracerebroventricularly amplified the feeding response over that evoked by NPY or MUS alone. Similarly, microinjection of either NPY or MUS into the PVN stimulated food intake in a dose-related fashion, and coinjection elicited a significantly higher response than that evoked by either individual treatment. These results suggest that GABA and NPY may coact through distinct receptors and second messenger systems in the PVN to augment food intake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CENTRAL administration of neuropeptide Y (NPY) in various species has been shown to stimulate robust feeding (1, 2, 3, 4, 5, 6). A large body of evidence suggests that NPY may be one of the physiological appetite transducers in the rat (7, 8, 9, 10). NPY produced in the neurons localized in the arcuate nucleus (ARC) and brain stem have been shown to act in the paraventricular nucleus (PVN) and neighboring sites to stimulate feeding (11, 12, 13). In addition, morphological and experimental evidence between neurons producing NPY and other orexigenic signals such as galanin and opioids suggests that NPY may stimulate feeding on its own and through the release of these orexigenic signals (10, 14, 15, 16, 17). {gamma}-Aminobutyric acid (GABA), a predominant inhibitory transmitter in the central nervous system (18, 19), has also been reported to participate in the stimulation of feeding in the rat (20, 21). Central administration of the GABAA receptor agonist muscimol (MUS) either intraventricularly or by microinjection into the PVN and other sites in the brain stimulated feeding (22, 23, 24, 25, 26, 27), a response blockable by the specific GABAA receptor antagonist, bicuculline (24, 27).

Recently, we reported that a subpopulation of NPY-producing neurons in the ARC coproduces GABA (28). This demonstration of the coexistence of a neuropeptide and an amino acid neurotransmitter in the ARC perikarya raised the following questions. Do NPY nerve terminals in the PVN contain GABA? What is the nature of the interaction of NPY and GABA in the hypothalamic stimulation of feeding if NPY and GABA are coreleased? To address these questions, first double labeling immunohistochemistry and electron microscopy were employed to determine whether GABA was colocalized in NPY-immunopositive nerve terminals in the PVN, and then the interaction of NPY and MUS on food intake was evaluated during the lights-on period when rats were satiated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Exp 1: morphological studies on colocalization of NPY and GABA in the PVN
These studies were performed at the Department of Obstetrics and Gynecology, Yale University School of Medicine (New Haven, CT). Adult male Sprague-Dawley rats (n = 5; 200–250 g BW) were used for immunocytochemical studies. Rats were kept under standard laboratory conditions, with tap water and regular rat chow available ad libitum, in a 12-h light, 12-h dark cycle. Rats were killed under ether anesthesia by transaortic perfusion with 50 ml heparinized saline, followed by 250 ml fixative. The fixative consisted of 4% paraformaldehyde, 15% picric acid, and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brains were dissected out, and coronal blocks were postaffixed for an additional 1–2 h in glutaraldehyde-free fixative. Tissue blocks were rinsed in several changes of PB, and then 60-µm Vibratome sections were prepared and rinsed four times for 15 min each time in PB.

For fluorescence microscopy, Vibratome sections were double immunostained for glutamic acid decarboxylase (GAD) and NPY (28). Sections were first incubated in a mixture of the primary antisera (anti-GAD, 1:1000; anti-NPY, 1:2000; diluted in PB containing 1% normal horse serum) and 0.3% Triton X-100, overnight at room temperature. After several rinses in PB, sections were further incubated in a mixture of rhodamine-conjugated antisheep IgG and fluorescein-conjugated antirabbit IgG (both diluted to 1:50 in PB; Jackson ImmunoResearch Laboratories, Inc.., West Grove, PA) for 2 h at room temperature, then thoroughly washed in PB five times for 10 min each time. Sections were mounted on slides and examined under an Olympus Corp. epifluorescence light microscope furnished with rhodamine, fluorescein, and combined rhodamine and fluorescein filters. In control double immunostaining experiments, in which one of the primary antisera was replaced with normal serum, only single immunostaining could be detected.

Preembedding immunostaining. For electron microscopy, sections were transferred into vials containing 0.5 ml 10% sucrose in PB and rapidly frozen by immersing the vial in liquid nitrogen to enhance antibody penetration. They were then thawed to room temperature and repeatedly washed in PB. Subsequently, sections were treated with sodium borohydride in PB for 10 min to eliminate unbound aldehyde from the tissue. Sections were incubated in the primary antibody generated in rabbit against neuropeptide Y diluted 1:14,000 (Peninsula Laboratories, Inc., Belmont, CA) in PB for 24 h at room temperature. After several washes in PB, sections were incubated in the secondary antibody (biotinylated goat antirabbit IgG, 1:250 in PB; Vector Laboratories, Inc., Burlingame, CA) for 2 h at room temperature, then rinsed in PB three times for 10 min each time. This was followed by incubation in avidin-biotin peroxidase (1:250 in PB; ABC Elite, Vector Laboratories, Inc.) for 2 h at room temperature, and the tissue-bound peroxidase was visualized with a nickel-diaminobenzidine (0.12 mg glucose oxidase, 12 mg ammonium chloride, 600 µl 0.05 M Ni ammonium sulfate, and 600 µl 10% ß-D-glucose in 30 ml PB; dark blue reaction product in the cytoplasm).

Postembedding immunostaining. Sections were postosmicated (1% OsO4 in PB) for 30 min, dehydrated through increasing ethanol concentrations (using 1% uranyl acetate in 70% ethanol for 30 min), flat embedded in Durcupan (ACM/Fluka), Electron Microscopy Sciences, Fort Washington, PA) between liquid release-coated slides (Electron Microscopy Sciences), and placed in an oven at 60 C for 48 h. After capsule embedding, blocks were trimmed and cut on a Reichert-Jung ultramicrotome (Leica, Inc., Deerfield, IL). Ribbons of ultrathin sections were collected on filtered Millipore Corp., Bedford, MA) solutions in humid chambers and processed as follows: 1) 10 min in periodic acid; 2) rinse in double distilled water (DDW); 3) 10 min in 2% sodium metaperiodate in DDW; 4) rinse in DDW; 5) three 2-min rinses in Tris-buffered saline (TBS), pH 7.4; 6) 30 min in ovalbumin in TBS; 7) three 10-min periods in normal goat serum in TBS; 8) first incubation for 1–2 h in rabbit anti-GABA (Incstar Corp., Stillwater, MN) (35) diluted 1:200 in normal goat serum-TBS; 9) two 10-min washes in TBS and a 10-min rinse in 0.05 M Tris buffer (pH 7.5) containing gold-conjugated goat antirabbit IgG diluted 1:10 in the same buffer; 10) two 5-min washes in DDW; and 11) contrasting with saturated uranyl acetate (30 min) and lead citrate (20–30 sec). Ultrathin sections were then examined using a Philips CM-10 electron microscope (Mahway, NJ). This approach has been widely used for postembedding labeling of GABA (29).

For a semiquantitative analysis, the same region of the parvocellular PVN was dissected out from three male rats, immunostained for NPY and GABA, and processed for electron microscopy as described above. From each sample (n = 3), in 10 ultrathin sections that were 5 µm apart, we counted 80 NPY-immunoreactive (proxidase-containing) axon terminals and noted how many of these were also immunopositive for GABA (immunogold).

Exp 2: effects of muscimol on NPY-induced food intake
The following experiments were performed at the Department of Neuroscience, University of Florida College of Medicine (Gainesville, FL). Normal female rats (HSD:SD, Harlan Laboratories, Indianapolis, IN), weighing 200–250 g, were housed under controlled light (lights on 0500–1900 h) and temperature (22–23 C) conditions. Food (Purina lab chow pellets, Ralston Purina Co., St. Louis, MO) and water were available ad libitum. All procedures were approved by the institutional animal use and care committee of the University of Florida.

The animals were permanently implanted with cannula aimed at either the third ventricle or the PVN of the hypothalamus under ketamine/xylazine anesthesia (100 and 15 mg/kg, respectively) as described previously (30). Rats were handled and mock injected daily during the 1-week recovery period to habituate them to the injection procedure. On the day of experiment, food was removed 1 h before central injection at 1100 h. Immediately after injection, a preweighed amount of food was provided to the animals. As previous studies showed that feeding in response to NPY or MUS is completed essentially within the first hour of administration (9, 12, 23, 24), we measured 2-h food intake in the current experiments. After 2 h of observation the test was terminated, and the remaining food and spillage were weighed. The possibility that cyclic ovarian steroid changes may confound results is unlikely, because each rat was treated in a counterbalanced design with a minimum of 48 h separating each test, and there is no documented evidence that food intake in response to NPY or GABAergic agonist changes during the lights-on period in sated rats.

Part 1. The effects of icv injection of NPY and MUS on food intake were examined in this experiment. All compounds were dissolved in artificial CSF (containing 127.6 mM NaCl, 2.5 mM KCl, 1.4 mM CaCl2, 1.0 mM MgSO4, and 12.0 mM phosphate buffer, pH 7.4) sterilized by filtering through a 0.22-µm pore size syringe filter, and delivered in 3-µl solutions through a 26-gauge stainless steel injection cannula that extended 0.5 mm beyond the guide cannula. The injection cannula was left in place for 15 sec and replaced with the stylet. NPY (Peninsula Laboratories, Inc., Belmont, CA) at doses of 0.06, 0.47, and 2.0 nmol and MUS (Sigma Chemical Co., St. Louis, MO) at doses of 0.44, 0.88, and 1.76 nmol were either injected individually or coadministrated. The effects of a competitive GABAA receptor antagonist, bicuculline methiodide (Sigma Chemical Co.; 1.0 nmol), and a NPY Y1 receptor antagonist, 1229U91 (a gift from Dr. A. J. Daniels, GlaxoWellcome, NJ; 4.2 nmol), on MUS- and NPY-induced feeding were also tested in additional groups of animals. The doses of antagonists were selected based on previous studies (31, 32) and preliminary experiments.

Part 2. The effects of PVN microinjection of NPY and muscimol on food intake were examined in this experiment. The rats were implanted with a 25-gauge stainless steel guide cannula aimed at the right side of the PVN (1.8 mm behind the bregma, 0.5 mm lateral from the midline, and 6.5 mm ventral from the dura) (33) under ketamine/xylazine anesthesia. A syringe pump (Autosyringe, Inc., Hooksett, NH) equipped with a 25-µl Hamilton syringe (Hamilton Co., Reno, NV) was used to deliver a 333-nl solution through a 33-gauge injection cannula that extended 1 mm beyond the guide cannula (30). The injection cannula was left in place for 1 min before replacement of the stylet. NPY at doses of 0.03 and 0.06 nmol and muscimol at doses of 0.03, 0.11, and 0.44 nmol dissolved in normal saline were injected either individually or in combination. At the conclusion of the experiment, rats were given an overdose of sodium pentobarbital and then perfused with formol saline. The brains were frozen and sectioned at 50 µm. All sections were stained with carbol fuchsin. The injection site was examined according to the Paxinos and Watson brain atlas (33). Only those rats with the injection site within the PVN were included in the analysis.

Statistical analysis
Data are presented as the mean ± SEM. One-way ANOVA followed by Newman-Keuls multiple comparison was used to detect significant differences among groups. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: morphological studies on colocalization of NPY and GABA in the PVN
In the PVN, an extensive network of neuronal processes was found to express both GAD and NPY immunoreactivity. Using oil immersion, a subset of green-fluorescent, NPY-containing axon terminals were also found to express red fluorescent GAD immunolabeling (Fig. 1Go, A and B). Although comparative quantitative analysis could not be carried out between the results of this approach and those obtained with electron microscopy (as described below), the overall expression and abundance of colocalization of NPY and GAD seemed to be the same regardless of the method used. It has to be noted, however, that the data gathered with epifluorescent microscopy serve only as a control for the electron microscopic studies, because without ultrastructural analysis, the coexistence of NPY and GAD at presynaptic profiles cannot be assessed.



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  		Figure 1. A and B, Color micrographs taken from the PVN after GAD (A) and NPY (B) immunolabeling. GAD axons are visualized with red fluorescent, rhodamine-conjugated secondary antiserum (A), whereas NPY-containing profiles (B) are labeled with green fluorescent, fluorescein-conjugated secondary antiserum. The numbered green arrowheads in A point to GAD-immunopositive axons in the PVN that were also immunolabeled for NPY (numbered red arrowheads on B). Blue stars on A and B indicate neuronal perikarya for orientation. The bar scale in A represents 10 µm. C–E are electron micrographs taken from the parvocellular region of the PVN after preembedding immunostaining for NPY (green arrows point to immunoperoxidase deposits) and postembedding labeling for GABA (red arrows point to immunogold). A population of NPY-immunoreactive axon terminals also expressed GABA immunolabeling (NPY/GABA; D and E), whereas other NPY boutons were free of immunogold. Bar scales in D and E represent 1 µm.

 
Preembedding immunostaining for NPY resulted in peroxidase-labeled neuronal processes throughout the hypothalamus. An abundant network of axons and axon terminals was detected in both the parvocellular and magnocellular divisions of the paraventricular nucleus. After embedding of the material for electron microscopy and postembedding immunolabeling for GABA, numerous axons and axon terminals were detected to contain immunogold indicating GABA content (Fig. 1Go, C–E) in both the parvo and magnocellular subdivisions of the PVN. An axon-like structure was determined to be GABAergic if the labeling could be found over the same process in consecutive ultrathin sections and the number of gold particles was at least three times higher than the background level. This latter was assessed in control experiments in which the primary antiserum for GABA was omitted. The synaptic membrane specializations established by the GABAergic boutons were symmetric. Within the parvocellular division of the PVN, immunogold labeling was detected on a population of NPY-immunoreactive, peroxidase-containing axons and axon terminals (Fig. 1Go, D and E). In an attempt to estimate the extent of colocalization of NPY and GABA, we counted 240 (80 + 80 + 80) NPY-immunoreactive axonal processes in the parvocellular PVN of three animals, of which 63 (25 + 17 + 21; 26%) axonal processes also contained immunolabeling for GABA. The synapses established by NPY boutons and unidentified dendrites and cell bodies were symmetrical. Both the light and electron microscopic appearances of NPY were in agreement with previous descriptions (14, 34, 35).

Exp 2: effects of muscimol on NPY-induced food intake
Effects of icv NPY and MUS alone on food intake (Fig. 2Go). As evidenced in Fig. 2Go, administration of NPY (Fig. 2AGo) and MUS (Fig. 2BGo) stimulated food intake in a dose-related fashion. Based on the results obtained with 0.47 nmol NPY and 0.44 nmol MUS, it appears that NPY is twice as potent as MUS in stimulating food intake.



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  		Figure 2. Effects of third cerebroventricular injection of NPY or muscimol on food intake. NPY (A) and muscimol (B) stimulated food intake in a dose-dependent manner at the doses tested. In this and the following figures, the numbers in each column represent the numbers of animals, and statistically significant (P < 0.05) differences between groups are denoted by different letters.

 
Figure 3Go shows the effects of a GABAA receptor antagonist, bicuculline, and a Y1 receptor antagonist, 1229U91, on MUS- and NPY-induced feeding. Although bicuculline (1.0 nmol) on its own produced little feeding, it completely blocked MUS-induced feeding, thereby suggesting the involvement of GABAA receptor in MUS-induced feeding. On the other hand, the same dose of bicuculline (1.0 nmol) produced no impact on feeding elicited by NPY. Similarly, 1229U91 produced little feeding on its own, but suppressed NPY-induced feeding and not that elicited by MUS.



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  		Figure 3. Coadministration of bicuculline icv completely abolished muscimol (0.88 nmol)-induced food intake, but failed to alter NPY (0.47 nmol)-evoked food intake (A). Similarly, 1229U91, a selective Y1 receptor antagonist, had no effect on muscimol-induced food intake, whereas it significantly reduced NPY-induced feeding (B).

 
Effects of combined administration of MUS and NPY. Because 0.47 nmol NPY and 0.44 nmol elicited submaximal food intake (Fig. 2Go), these doses were selected for evaluation of the combined effects of NPY and MUS on food consumption. As shown in Fig. 4Go, a combination of NPY (0.47 nmol) and MUS (0.44 nmol) stimulated a feeding response that was markedly higher than that elicited by these compounds administered alone. Coadministration of either a higher dose of MUS (0.88 nmol) with the same dose of NPY (0.47 nmol) or a higher dose of NPY (2.0 nmol) with the same dose of MUS (0.44 nmol) did not improve the feeding response evoked by individual treatments. Also, coinjection of higher dose of MUS (1.76 nmol) with 2.0 nmol NPY did not further augment the feeding response (data not shown).



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  		Figure 4. Effects of coadministration of NPY and muscimol icv at various doses on food intake. Note that the combination of 0.44 nmol muscimol and 0.47 nmol NPY resulted in significantly higher feeding responses than those evoked by NPY or MUS alone.

 
Effects of microinjection of NPY and MUS alone or together into the PVN on food intake (Figs. 5Go and 6Go). As expected, much smaller doses of NPY and MUS also stimulated feeding in a dose-related fashion when microinjected into the PVN (Fig. 5Go). In the case of MUS, the higher dose of 0.44 nmol produced a smaller response than that evoked by 0.11 nmol (Fig. 5BGo).



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  		Figure 5. Effects of microinjection of NPY (A) or muscimol (B) into the PVN on food intake. PVN microinjection of either NPY or muscimol stimulated food intake at much lower doses than those used for icv injection.

 


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  		Figure 6. Effects of coadministration of NPY and muscimol into the PVN on food intake. A combination of 0.03 nmol NPY with either 0.11 or 0.44 nmol muscimol augmented food intake over that induced by either NPY or MUS alone.

 
The results of the experiments with combinations of NPY and MUS doses are summarized in Fig. 6Go. Two combinations produced an augmented response. The feeding response elicited by 0.03 nmol NPY plus 0.11 nmol MUS and by 0.03 nmol NPY plus 0.44 nmol MUS were higher than that produced by individual doses. However, the feeding response to a high dose of NPY (0.06 nmol) was not further augmented by the addition of 0.44 nmol MUS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of neurotransmitters/neuromodulators has been shown to stimulate feeding in the rat (9, 16). However, there is limited information regarding the interrelationship among them in the elicitation of feeding behavior. The current study for the first time provides experimental evidence in support of a possible interaction between NPY and GABA systems in stimulation of feeding. A subpopulation of NPY-producing neurons in the ARC has been shown to project into the PVN (11), a primary site of action of NPY in evoking feeding behavior (12). A subgroup of NPY neurons in the ARC was also shown to express GABA (28). We have extended these studies to show that NPY-immunopositive axons and neuroterminals in the PVN displayed immunogold labeling indicative of the presence of GABA. These neural processes containing NPY and GABA accounted for about 26% of the axonal processes containing NPY in the PVN. These findings suggest the possibility that a network of axons emanating from the NPY- and GABA-coexpressing cells in the ARC (11, 28) innervate the PVN. However, it is known that NPY-producing neurons in the brain stem also innervate PVN (13, 36). As it is not clear that whether these PVN projective cells coproduce GABA along with adrenergic transmitters (10, 17, 36), additional studies are warranted to precisely determine the origin of NPY- and GABA-coexpressing nerve terminals in the PVN.

Nevertheless, visualization of axon and axon terminals containing NPY and GABA in the PVN underscores a possible coaction of these signals in the hypothalamic regulation of ingestive behavior. Indeed, the results of our experimental study argue for an interaction between NPY and GABA in the stimulation of feeding. Although several laboratories have previously shown that central injection of either NPY or MUS stimulates feeding (1, 2, 3, 4, 5, 6, 20, 21, 22, 23, 24), the current study has extended these observations by documenting that coinjection of NPY and MUS either icv or directly into the PVN evoked significantly higher food intake than that produced by either NPY or MUS alone. These results demonstrated that NPY and MUS can act together to evoke feeding, and one of the sites of coaction may be resident in and around the PVN. However, the coaction was evident within a narrow dose range in two experimental paradigms. A plausible explanation may be related to the pleiotropic nature of NPY and GABA (16, 18, 19). Aside from stimulation of food intake, injection of these neurotransmitters is likely to simultaneously stimulate and/or inhibit several neuroendocrine and behavioral responses that are likely to affect the stimulation of ingestive behavior and thereby allow only a narrow range of dose-dependent coaction. Nevertheless, the consistency in demonstrating the existence of positive interaction between NPY and MUS on food intake by icv and direct microinjection into the PVN argues strongly for a physiological role for these coexpressing neurotransmitters/neuromodulators in the hypothalamic control of ingestive behavior.

The selectivity of the effects of bicuculline and 11229U91 to inhibit MUS- or NPY-induced feeding, respectively, further implies that the augmented effect on feeding may be attributable to activation of distinct receptors by NPY and GABA, which, in turn, triggers common or differing intracellular signal transduction cascades that promote an increased drive for food. In this regard, the interaction of norepinephrine, the other orexigenic signal coexpressed with NPY in the brain stem neurons projecting to PVN, is quite different. Experimental findings showed that the {alpha}2-adrenergic receptor antagonist attenuated NPY-induced feeding (37), and the {alpha}2-adrenergic agonist competitively displaces NPY from plasma membranes (38, 39). These observations suggest that a cross-talk between NPY and adrenergic signals existed at the membrane receptor level. Taken together, it seems that neurotransmitters of different chemical compositions, amino acid GABA and monoamine norepinephrine, coact with NPY by employing different cellular and subcellular mechanisms.

The results of these and previous studies show that 1229U91, a putative Y1 receptor antagonist (40), inhibited NPY-induced feeding in the rat (31, 32). These observations imply participation of Y1 receptors in the drive for food intake evoked by NPY (30, 41). However, recent reports also suggested that the hypothalamic Y5 receptor subtype may mediate NPY-induced feeding (42, 43, 44). 1229U91 has high affinity for Y1 and Y4 receptors, a subtype not found in the hypothalamus (45), and it displays very little affinity for the Y5 receptor (46, 47). Additionally, we observed that Y1 receptor messenger RNA (mRNA), and not Y5 receptor mRNA, in the hypothalamus was up-regulated in conjunction with increased appetite in fasted rats and in rats displaying hyperphagia produced experimentally (48, 49). Blockade of up-regulation of Y1 receptor mRNA with the cytokine, ciliary neurotropic factor, diminished feeding in fasted rats (49). Consequently, it is reasonable to conclude that regardless of which NPY receptor subtype is involved in mediation of NPY-induced food intake, our current findings with 1229U91 and bicuculline are in accord with the idea that NPY and GABA can act together, possibly mobilizing a common intracellular signal transduction cascade in part through the Y1 receptor subtype, a conclusion supported by findings from mice lacking Y1 or Y5 receptors (50, 51).

There is a general consensus that GABA serves as a major inhibitory neurotransmitter in the brain (18, 19). In agreement with numerous previous reports (22, 23, 24, 25), we observed that stimulation of GABAA receptors resulted in a dose-dependent feeding response. Microinjection of MUS in extrahypothalamic sites also elicited feeding (26, 27). Additionally, it is important to note that there was no evidence of inhibition of NPY-induced feeding by MUS in our studies. However, it remains possible that GABAA receptor activation may inhibit the effects of inhibitory interneurons that tonically exert an inhibitory control on feeding, thereby resulting in stimulation of feeding. If this is the likely scenario, then it is reasonable to envision that either corelease of GABA with NPY or release of GABA at different times before or after NPY release may amplify the feeding response induced by NPY through a disinhibition process locally in the PVN and surrounding sites. On the other hand, there are several reports showing GABA acting as an excitatory signal (52, 53, 54, 55). Consequently, the alternate possibility that GABAA receptors are directly engaged in the stimulation of feeding cannot be completely ruled out at present. Nevertheless, demonstration of coexistence and coaction of NPY and GABA together with previous similar findings with NPY and norepinephrine (10) represent another level of redundancy and a fail-proof mechanism in the brain control of appetite behavior. Seemingly, the coexistence of neural transmitters represents a modality in the neural mechanism to compensate for a malfunction or loss of NPY, as demonstrated in NPY gene knockout mice (56).

In summary, these results show that GABA is colocalized with NPY in a subpopulation of axon terminals in the PVN. The incremental responses of central coadministration of NPY and MUS on food intake and a lack of cross-talk at the receptor level between Y1 and GABAA receptor systems suggest that NPY and GABA systems can function independently or together by mobilizing common or differing intracellular signal transduction pathways in the target sites to enhance feeding. It is also possible that each of these neurotransmitters may activate a distinct orexigenic network within the hypothalamus to stimulate feeding, and combinations of NPY and MUS may result in augmented responses. Elucidation of the underlying cellular and molecular events involved in the interaction of NPY and GABA in the hypothalamus warrants further investigation.


   Footnotes
 
1 This work was supported by NIH Grants NS-32727 and DK-37273. A portion of this study was presented at the 27th Annual Meeting of the Society for Neuroscience, New Orleans, Louisiana, October 25–31, 1997. Back

Received June 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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