Distribution of GABA-like immunoreactivity in the song system of the zebra finch

BRAIN RESEARCH ELSEVIER

Brain Research 651 (1994) 115-122

Research Report

Distribution of GABA-like immunoreactivity in the song system of the zebra finch William Grisham *, Arthur P. Arnold Department of Psychology and Laboratory of Neuroendocrinology of the Brain Research Institute, University of California, Los Angeles, Los Angeles, CA 90024-1563, USA

Accepted 22 March 1994

Abstract

GABA-like immunoreactivity (GABA-LIR) was mapped in the male and female zebra finch song system using a polyclonal antibody to GABA. GABA-LIR was found throughout the song system in neurons and neuropil of the robust nucleus of the archistriatum (RA), the higher vocal center (HVC), Area X, the magnocellular nucleus of the neostriatum (MAN), and the dorsomedial portion of the nucleus intercollicularis (DM of ICo). Puncta present in the lateral division of MAN (1MAN) may be local interneurons since the only known afferents of 1MAN are from the dorsolateral nucleus of the anterior thalamus (DLM), which did not appear to have any cell bodies with GABA-LIR. Distinct and dense puncta with GABA-LIR were present in DLM, and may be projections from Area X/lobus parolfactorius (LPO). Dramatic sex differences in GABA-LIR distribution were found. Females did not appear to have any GABA-LIR above background in either RA or HVC. Females also did not appear to have a distinct Area X, although they did have many small, lightly staining cell bodies in the corresponding LPO. The distribution of GABA-LIR and sex differences in its distribution suggests that GABAergic neurons may play a role in the acquisition and/or production of song in the zebra finch. Key words: GABA; Immunohistochemistry; Bird song; Higher vocal center; Sex difference; Robust nucleus of the archistriatum

1. Introduction

Evidence suggests that cholinergic, monoaminergic, peptidergic, and amino acid neurotransmitters are present in the song control system, a set of interconnected nuclei that have been implicated in the acquisition and production of birdsong. The main descending song system, which has been defined by sites where lesions produce deficits in song production and by the pattern of projections, consists of the higher vocal center (HVC), the robust nucleus of the archistriatum (RA), and the dorsomedial nucleus of the nucleus intercollicularis ( D M of ICo) [47,60,63]. Those nuclei in which

* Corresponding author. Department of Psychology, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 900241563, USA. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00415-9

lesions prevent song acquisition include A r e a X [59,62], the medial portion of the dorsolateral nucleus of the anterior thalamus (DLM) [25], and the magnocellular nucleus of the neostriatum (MAN) [14,59]. Evidence of acetylcholine in the song system comes from studies using micropunches and studies of the distribution of enzymes and receptor sites. Acetylcholine (ACh) has been detected in micropunches of MAN, HVC, and R A [57]. MAN, HVC, RA, ICo, and the nXIIts have cell bodies that express the degrative enzyme acetylcholinesterase (ACHE); and these areas, along with A r e a X, show evidence of A C h E in the neuropil [53]. Choline acetyltransferase (CHAT), an enzyme responsible for the synthesis of ACh, has been identified in RA, HVC, IMAN, and Area X of the lobus parolfactorius (LPO); but cell bodies with C h A T were only identified in the L P O and nXIIts [58]. Muscarinic cholinergic receptor sites have been found in

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A r e a X, ICo, a n d H V C [5,10,53]; and nicotinic A C h receptors have b e e n f o u n d in HVC, M A N , ICo, a n d n X I I t s [68]. M o n o a m i n e s are also p r e s e n t in the song system. Tyrosine hydroxylase is distributed in A r e a X, M A N , HVC, a n d R A [12]. T h e s e areas, as well as the D M of ICo, c o n t a i n both d o p a m i n e a n d n o r e p i n e p h r i n e [6,7,35,57]. a 2 A d r e n e r g i c receptors have b e e n f o u n d in A r e a X, RA, a n d ICo [42]. S e r o t o n i n has b e e n f o u n d in A r e a X, M A N , HVC, RA, a n d the D M of ICo by m e a n s of H P L C analyses of m i c r o p u n c h e s [6,7]. I m m u n o h i s t o c h e m i c a l t e c h n i q u e s have revealed peptidergic n e u r o t r a n s m i t t e r s in the song system. E n k e p h a l i n e r g i c fibers a n d cell bodies have b e e n identified in M A N , HVC, RA, a n d ICo [4,19,54]. Vasoactive intestinal p e p t i d e (VIP) fibers have b e e n f o u n d in M A N a n d HVC. Vasotocin fibers a n d VIP, cholecyst o k i n i n (CCK), a n d s u b s t a n c e P cells a n d fibers have b e e n f o u n d in ICo [4,64]. Excitatory a n d inhibitory a m i n o acid n e u r o t r a n s m i t ters also exist in the song system. G l u t a m a t e a n d aspartate are released by electrically stimulating afferents of R A in in vitro slices [56]. S t i m u l a t i o n of the M A N - t o - R A pathway or the H V C - t o - R A pathway of in vitro slices p r o d u c e s excitatory post-synaptic p o t e n tials (EPSPs) in R A that are blocked by N-methyl-Daspartate ( N M D A ) or n o n - N M D A receptor antagonists, respectively [34,39,40]. N M D A receptors have b e e n f o u n d in HVC, A r e a X, R A , a n d 1MAN [1]. S t i m u l a t i o n of slices c o n t a i n i n g R A in vitro results in the release of y - a m i n o b u t y r i c acid ( G A B A ) [39,55], a n d G A B A has b e e n identified in the canary H V C by m e a n s of i m m u n o h i s t o c h e m i s t r y [16,51]. We were particularly i n t e r e s t e d in the d i s t r i b u t i o n of G A B A e r g i c n e u r o n s in the zebra finch song system since estrogen has d r a m a t i c effects on the d e v e l o p m e n t a n d f u n c t i o n of the song system a n d has d r a m a t i c effects o n G A B A e r g i c n e u r o n s in other systems. For example, e s t r o g e n can regulate the rate of G A B A p r o d u c t i o n [38,65], the release of G A B A [30], the n u m ber of G A B A e r g i c axo-somatic synapses [50], a n d the n u m b e r of G A B A receptors [17,18,48,52]. E s t r o g e n also seems to be the h o r m o n e responsible for masculinizing the sexually d i m o r p h i c aspects of the song control system d u r i n g d e v e l o p m e n t [22,23,24,36,43,44,61], a n d it is essential for activating song behavior [26,66,67]. Since the effects of estrogen o n sexual differentiation a n d / o r song p r o d u c t i o n might be m e d i a t e d in part by e s t r o g e n ' s effects o n G A B A e r g i c n e u r o n s , we m a p p e d the d i s t r i b u t i o n of G A B A - I i k e i m m u n o r e a c t i v ity ( G A B A - L I R ) in the song system of the zebra finch. W e f o u n d n e u r o n s with G A B A - L I R in all of the telencephalic song areas (RA, HVC, M A N , a n d A r e a X) a n d distinct p u n c t a with G A B A - L I R in I M A N a n d DLM. W e also f o u n d m a l e - b i a s e d sex differences in G A B A - L I R in RA, HVC, a n d A r e a X.

2. Materials and methods Data were collected from 11 male and 5 female zebra finches (Poephilia guttata) obtained from our breeding colony at UCLA. The birds were judged to be fully adult and sexually mature on the basis of plumage, beak color, and inspection of the gonads at sacrifice. Sexual maturity in this species occurs by about 90 days of age [29]. The birds were anesthetized with Equithesin, then perfused on ice with ice-cold phosphate buffered saline (PBS) followed by an ice-cold solution of 1% paraformaldehyde and 1.25% glutaraldehyde. After dissection, the brain was post-fixed for one hour in the same fixative and placed in refrigerated 30% sucrose overnight. The next day the brain was frozen-sectioned at 40 ~m, and every second or third section was stored in ice-cold PBS. In some animals, adjacent sections were stained with thionin. A rabbit polyclonal antibody to GABA conjugated to keyhole limpet hemocyanin complex (Chemicon AB141) was used as the primary antibody. Several concentrations of primary antibody from 1:500 to 1:2000 were tried, and a solution of 1:500 proved most satisfactory. Sections were incubated for 1 h in 3% normal goat serum (NGS Vector S-1000) as a blocker and then incubated overnight with the primary antibody in 3% NGS. After incubation with the primary antibody, the sections were given three PBS rinses and incubated with the secondary biotinylated antibody (goat anti-rabbit IgG, Vector BA-1000) for 1 h. The sections were then given three more PBS rinses followed by an avidinbiotin-complex reaction (Vector Laboratories Vectastain Elite ABC kit). After three more PBS rinses, the sections were briefly incubated in a 0.05% solution of 3,3'-diaminobenzidine (Sigma) in tris buffer containing 0,01% hydrogen peroxide. Sections were washed with PBS, mounted, air-dried and coverslipped using Permount. Because we had difficulty identifying some song nuclei in females with the GABA antibody (see below), the coverslips were removed and the female brain sections were counterstained with thionin. A subset of 8 males and 4 females was were selected for morphometric analyses. The soma size of neurons with GABA-Iike immunoreactivity (GABA-LIR) was determined by tracing 25 immunoreactive neurons in IMAN, Area X/LPO, HVC, RA, and ICO of each bird using a digitizing pad connected to a computer. The mean of these 25 cross-sectional areas was then taken as the soma size. Data presented are the means of each bird's mean with n = the number of birds. To determine if there were sex differences in one of these song control nuclei, t-tests comparing the male and female means were employed. Control procedures consisted of (1) eliminating the primary antibody; (2) eliminating the secondary antibody; (3) preadsorbing the antibody by incubating the antibody (1:500 dilution) with GABA overnight at concentrations of 2 mg GABA/ml, 20 mg/ml, or 200 mg/ml; and (4) substituting rabbit pre-immune serum of the type for the primary antibody in dilutions of 1 : 500, 1 : 1000, and 1 : 5000.

3. Results 3.1. Positiue controls Two areas of well-established G A B A e r g i c n e u r o n s , the c e r e b e l l u m a n d the optic tectum, showed G A B A L I R in all of o u r zebra finches. G A B A - L I R in the c e r e b e l l u m of the zebra finch was identical to that described in the chicken [21]; m a n y n e u r o n s with G A B A - L I R were p r e s e n t in the m o l e c u l a r layer, a n d P u r k i n j e cells varied in the intensity of reaction product from light to fairly intense. T h e s e cerebellar cell

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populations have also been shown to be GABAergic in rats [41,49]. All layers of the optic tectum had GABALIR in our zebra finches, which is consistent with G A B A - L I R with other avian species [3,20,21]. Cells of the optic rectum of birds have been confirmed to be GABAergic by uptake of [3H]GABA [27,28] and the effects of locally applied bicuculline (a G A B A A antagonist) on intertectal inhibition [8]. The superior colliculus, which is homologous to the optic tectum, has been shown to have GABAergic cells in the rat [41]. Thus, this antibody identified cells which are likely to be GABAergic. 3.2. Negative controls ~!~ ~ i ~ :

Incubating the sections without the primary antibody resulted in a complete absence of reaction product. Incubating the sections with the primary antibody but not the secondary antibody resulted in no reaction product, except for some very light reaction product in cells in the nucleus isthmi, pars magnocellularis, which we and others have identified as having large neurons with intense G A B A - L I R [3,20,21]. Preadsorbing the antibody with G A B A did not diminish G A B A - L I R even when the antibody was incubated with G A B A at a concentration as high as 200 m g / m l . This result is not unexpected because antibodies to small molecules (such as GABA) may not bind with their antigens so as to prevent reaction product in tissue sections [9]. This failure to eliminate GABA-LIR by preadsorption with GABA, which has been previously reported [21], is likely due to the fact that the antibody is raised against G A B A conjugated to a protein (keyhole limpet hemocyanin). Presumably the antibody recognizes G A B A in the tissue because G A B A is conjugated to endogenous proteins during fixation with glutaraldehyde, and these GABA-protein conjugates are capable of being recognized by the antibody. Controls in which normal rabbit serum was substituted for the primary antibody had extremely high background at all concentrations but never showed the same pattern of reaction product as when we used the primary antibody. Presumptive GABAergic cells in the tectum, cerebellum, or song system rarely showed any more reaction product than did any other part of the brain when rabbit serum was used in place of the primary antibody. Altogether the negative controls suggest that the reaction product found when using the primary antibody is due to the antibody recognizing G A B A conjugated to endogenous proteins. 3.3. Song system

In males, the entire robust nucleus of the archistriaturn (RA) was quite distinct from the background and had large, widely scattered somata with intense

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Fig. 1. Top: photomicrograph of the RA of a male zebra finch showing clear neurons and neuropil with GABA-LIR. Medial is to the right, dorsal is toward the top. Bottom: photomicrographof the HVC of a male showing neurons with GABA-LIR and some GABALIR in the neuropil. Medial is to the left, dorsal is toward the top. Bars = 0.2 mm.

GABA-LIR and moderate to intense GABA-LIR in the neuropil (Fig. 1, top). All of the male R A had G A B A - L I R so that the boundaries of R A were the same as in Nissl stained material [2]. The mean soma size of neurons with G A B A - L I R was 78.6/zm 2 (range 11.7-221.1 /~m2), somewhat smaller than that usually reported for male RA neurons [15,36,37] (Fig. 2). Although the upper size limit of GABA-LIR neurons in RA was only slightly smaller than the size limit reported with Nissl stain (240/.~m 2 versus 300/zm 2) [15], the modal size of R A neurons with G A B A - L I R was much smaller than Nissl-stained neurons (30-60 /~m 2 versus 180-210 ~m2). Neurons with GABA-LIR may have appeared to be smaller if the G A B A - L I R did not extend throughout the whole cell body, however. No

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GABA-LIR was found in the female RA; neither the somata nor the neuropil of the female R A were readily distinguishable from background. In males, the HVC had a large number of loosely packed somata and neuropil with light to moderate GABA-LIR (Fig. 1, bottom). This pattern completely filled HVC, so that the boundaries in GABA immunoreacted sections were the same as HVC boundaries in Nissl stained material [2]. The intensity of GABA-LIR in the HVC of males was variable among individuals. As was the case in RA, the size of HVC neurons with GABA-LIR was somewhat smaller than that usually reported for male HVC neurons (mean = 59.2 /xm 2, range 11-215 /~m 2) (Fig. 2). The HVC of females never showed any evidence of GABA-LIR. In males, Area X contained numerous small neurons (range 6-146 t~m 2) and neuropil with light GABA-LIR so that it could be easily distinguished from the surrounding LPO, which had less intense GABA-LIR (Fig. 3, top). As with Nissl-stained material, the G A B A antibody did not reveal a recognizable Area X in females. Nevertheless, numerous small somata (range 13-91 /xm 2) with light GABA-LIR were found in the corresponding lateral LPO of females. Almost every neuron appeared to be labeled in both the male Area X and the female LPO. In both males and females these neurons often occurred in clusters of 4 to 6 cells, and there was no sex difference in soma size, tl0 = 0.79, P > 0.10 (Fig. 2). Numerous small cells

I • Males []

Females

80--

Fig. 3. Top: photomicrograph of G A B A - L I R in Area X (demarcated on the medial and ventral borders by open arrows), IMAN (demarcated by small thin solid arrows), and scattered cells in medial M A N (indicated by solid arrow with large arrowhead) of a male. Medial is to the left (lateral ventricle can be seen), dorsal is toward the top. Bottom: photomicrograph of G A B A - L I R in the D M of the ICO (indicated by the open arrow) and the MLd (indicated by the filled arrow). Dorsal is up and medial is right. Bars = 1 mm.

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~4020-

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m A R E A X/ LPO

IMAN

ND HVC

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Fig. 2. Mean soma area of G A B A - L I R neurons of both males and females as a function of various regions. ND = somata not detectable with G A B A - L I R in the H V C and R A of females. Bars represent the S.E.M.

and neuropil with GABA-LIR have also been reported in the pigeon LPO [20]. In both males and females, MAN was difficult to distinguish from background since GABA-LIR was barely above background in the neuropil, although occasional puncta were seen in IMAN. Large neurons with intense G A B A - L I R were widely scattered throughout both the lateral and medial divisions of MAN (Fig. 3, top). MAN in males and females did not appear notably different. Only neurons in the lateral

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division of MAN were selected for morphometric analyses. The ranges of soma sizes were quite similar between the sexes (male's range 12.9-157.8 tzm 2, female's range 16.7-160.3 tzm2), and there was no sex difference in the soma size of neurons with GABA-LIR, tx0 = 0.61, P > 0.10 (Fig. 2). The relatively small size of IMAN neurons with GABA-LIR probably was not due to the type of fixative or histological procedures used since 1MAN neurons as large as the largest identified by Bottjer [11] (330/zm 2) could be found after counterstaining with thionin. In both males and females, the DLM had no detectable cell bodies with GABA-LIR but did have extensive puncta with intense GABA-LIR in both males and females. The dorsomedial nucleus (DM) of the ICo could sometimes be distinguished from the surrounding ICo and from the dorsal nucleus of the lateral mesencephalon (MLd), an auditory nucleus contiguous with the DM of the ICo. Large neurons and neuropil with GABA-LIR were clearly present in both the DM of the ICo and the MLd (Fig. 3, bottom). Studies of the pigeon brain also report large cell bodies with GABA-LIR in the MLd and fibers with GABALIR in the ICo [20,21].

4. Discussion The data clearly show that GABA-LIR is present throughout the song system of the male zebra finch. Males have much more intense GABA-LIR than do females in RA, HVC, and Area X. Since only zebra finch males acquire song during development and sing as adults, GABAergic neurons may be involved in either song acquisition a n d / o r production of song. GABA-LIR in the song system in RA is dramatic and distinct in males and appears to be absent in females. The presence of GABA has also been detected in media bathing zebra finch RA slices after stimulating the presumptive HVC-to-RA pathway [55]. Chemical stimulation of slices containing RA in vitro produced a sex difference in the absolute amount of GABA released; males released a greater amount [55]. Although we found no evidence of GABA-LIR in the female RA, slices containing the female RA were found to release GABA upon chemical stimulation [55]. It is possible that our histochemical methods were not sufficient to detect GABA in the female RA. Alternatively, the GABA collected by stimulation of slices may not have come from RA but from surrounding tissue, perhaps from nucleus taeniae which has a high level of GABA-LIR. The presence of GABA in RA also has been confirmed electrophysiologically in slices of male zebra finch brain [39]. Stimulation of either the HVC-to-RA pathway or the MAN-to-RA pathway results in an EPSP followed by an IPSP in RA

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neurons. These IPSPs can be eliminated by bicuculline, a GABA A antagonist, suggesting that there are GABAergic interneurons in RA. The finding of GABA-LIR in the male zebra finch HVC parallels a similar finding in the canary HVC [16,51]. The size of HVC neurons with GABA-LIR is smaller than that usually reported for male HVC neurons [11,14,37,45], and corresponds well with the size of HVC neurons that do not accumulate androgens [11]. Although the HVC neurons that project to RA tend to be smaller in size [32], the neurons with GABA-LIR were probably not projection neurons. In the canary, HVC neurons with GABA-LIR are not found to project to either RA or Area X and are believed to be interneurons [16]. Secondly, stimulation of the HVCto-RA pathway in in vitro slices results in EPSPs followed by IPSPs [39]. The application of 6-cyano-7nitoquinoxaline-2,3 dione (CNQX), an antagonist of the quisiqualate glutamate receptor, abolishes both the early EPSP and the later IPSP [39]. This effect is consistent with the interpretation that excitatory HVC projection neurons excite inhibitory interneurons in RA but is not consistent with the interpretation that there are GABAergic projection neurons from HVC to RA. Our data also indicate that there is a profound sex difference in GABA-LIR in HVC; we found no GABA-LIR in females. In both RA and HVC, there are bigger sex differences revealed by GABA immunocytochemistry than by Nissl stains. In Nissl-stained material, RA and HVC are smaller but still distinctly present in females [2,46], whereas we found a complete absence of GABA-LIR in both the RA and HVC of females. Several possible mechanisms could have led to the sexual dimorphism of GABA-LIR in these nuclei. This dimorphism could be caused by differential generation or survival of GABAergic neurons as a result of steroid hormone actions during development. Indeed, this sex difference in GABA-LIR may actually be a part of the causal chain that ultimately gives rise to the dramatic sex differences in these nuclei. The presence of GABAergic cells in males may influence development so that the RA and HVC grow much larger in males than females. Since GABAergic neurons are influenced by steroid hormones in other systems, hormones may also play a role in this process [17,18,30,38,48,50,52,65]. Finally, the extreme differential expression of GABALIR in these nuclei may be due to the activational effects that male and female steroid hormones have in adulthood. There was also a profound sex difference in Area X / L P O in that the neurons had greater GABA-LIR in Area X relative to the rest of the LPO in males but not females. Since Area X projects to DLM [13,31], the puncta that we observed with GABA-LIR in DLM may be projection neurons from Area X. Nevertheless, we

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also found puncta in the DLM of females. Whether or not the corresponding LPO of females also projects to DLM is unknown. In any case, the puncta present in DLM are not likely to be projections from local interneurons since we never found any cell bodies with GABA-LIR in DLM. Scattered neurons with GABA-LIR were present in the lateral division of MAN (IMAN), which projects to RA in males [13], and the medial division of MAN, which projects to HVC [13]. The presence of puncta in 1MAN suggest that there may be terminals of GABAergic neurons in IMAN, probably from interneurons within MAN since the only known source of fibers afferent to the 1MAN is the DLM [13,31], in which we found no somata with GABA-LIR. The size of 1MAN neurons with GABA-LIR is smaller than that usually reported [36,45], but it corresponds well to the size of MAN neurons that do not accumulate androgen and do not project to RA [11,33]. Although sex differences in the size of 1MAN neurons seem to be rather robust [36], we did not find any sex difference in the size of IMAN neurons with GABA-LIR. Sex differences in the distribution of GABA-LIR are paralleled in various degrees by sex differences in other neurotransmitter systems. The closest similarities are those found in tyrosine hydroxylase distribution [12]. Like GABA-LIR, tyrosine hydroxylase is found in the male but not female RA, is much more prominent in Area X of males than females, and is much more pronounced in the HVC of males than females. Nicotinic ACh receptors also parallel the sexually dimorphic distribution of GABA-LIR in HVC; they are present in the HVC of males but not females [68]. The sexually dimorphic distribution of GABA-LIR in Area X / L P O corresponds to sexually dimorphic distributions of catecholamines, cholinesterase, and muscarinic receptors [5,12,35,53]; in each of these, Area X can be seen in males but not females. GABA-LIR distribution did not conform to all reported sexual dimorphisms in neurotransmitter system distributions. Tyrosine hydroxylase is found in the male but not female MAN [12], and the terminal field of enkephalinergic and V1P neurons is smaller in the female MAN [4]. In contrast, GABA-LIR in MAN seemed to be identical in both males and females. Our data suggest that since GABA-LIR is found throughout the male song system, GABAergic neurons may play a role in the acquisition a n d / o r production of song. Lesion studies have shown that lesions of MAN, Area X, and DLM interfere with the acquisition of song and that RA, HVC, and the DM of the ICo are involved in the production of song [14,25,47,59,60,62]. Since our data show GABA-LIR in these nuclei, GABAergic neurons may play a role in the acquisition and production of song.

Acknowledgements We would like to thank Lori Miyasato for expert technical assistance and Anne-Marie Schaaf for assistance in editing the manuscript. This research was supported by NIH NRSA 1 F32 NS09040 to W.G. and USPHS DC00217 to A.P.A..

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