THE JOURNAL OF COMPARATIVE NEUROLOGY 314:467477 (1991)

The Distribution of Neurotransmitters and Neurotransmitter-Related Enzymes in the Dorsomedial Telencephalon of the Pigeon (Columba livia) JOHN R. KREBS, JONATHAN T. ERICHSEN, AND VERNER P. BINGMAN Edward Grey Institute of Field Ornithology, Department of Zoology, Oxford OX1 3PS, England (J.R.K.); Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, New York 11794 (J.T.E.); and Department of Psychology, Bowling Green State University, Bowling Green, Ohio 43403 (V.P.B.)

ABSTRACT Immunoreactivity to four neurotransmitters/ transmitter-related enzymes was found in the dorsomedial telencephalon (hippocampal region) of the pigeon. Putative afferent fibers containing choline acetyltransferase-like, serotonin-like, and tyrosine hydroxylase-like immunoreactivity were seen in a fiber tract passing through the septo-hippocampal junction and along the medial wall of the hippocampal region. The most intensive labeling of neuropil and terminals of all four substances was found in the dorsomedial area of the hippocampal region. Glutamic acid decarboxylase-like immunoreactivity was seen in sparsely scattered cells throughout the region. These results are discussed in relation to hypotheses about the boundaries and subdivisions of the hippocampal region of the pigeon. Key words: avian hippocampal region, choline acetyltransferase, glutamic acid decarboxylase,

serotonin, tyrosine hydroxylase, immunohistochemistry

The hippocampal complex is now recognized as being crucial for the formation or processing of certain kinds of memory in mammals (Rawlins, '85; Mishkin and Appenzeller, '87; Barnes, '88; Greenough and Bailey, '88). The nature of behavioral deficits in memory resulting from hippocampal lesions is still a matter of debate, but many authors have suggested that the hippocampus at least plays an important role in spatial memory (O'Keefe and Nadel, '78; Morris et al., '86a,b). Recently, a number of lesion studies have indicated that the avian dorsomedial telencephalon (hippocampal and parahippocampal regions of Karten and Hodos, '67) plays a role in memory and thus has some functional similarity to the mammalian hippocampus (Bingman et al., '84, '88a,b; Good, '88; Sherry and Vaccarino, '89). Further, anatomicaI evidence that the avian hippocampal region may be homologous with the mammalian hippocampal formation includes similarities in a) topological organization with respect to the lateral ventricle (Craigie, '351, b) the three-layered organization (Durward, '32; Craigie, '35) and the diversity of cell types (Molla et al., '86; Pisana, '86; Smith, '891, and c) the sources of both afferents and efferents (Benowitz and Karten, '76; Casini et al., '86; Bingman et al., '89). In spite of this evidence for functional similarity and anatomical homology, cursory examination of the avian O 1991 WILEY-LISS, INC.

dorsomedial telencephalon (hereinafter referred to as the hippocampal region, essentially equivalent to the wulst regio hippocampalis of Shimizu and Karten, '90) reveals a structure whose cytoarchitectural organization does not closely resemble the hippocampal formation of mammals. The areas in the avian hippocampal region that might correspond to subdivisions such as the dentate gyrus, hilar region, and Ammon's Horn of the mammal are not immediately apparent. Some early workers attempted a characterization of subdivisions of the avian hippocampal region (e.g., Craigie, '30, '35, '40;Durward, '32; Ariens-Kappers et al., '361, but no consensus was reached. Thus, the extent and significance of cytoarchitectural differences between the avian and mammalian hippocampal formations remain obscure. If the avian hippocampal region has a similar function to that of mammals but a different regional organization, this might lead to new insight about the relationship between structure and function in this region. Immunohistochemistry for neuroactive substances has been used extensively to analyze the organization of the mammalian hippocampal formation (Amaral and Campbell, '86; Sloviter and Nilaver, '87; Frotscher et al., '88). Although this approach has been helpful to demonstrate Accepted September 4, 1991.

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additional features of specialization in this region, equivalent data are not currently available for birds. In this and a companion paper (Erichsen et al., ’911, we describe the distribution of neurotransmitters and other neuroactive substances in the hippocampal region and some adjacent areas. This characterization of the neurochemistry of the hippocampal region will enable us to provide a basis for a) defining subdivisions and boundaries, b) suggesting afferent and efferent systems containing specific neurochemicals, and c) investigating possible homologies between the regions of the hippocampal formation of birds and mammals. The procedure of using immunohistochemical evidence to suggest hypotheses about homologies between avian and mammalian brain regions has previously been used in other systems (e.g., Cassone and Moore, ’87). In the present paper, we examine the distribution of a neurotransmitter (serotonin [5HTJ) and two transmitterrelated enzymes (choline acetyltransferase [ChAT] and tyrosine hydroxylase [TH]) which have been found in mammals to be associated with afferents to the hippocampus (Frotscher and Leranth, ’86, ’88; Zhou et al., ’88)and one transmitter-related enzyme (glutamic acid decarboxylase [GAD])which in mammals is associated primarily, but not exclusively, with local connections (Somogyi et al., ’83; Kohler et al., ’84; Frotscher et al., ’84). In the second paper, we describe the distribution of six neuropeptides. A preliminary summary of these two papers was presented elsewhere (Krebs et al., ’87).

METHODS Animals Four adult homing pigeons (Columba liuia) of both sexes were deeply anesthetized with pentobarbital intravenously. When the corneal reflex was absent, the heart was exposed and the animal was transcardially perfused with 100-200 ml of 0.75% (avian) saline followed by 500 ml of either 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) [PB] with 0.1% glutaraldehyde added in one bird. The brains were then removed, placed in the perfusion fixative for an additional 3-4 hours at 4”C,and transferred to 30% sucrose in PB containing 0.01% sodium azide (as a preservative) for 3-4 days.

Immuno histochemistry The brains were cut frozen on a sliding microtome at a thickness of 30 pm in the transverse plane defined by the brain atlas of Karten and Hodos (’67). Sections were collected serially into PB containing 0.05-0.1% sodium azide. Sections from the brain fixed with 0.1% glutaraldehyde (see above) were pretreated in 1%sodium borohydride (in 0.1 M PB) for 5 minutes and then given three 10 minute washes in 0.1 M PB. For each antibody, a 1 in 12 series of sections through the brain was washed in PB and then in 0.05 M and 0.025 M PB for 2-3 minutes. This was followed by a 10 minute wash in 0.3% H,O, and successive brief washes in 0.025 M, 0.05 M, and 0.1 M PB. Groups of several

Abbreviations

APH CDL HP HA TSM

area parahippocampalis area corticoidea dorsolateralis hippocampus hyperstriatum accessorium tractus septomesencephalicus

sections were then incubated in 1.5%normal serum ( C U T , 5HT, TH: goat; GAD: rabbit) in 0.1 M PB containing 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO) [PBX] for 30 minutes at room temperature [RT], by using 1.5 ml plastic centrifuge tubes on a rotator. Following a brief wash in PB, the sections were incubated in primary antibody (see below) diluted in PBX with 1.5% normal serum for 65-89 hours at 4°C. Three 10 minute washes in PB preceded a 1 hour incubation at RT in a biotinylated IgG (Vector Labs, Burlingame, CAI (diluted 1:200 in PBX) directed against the species in which the primary antibody was made (see below). After washing in PB, the sections were incubated in ABC solution (Vector Labs, Burlingame, CA; 1:50 dilution) for 1 hour, washed in PB, and placed in a solution of 3-3’ diaminobenzidine tetrahydrochloride (DAB) (55 mgi 100 ml PB) for 15 minutes and then in DAB with 0.03%H,O, for an additional 15 minutes. Finally, the sections were washed in PB and mounted sequentially onto gelatin-coated glass slides. When completely dry, the slides were placed in 0.05% osmium tetroxide for 15 seconds, rinsed in distilled water briefly, dehydrated, cleared, and coverslipped with Permount. The specificity of immunostaining was investigated in several different ways. First, a standard dilution series was carried out for each antiserum on selected sections to ascertain the optimal dilution at which maximum contrast was obtained between “signal” and diffuse, background staining. Second, occasional sections were incubated either 1) without the primary antiserum, or 2 ) with a primary antiserum made in the same animal (e.g., rabbit) but directed against an antigen not present in the brain (e.g., wheat germ agglutinin). In the absence of any primary antiserum, the sections were invariably devoid of any immunoreaction product; in fact, the tissue was virtually translucent. When an inappropriate primary antiserum was used, the tissue was slightly colored but never displayed any of the distinctive staining patterns observed in the case of any of the antibodies used in this or the companion study (Erichsen et al., ’91). In any event, the biochemical identity of the antigen for each of these antibodies cannot be established for certain from immunohistochemical analysis alone. Therefore, we use the term “-like immunoreactivity” to indicate that the substance present in the brain is likely to be structurally similar to the intended antigen.

Antibodies The initial range of dilutions used was 1500-1:4,000. Optimal staining patterns were usually obtained at a dilution of 1:1,000-2,000 ( C U T , 5HT, TH) or 1:3,0004,000 (GAD). The localization of ChAT in the hippocampal region was carried out with a polyclonal antiserum directed against chicken choline acetyltransferase (Johnson and Epstein, ’86).The two different bleeds used (B9 and 2017) produced virtually identical immunoreactive staining patterns. In some cases, antisera were purified by a preadsorption step prior to the incubation with normal goat serum. Sections from the cerebellum of the same brain were incubated with the antiserum (at one half the final dilution used in our study) for 1 hour on a rotator. The antiserum was then removed from the Centrifuge tube with a pipette and diluted 1:l with additional PBX to make the final dilution for use on the experimental brain sections. Glutamic acid decarboxylase (GAD)-likeimmunoreactivity was localized using an antiserum (number 1440-4; NIMH)

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DORSOMEDIAL TELENCEPHALON OF PIGEON characterized previously by Oertel et al. ('81a,b; '82). Serotonin (5HT)-like immunoreactivity was localized with a polyclonal antiserum (20080; INCSTAR), which has been characterized previously by Beitz ('821, Willcockson et al. ('87), and McLean and Shipley ('87). Tyrosine hydroxylase (TH)-like immunoreactivity was localized with an antiserum (TE101; Eugene Tech) with well-established specificity (Pickel et al., '75; Hervonen et al., '80; Armstrong et al., '81).

Mapping the distribution of immunoreactive staining The areas under study primarily include the hippocampus (Hp) and parahippocampus (AF'H) as described by Karten and Hodos ('67) (Fig. 1).We also include portions of areas immediately lateral to APH, i.e., hyperstriatum accessorium (HA) and the dorsolateral cortex (CDL). For descriptive purposes only, we have arbitrarily defined three regional subdivisions: ventromedial (VM), dorsomedial (DM), and dorsal (D) areas (Fig. 2). VM is characterized by a v-shaped arrangement of cells that stain heavily for Nissl substance (we refer to this as the V) and is essentially the region defined as Hp by Karten and Hodos ('67). At the dorsal end of the lateral arm of the V, horizontal and vertical lines are drawn parallel to the horizontal and vertical axes of the atlas. The ventromedial area (VM) includes the V and all adjacent areas below the horizontal line and medial to the vertical line. The dorsomedial area (DM) is the area dorsal to the horizontal line and medial to the vertical line. In addition, DM is further subdivided into superior (DMs) and inferior (DMi) halves. DM corresponds to the more medial part of APH. The dorsal region (D) is lateral to the vertical line and dorsal to the ventricle. Only that portion of D medial to lateral 6.0 (Karten and Hodos, '67) has been included in the chartings of the immunoreactive staining presented here. D consists of the lateral extent of APH as well as portions of HA rostrally and CDL caudally. For each antibody series, four sections were selected that corresponded most closely to the levels of A9.00, A7.50, A6.00, and A3.75, which denote millimeters anterior to the external auditory canals, the posterior point of fixation for the atlas of Karten and Hvdos ('67) (Figs. 1 and 2). These levels, which were chosen so as to span the rostral-caudal extent of the hippocampal region, were used to illustrate the distribution of immunoreactive staining for all the antibodies examined. Our results are summarized in two series of four schematics, each showing the distribution of immunoreactive staining for two antibodies at the four levels we chose to illustrate. For reasons of clarity, only intermediate and heavily labeled areas are depicted in the schematics. Very sparsely or lightly labeled areas are described in the text. In addition, the V is omitted so as to avoid obscuring the details of the immunoreactive staining patterns.

RESULTS Choline acetyltransferase (ChAT) CUT-like immunoreactivity is found in fine, unbeaded fibers passing through the septohippocampal junction and continuing in a fiber tract that runs dorsally, close to the medial wall, in VM and DMs (the "medial fiber tract") (Fig. 3A), shown by Bagnoli and Burkhalter ('87) to be part of the tractus septomesencephalicus (TSM). These fibers also

occur throughout the area of the hippocampus in the anterior region (A9.00-A7.50) and in a fiber tract that runs close to the dorsal surface of DMs and D (the "dorsal fiber tract") at A7.50 and A6.00. In certain instances, the fibers of the medial and dorsal fiber tracts are seen to be continuous. There is a lightly labeled neuropil with scattered fine beaded fibers in DMs and the mediodorsal portion of D, this latter region being most densely labeled in the caudal section (A3.75). The field of labeled axons extends further laterally and medially in the rostral sections (Fig. 4A-D). Prominent ChAT-like immunoreactivity is also observed in cells of the diagonal band of the ventral septum (Fig. 3B); these cells are a possible source of cholinergic afferents to the hippocampal region (see Discussion).

Glutamic acid decarboxylase (GAD) In the rostral portion (A 9.001, GAD-like immunoreactivity is seen in two areas of densely stained neuropil (Fig. 4A-D), the larger of which is situated in DMs and D (adjacent to the dorsal surface) and the other in DMi extending into VM on the medial wall. In these areas, the densely stained neuropil is interspersed with labeled short beaded fibers and small unstained cell bodies (see Fig. 6A). Proceeding caudally, the medial area appears to move dorsally into DMs where it becomes continuous with the larger, more dorsolateral, densely stained neuropil area. The densely labeled neuropil is surrounded by an area of less dense staining, which in A7.5 shows an obvious gap extending dorsally from the dorsal surface of the ventricle (Fig. 4B). In the caudal-most portion, labeling of neuropil is restricted to a small region in DMs, and there is an additional lightly stained area of neuropil close to the medial wall. In addition to the areas of dense and light staining of neuropil indicated in Figure 4A-D, staining for GAD-like immunoreactivity throughout the hippocampal region is more marked than in many other dorsal forebrain areas. Throughout the hippocampal area, there are small to medium-sized cells with diffusely labeled cytoplasm (see Fig. 6B,C), a small proportion of which are surrounded by very densely stained bouton-like terminals. Associated with these cells are two kinds of processes: thin beaded processes and much thicker processes also covered in bead-like terminals or spines. In the caudal section (A3.751, the stained cells tend to be more abundant near the dorsomedial neuropil area, while in the rostral sections (A9.00-A6.00), they tend to be found in VM between the arms of the V.

Serotonin (5HT) 5HT-like immunoreactivity is seen in thick axons passing through the septohippocampal junction and in the medial fiber tract (Fig. 3C). Four aspects of these axons are worth noting: a) Some appear to give off collaterals which may terminate on cells in VM, between the arms of the V, and on cells in the lateral arm of the V. b) Many of the fibers appear to ascend in the medial fiber tract and terminate in DMs (also in DMi rostrally, A9.00) and in medial D, where a region of densely stained neuropil and fine beaded fibers is seen (Figs. 5A-D, 6D). c) Some fibers pass through the septohippocampal junction and then diverge laterally from the medial fiber tract to enter the area of densely labeled neuropil in DMs and D. d) The medial fiber tract changes its orientation by the caudal-most section (A3.75); instead of

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Fig. 1. Photomicrographs of four transverse Nissl- and KluverBarrera-stained sections (A-D) through the left hippocampal region of the pigeon, chosen to correspond, respectively, to four rostral-caudal levels (k, A9.00, A7.50, A6.00, and A3.75) defined by the atlas of Karten and Hodos ('67). In Figures 4 and 5, the distribution of

J.R. KREBS ET AL.

immunoreactivity for each neurotransmitteritransmitter-relatedenzyme is presented at each of these levels (see text and Fig. 2). Note the location of the ventricle (v).Arrowheads indicate the cells of theV of the hippocampus (Hp). Bar, 500 km.

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beled cells. These varicosities are much more numerous in a second area of heavier staining in the lateral part of D (Fig. 3E inset) which continues throughout more lateral cortical areas. Because this area of staining extends into lateral cortical areas, we suggest that its medial limit marks the transition between the hippocampal region and lateral cortex. Labeled beaded terminals are sparsely distributed in VM. Labeled cells are seen in the locus coeruleus and in the midbrain area ventralis of Tsai (Fig. 3F), one or both of which could be the source of catecholaminergic afferents to the hippocampal region.

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The major features of our results are as follows: a) All four transmitter/ transmitter-related enzymes examined in this study occur in the hippocampal region of the pigeon. b) Putative afferent fibers containing ChAT-like, 5HT-like, or TH-like immunoreactivity are seen in a fiber tract that passes through the septohippocampal junction. c) All four substances are found in neuropil and terminals, especially in more dorsal areas (DM and D), although sparse terminals are found throughout the hippocampal region. d) Of the four substances, only GAD-like immunoreactivity is seen in cell bodies, and these are found scattered throughout the hippocampal region.

Subdivisions of the avian hippocampal region Fig. 2. A schematic showing the arbitrary subdivisions of the left hippocampal region of the pigeon and some adjacent areas (see text). The four panels (A-D) refer, respectively, to four levels of the Karten and Hodos ('67) atlas (A9.00, A7.50, A6.00, and A3.75) (see also Fig. 11, and the scale marked around the edge refers to the atlas coordinates (in mm). D, dorsal region; DMs, superior part of dorsomedial region; DMi, inferior part of dorsomedial region; VM, ventromedial region.

being aligned in a ventral-dorsal direction, it is oriented in an oblique plane (Fig. 3C inset). There is a second, smaller area of densely labeled neuropi1 close to the medial wall in DMi at the point where the medial fiber tract becomes more diffuse (where fibers diverge laterally) in A9.00. This dense neuropil area appears to move dorsally and laterally into DMs in more caudal sections (A7.50-A3.75) (Fig. 5A-D). It remains distinct from the larger area of dense neuropil in D. Both densely stained neuropil areas are surrounded by a continuous area of less heavily labeled neuropil (Fig. 5A-D). Between the medial fiber tract and the medial wall, there are labeled beaded fibers and terminals throughout the rostral-caudal extent. Prominent staining of cells with 5HT-like immunoreactivity is seen in linearis caudalis (medial raphe nucleus) (Fig. 3D). These cells are possibly the source of afferents to the hippocampal region (see Discussion).

Tyrosine hydroxylase (TH) In A9.00 and A7.50, TH-like immunoreactivity is seen in fine fibers passing along the medial fiber tract and running along the lateral arm of the V (Fig. 3E). Diffuse terminations are seen in the mediodorsal portion of D rostrally (A9.00 and A7.50) and in DMs in more caudal sections (Fig. 5A-D). This area also contains some THimmunoreactive bouton-like varicosities surrounding unla-

The immunohistochemical evidence presented here suggests at least four major subdivisions of the hippocampal region of the pigeon: i) The medial fiber tract, containing putative afferents, some of which pass through the septohippocampal junction, extending to the dorsal surface. ii) A dorsal area (including dorsal DMs and dorsomedial regions of D) rich in terminals and neuropil. iii) The v-shaped area of large cells in VM, readily visible in Nissl material but not particularly highlighted by any of the substances studied here. iv) An area including DMi and VM between the arms of the V which contains sparse terminals immunoreactive for all four substances and cells immunoreactive for GAD. A fifth area, in lateral D, identified by TH-positive basket-like terminals around cells, is tentatively considered to be outside the hippocampal region (see below). There is some evidence to suggest further subdivisions of the dorsomedial area of neuropil. In A6.00 and A3.75, the location of the two areas of neuropil immunoreactive for 5HT is almost exclusive of that of areas containing TH- and ChAT-positive neuropil, while these latter two overlap considerably in their distribution. Thus, there are three bands, proceeding from medial to lateral, containing a) 5HT, b) TH and ChAT, and c) 5HT, respectively.

Boundaries of the hippocampal region The distribution of 5HT-like and TH-like immunoreactivity suggests a lateral boundary of the hippocampal complex. Medial to the boundary is a field of dense neuropil and terminations containing 5HT-like immunoreactivity, while lateral to the boundary, the density of TH-positive boutons surrounding cells is much greater than in areas medial to the boundary. The dense TH-like labeling continues laterally and ventrally well into the dorsolateral cortical region, suggesting that it is not part of the hippocampal formation. These two areas are contiguous in A6.00, but separated by a gap at more rostra1 and caudal levels. This putative lateral boundary is also indicated by the size distribution of cells

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Fig. 3. Photomicrographs of (A) ChAT-immunoreactive fibers in the left medial fiber tract (i.e.,TSM; see text) of the hippocampal region (A9.00); (B) CUT-positive cells in the diagonal band of Broca (A9.00); (C) 5HT-positive fibers in the right medial fiber tract (A7.00); inset: 5HT-positive fibers in the medial fiber tract showing oblique orientation in caudal sections (A3.75); (D) 5HT-positive cells in linearis

caudalis (A2.00); (El TH-positive fibers in the left medial fiber tract (A9.00) (indicated by arrowheads); inset: TH-positive varicosities surrounding cells in lateral D (A9.00) (small arrows); and (F) TH-positive cells in the left area ventralis of Tsai (A4.50).Bars, 50 pm (A-C,Ej; 100 pm (D,F); 25 pm (both insets).

seen in Nissl material (Krebs et al., '89) and by the distribution of neuropeptides (Erichsen et al., '91). With regard to the rostral-caudal limits of the hippocampus, our results show that the dorsal area of neuropil (region (ii) listed above) appears to move medially and

ventrally in more rostra1 sections. At the same time, the lateral field of TH-positive boutons is located further medially and ventrally, suggesting possible changes in the position of the lateral boundary of the hippocampal region. This might suggest that towards its anterior limit, the

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Fig. 4. (A-D) Schematics to show the distributions of C U T - and GAD-like immunoreactivity in neuropil and terminations in the hippocampal region at four different levels beginning at the rostra1 end and moving caudally (see Fig. 2). ChAT is indicated by horizontal shading,

and GAD by irregular dots and short lines. Two levels of GAD staining are distinguished: light and heavy. The distribution of GAD-positive cells is indicated by stars. CUT-positive fibers discussed in the text are not shown.

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Fig. 5. (A-D) Schematic distribution of neuropil and terminals immunoreactive for 5HT and TH. The diagonal stripes refer to TH-like immunoreactivity and the stippling to 5HT. Two levels of intensity of stippling are shown: dark dots correspond to heavy staining, light dots to light staining. Fibers are not shown.

DORSOMEDIAL TELENCEPHALON OF PIGEON

475 hippocampal region is reduced to a small region close to the medial wall in V or DMi. This occurs at a level of A11.00A11.50. The caudal limit appears to be at the posterior pole of the telencephalon.

Comparison with mammals All four substances studied here are also found in the mammalian hippocampal formation (Amaral and Campbell, '86). The most striking parallel with the mammalian hippocampal formation revealed by the present study is in the serotonergic, cholinergic, and catecholaminergic afferents within the medial fiber tract (subdivision i) listed above). Similar afferents are seen in mammalian fimbriafornix and in the alvear layer of the hippocampus, suggesting that the medial fiber tract is equivalent to the fimbriafornix and/or the alvear layer of mammals. The possible cells of origin of these afferents in the pigeon also show parallels with mammals. In mammals, cholinergic afferents are known to originate in the medial septum, diagonal band of Broca, and the basal nucleus of Meynert (Clarke, '85; Frotscher and Leranth, '861, serotonergic fibers arise from the medial raphe nucleus (Amaral and Campbell, '86; Zhou et al., '88), and catecholaminergic (TH-like immunoreactive) inputs to the hippocampal formation have been reported to come from various brainstem regions including the locus coeruleus (noradrenergic) (Loy et al., '80) and ventral tegmentum (dopaminergic) (Verney et al., '85; Frotscher and Leranth, '88). In the present study, we have observed cells containing CUT-like immunoreactivity in the diagonal band, 5HT in cells of linearis caudalis (medial raphe nucleus), and TH in cells of the locus coeruleus and the area ventralis of Tsai. Connectivity studies have shown that afferents from these areas are also found in the avian hippocampal region (Benowitz and Karten, '76; Casini et al., '86). Together with the present immunohistochemical evidence, this suggests that the major ascending afferents are similar in birds and mammals. The terminals and neuropil in the dorsal area of the pigeon hippocampal region (subdivision ii) presumably include terminations of the ascending afferents in the medial fiber tract, in addition to the GAD-positive terminals, which may primarily reflect intrinsic connections, as in the mammalian hippocampus (Somogyi et al., '83). Drawing a close parallel between the region of dense terminations in the pigeon and a particular part of the mammalian hippocampus is not straightforward, since GAD-, 5HT-, TH-, and CUT-positive terminations are distributed throughout the hippocampal formation of mammals (Amaral and Campbell, '86). Similarly, the occurrence of GAD labeling in cells scattered throughout the avian hippocampal region, while resembling the distribution in mammals (Somogyi et al., '83; Amaral and Campbell, '86), does not help to specify particular regional subdivisions. The occurrence of dense GAD-like immunoreactive terminals surrounding small cells in the dorsal area might suggest that this is equivalent to the granule layer of the dentate gyrus of mammals (Amaral and Campbell, '86). However, the present findings offer a somewhat limited data base for defining regional subdivisions of the pigeon hippocampus or making comparisons with the mammalian Fig. 6 . Photomicrographs of (A)GAD-positive neuropil surrounding small unstained cells in left DMs (A7.00); (B and C)GAD-positive hippocampus. A more thorough consideration of these cells in DMs (A6.00); and (D) 5HT-positive neuropil in right D M ~ issues would be facilitated by an investigation of the occurrence and distribution of neuropeptides in the pigeon (A4.50). Bars, 25 km (A-c); 100 Km (D). D M ~ ,superior part of dorsomedid region. hippocampus.

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ACKNOWLEDGMENTS We thank Angela K. Levine and Anne F. Bushnell for technical help and the following for financial support: SERC and Royal Society (JRK),NIH grant EY04587 (JTE), and NSF grant BNS8611204 (VPB). Dr. Peter Somogyi was most helpful in discussing the interpretation of the results and in commenting on a n earlier draft of the manuscript. Dr. Hugh Perry helped in the early phases of the project and commented on the manuscript. Dr. P. Bagnoli, Dr. C. Gall, Dr. R.O. Kuljis, Dr. R.Y. Moore, and Dr. A. Reiner commented on a draft of the manuscript. The antiserum to ChAT was generously provided by Dr. Miles Epstein (University of Wisconsin, Madison). Antiserum to GAD was provided through the laboratory of Clinical Science, NIMH where it was developed under the supervision of Dr. Irwin Kopin with Dr. Wolfgang Oertel, Dr. Donald E. Smechel, and Dr. Marcel Tappaz. Effective use in immunohistochemistry was greatly aided through the laboratory of Dr. Enrico Mugnaini (University of Connecticut, Storrs). Part of the work was done while JRK was a Visiting Professor in the Departments of Ecology and Evolution and Neurobiology and Behavior a t SUNY, Stony Brook. Dr. Jeff Levinton and Dr. Lorne Mendell are gratefully acknowledged for their hospitality.

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