Immunohistochemical analysis of the visual wulst of the pigeon (Columba livia).

THE JOURNAL OF COMPARA'I'IW NEUROLOGY 300.346369 (1990)

hnmunohistochemical Analysis of the Visual Wiilse of the Pigeon (Columbuliviu) TORU SHIMlzu AND HARVEY J. KARTEN Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0608

ABSTRACT The avian wulst, a laminated "bulge" in the dorsal telencephalon, contains several distinct regions. The posterolateral portion (visual wulst) has been proposed to be an avian equivalent of the mammalian striate cortex. The present study examines specific neurotransmitters and neuropeptides within the visual wulst by immunohistochemical techniques. Antisera and monoclonal antibodies against choline acetyltransferase ( C U T ) , nicotinic acetylcholine receptor (nAChR),tyrosine hydroxylase (TH), serotonin (5-HT),glutamic acid decarboxylase (GAD), gamma-aminobutyric acid A receptor (GABA,R), cholecystokinin (CCK), substance P (SP), leucine-enkephalin (L-ENK), neurotensin (NT), neuropeptide Y (NPY), somatostatin (SRIF), corticotropin-releasing factor (CRF), and vasoactive intestinal polypeptide (VIP) were used. Somata and neuropil displaying specific immunoreactivity were generally distributed in accordance with the laminar cytoarchitectonic organization of the wulst. The superficial layer of the wulst, the hyperstriatum accessorium, contained the highest densities of TH-, 5-HT-, SP-, NPY-, SRIF-, CRF-, and VIP-positive neuropil in the wulst, whereas the highest density of CCK- and NT-staining was found in the deepest layer of the wulst, the hyperstriatum dorsale. In addition to the traditionally defined four laminae of the wulst, the immunoreactive staining revealed several subregions within each lamina. The most dorsolateral portion of the wulst contained the highest densities of C U T - and L-ENK-stained fibers in the wulst, as well as moderately dense staining of neuropil for 5-HT-, TH-, SP-, and CCK-like immunoreactivity. The nAChR-immunoreactivity was faint and distributed rather uniformly throughout the wulst. The results suggest that the wulst consists of multiple regional variations within layers comparable to laminar variations found within different cytoarchitectonic areas of the mammalian neocortex. Key words: striate cortex, chemoarchitecture,birds

Comparative studies of the organization of the avian and mammalian telencephalon may provide valuable clues for understanding the evolution of neocortex. The avian forebrain contains two major regions that appear directly comparable to the mammalian neocortex: the dorsal ventricular ridge and the wulst. These two regions appear to have different evolutionary histories and morphologies, yet functionally, both closely resemble the mammalian neocortex (Karten and Shimizu, '89). Ariens Kappers et al., ('36) suggested that the lateral portion of the wulst is the "vicerious neocortex" of birds on the basis of its position, connections, and cytoarchitectonic organization. More recent studies have provided evidence that a major portion of the wulst is directly comparable to the striate cortex. The wulst ("bulge") is a parasagittal elevation located in the dorsomedial region of the avian hemisphere. The wulst csnsists of at least two distinct regions (see Fig. 1): 1) a medial portion purportedly similar to the hippocampus and associated areas (wulst regio hippocampalis, Wrh); and 2 ) a lateral portion, including regions comparable to some senO 1990 WILEY-LISS, INC.

sory areas of the mammalian neocortex (wulst regio hyperstriatica, Whs). The Whs is similar to the neocortex in that it has a laminar configuration (Karten et al., '73; Pettigrew, '79; Reiner and Karten, '831, equivalent afferent and efferent connections (Adamo, '67; Karten, '71; Delius and Bennetto, '72; Hunt and Webster, '72; Karten et al., '73; Meier et al., '74; Miceli et al., '75, '79, '87; Bagnoli et al., '80, '82b; Streit et al., '80a,b; Bravo and Pettigrew, '81; Meatres and Delius, '82; Miceli and Reperant, '82, '83, '85; Nixdorf and Bischof, '82; Bagnoli and Burkhalter, '83; Reiner and Karten, '83; Wild, '87), similar single-unit responses (Revzin, '69; Perisic et al., '71; Pettigrew and Konishi, '76; Miceli et al., '79; Wilson, '80; Denton, '81), and extensive monoaminergic innervation (Bagnoli and Casini, '85; Yamada and Sano, '85). The anterior portion of Whs is considered to be the avian equivalent of the somatosensory/motor cortex of mammals (Delius and Bennetto, '72; Karten et al., '78; Wild, '87; Funke, '89a,b), the posterior portion to the Accepted June 27,1990.

IMMUNOHISTOCHEMICAL STUDY OF THE AVIAN WULST striate cortex (Karten et al., '73; Karten, '79; Bagnoli et al., '82b) and the medial portion of Whs may be comparable to the limbic cortex (Karten et al., '73; Berk and Hawkin, '85). The topological relationships of these areas in the wulst are schematically shown in Figure 1. There are, however, major differences between the avian m'hs and the mammalian neocortex. For example, the topological relationships of laminae with particular afferent and efferent connections is partially inverted in the wulst. In both mammals and birds, specific thalamic projections terminate mainly, but not exclusively, in a granular layer (layer IV of the mammalian cortex). However, in the mammalian cortex, the supragranular cortical layers (I1 and 111) are the major source of the intratelencephalic projections of the neocortex, while neurons of infragranular cortical layers (V and VI) are the source of both extratelencephalic and intratelencephalic efferents (Lund et al., '75; Gilbert and Kelly, '81; Gilbert, '83). In contrast to the mammalian neocortex, the supragranular layer of the wulst is the source of both the direct extratelencephalic projection (Karten et al., '73; Bagnoli et al., '80; Bravo and Pettigrew, '83 ; Reiner and Karten, '83) and an intratelencephalic projection (Shimizu et al., 'go), and the infragranular layer appears to send an output to the intratelencephalic targets (Ritchie, '79; Shimizu et al., '89, '90). In order to further analyze the characteristics of the wulst, immunohistochemical techniques were used in the present study to examine the distribution of a wide variety of neurotransmitters and neuropeptides within the wulst of the pigeon. These techniques have provided a powerful tool for the study of characteristics of the specific neuronal populations within cortical laminae of mammals, particularly of the visual cortex (see Foote and Morrison, '87, for a review). Assuming that the avian wulst and mammalian neocortex are directly comparable, analysis of the similarities and differences of chemical anatomy between the two structures may be of value in clarifying the potential diversity and nature of the evolutionary process within the brain (Karten, '69; Nauta and Karten, '70). We surveyed the distribution of the following substances: one neurotransmitter-serotonin (5-hydroxytryptamine, 5-HT); three enzymes-1) choline acetyltransferase (ChAT; the rate limiting enzyme in the synthesis acetylcholine),2) tyrosine hydroxylase (TH; the first enzyme in the biosyn-

347

thetic pathway for the formation of catecholamine), 3) glutamic acid decarboxylase (GAD; the enzyme for gammaaminobutyric acid); two receptors-1) nicotinic acetylcholine receptor (nAChR),2) GABAAreceptor (GABAARthe GABA, subtype of the gamma-aminobutyric acid receptors); and seven neuropeptides-1) substance P (SP),2) leucineenkephaline (L-ENK), 3) neuropeptide Y (NPY), 4) neurotensin (NT), 5) somatostatin (somatotropin release-inhibiting factor, SRIF), 6) corticotropin-releasing factor (CRF), and 7 ) vasoactive intestinal polypeptide (VIP). All the substances surveyed have been previously observed in the mammalian telencephalon. On the other hand, only limited information is available concerning the distribution of these substances in the wulst. We have emphasized potential similarities of the posterior Whs and the visual cortex mainly because of the large body of information available on the visual cortex. However, the readers should keep in mind that both Wrh and Whs contain several subdivisions representing other cortical areas. Only a few of the subdivisions of the wulst have been characterized to date. Cytoarchitectonically,the Whs is a laminated structure with at least four constituents. These include, from the dorsal surface inward, the hyperstriatum accessorium (HA), the intercalated nucleus of the hyperstriatum accessorium (IHA),the hyperstriatum intercalatus superior (HIS), and the hyperstriatum dorsale (HD) (Karten et al., '73). The Wrh contains hippocampus (Hp), area parahippocampalis (APH), entorhinal area of Rose ('14),and/or subiculum of Cragie ('30, '32), although the boundary with Whs has not been previously sharply defined (Karten and Hodos, '67). Ventrally, the wulst is separated rather distinctively from the hyperstriatum ventrale ( H V ) by the lamina frontalis superior (LFS) (Karten et al., '73). Laterally, the area extends from the midline to the uallecula (Val, a shallow groove separating the wulst from the area corticoidea dorsolateralis (CDL) (Karten and Hodos, '67). A preliminary report of the following findings have been presented elsewhere (Shimizu et al., '87).

MAWSANDMETHODS The results presented here are part of a large study of the chemical anatomy of the telencepahlon in white Carneaux

Abbreviations 5-HT APH APHd APHv

CCK CDL ChAT

CRF GABA,R GAD HA HAd HAV HDI HDm HD-shell HIS HL HP

serotonin area parahippocampalis dorsal portion of the area parahippocampalis ventral portion of the area parahippocampalis cholecystokinin area corticoideadorsolateralis choline acetyltransferase corticotropin-releasing factor gamma-aminobutyric acid A receptor glutamic acid decarboxylase hyperstriatum accessorium dorsal portion of the hyperstriatum accessorium ventral portion of the hyperstriatum accessorium lateral portion of the hyperstriatum dorsale medial portion of the hyperstriatum dorsale area, with an intense substance P-like immunoreactivity, adjacently dorsal to the caudal hyperstriatum dorsale hyperstriatum intercalatus superior hyperstriatum laterale hippocampus

Hv IHA L-ENK LFM LFS LFSl LFSm nAChR NPY NT

SP SRIF

sv

TH

V Va VIP WhS

Wrh

hyperstriatum ventrale intercalated nucleus of the hyperstriatum accessorium leucine-enkephalin lamina frontalis superema lamina frontalis superior lateral portion of the lamina frontalis superior medial portion of the lamina frontalis superior nicotinic acetylcholine receptor neuropeptide Y neurotensin substance P somatostatin supraven tricular area tyrosine hydroxylase ventricle vallecula vasoactive intestinal polypeptide wulst, regio hyperstriatica wulst, regio hippocampalis

T. SHIMIZU AND H.J. KARTEN

348

nia

x

B

Fig. 1. Schematic drawings of a dorsal view of the pigeon brain (A), and transverse sections of the rostra1 !B, C) and caudal (D, El wulst. B , 9 The topological relationships of Wrh, the medial Whs, and the posterior Whs (the boundaries of which are not clear; see text). C,E:

I

LFM

CDL

\

C

Cytoarchitectonically defined subdivisionsof the wulst. Different hatchings indicate subregions of the wulst: area with dots = Wrh; area with horizontal lines = anterior Whs; area with vertical lines = posterolateral Whs; area with diagonal lines = posteromedial Whs.

IMMUNOHISTOCHEMICAL STUDY OF THE AVIAN WULST pigeons (Columba liuia). To date, 40 adult pigeons obtained from Palmetto Pigeon Plant, South Carolina, have been used. Brain tissues for at least three pigeons were used for each immunohistochemical experiment. The pigeons were deeply anesthetized with ketamine (5 mg/100 g of body weight, i.m.) and xylazine (1 mg/l00 g, i.m.1, and perfused transcardially with 6% dextran in 0.1 M phosphate buffer (PB) (pH 7.4) or 0.9% saline, and then generally followed by a fixative of 4% paraformaldehyde in PB. For fixative-sensitive antigens, 1.0% paraformaldehyde in PB was used instead. The brains were removed and stored in the same fixative at 4°C for 4-6 hours, and then transferred into 30% sucrose-PB and stored at 4°C for 12-16 hours. They were then sectioned 30-35 pm thick on a sliding microtome in either the transverse or sagittal stereotaxic planes (Karten and Hodos, '67). The sections were washed three times in PB at room temperature for at least 30 minutes, and then incubated in the primary antisera or antibodies diluted in PB with 0.3% Triton X-100 at 4°C for 15-20 hours. Several of the antisera and antibodies used in the present experiments were generously provided by the following individuals: anti-ChAT (rabbit polyclonal), Dr. M. Epstein, (Johnson and Epstein, '86); anti-nAChR (mouse monoclonal antibodies mAb 270 and mAb 2101, Dr. J. Lindstrom, (Whiting and Lindstrom, '86);anti-GAD (GAD-1440)(sheep polyclonal), Dr. W. Oertel, (Oertel et al., '81); anti-GAD (GAD-1,GAD-2, and GAD-5) (mouse monoclonal), Dr. D.I. Gottlieb, (Gottlieb et al., '86); anti-GABAAR(mouse monoclonal), Dr. A. de Blas, (Vitorica et al., '88); anti-SRIF (rabbit polyclonal), Dr. R. Benoit, (Benoit et al., '82); and anti-CRF (rabbit polyclonal), Dr. W.W. Vale, (Vale et al., '81). The anti-CCK (rabbit polyclonal), anti-L-ENK (rabbit polyclonal), anti-NT (rabbit polyclonal), anti-5-HT (rabbit polyclonal), and anti-VIP (rabbit polyclonal) antibodies were purchased from Incstar. Anti-SP (rat monoclonal), anti-5-HT (rat monoclonal), and anti-L-ENK (mouse monoclonal) were purchased from Accurate Chemical Scientific. Anti-NPY (rabbit polyclonal) and anti-TH (rabbit polyclonal) were purchased from Amersham and Eugenetech, respectively. Sections were then washed three times with PB for at least 30 minutes and processed according to the imrnunofluorescence method or the avidin-biotin (ABC) method. For the immunofluorescence method, the sections were incubated in microcentrifuge vials with appropriate secondary antiserum labeled with fluorescein isothiocyanate (FITC) (Boehringer Mannheim Biochemicals). In a few instances, when the sections were stained for two different antigens with antibodies generated in different species (ie., mouse and rabbit), secondary antisera labeled with rhodamine isothiocyanate (RITC) (Boehringer Mannheim) were also used to differentiate the immunoreactivity in the same tissue. The incubation in the second antisera was carried out a t a dilution of 1 : l O O in 0.3% Triton X-100 in PB at room temperature for 1 hour. The sections were washed in three changes of PB for a total of at least 30 minutes, mounted on gelatin-coated slides, and air dried. After a brief rinse with deionized water, the slides were coverslipped with a glycerin-carbonate buffer mixture (9:1). For the ABC processing, the sections were incubated in various biotinylated secondary antibodies. The incubation was carried out at a dilution of 1:200 in 0.3% Triton X-100 in PB at room temperature for 1hour. The tissue was then washed three times for 30 minutes, drained, and incubated in avidin-coupled peroxidase (Vector ABC kit) at a dilution

349

of 1 : l O O in 0.3% Triton X-100 in PB at room temperature for 1hour. After washes in PB, the sections were incubated with 0.025% 3,3'-diaminobenzidine (Sigma) in PB for 15 minutes. Hydrogen peroxide was added to the medium to a final concentration of 0.01%. The reaction proceeded for 15-20 minutes. Sections were then washed several times in PB, mounted on gelatin-coated slides, dried, and the reaction product was intensified with 0.05% OsO,. The sections were dehydrated and coverslipped with Permount. Several pigeons received a pressure injection of a solution of colchicine in buffered saline to inhibit axonal transport and enhance the visualization of staining in cell bodies. Although the colchicine treatment has had some effect on the staining of cell bodies in the injection area, the distribution of neuropil and cell bodies itself was not apparently different between normal and treatment cases. Thus, the data presented here were all based on analyses of normal tissues. Sections were examined with darkfield, standard brightfield, and Nomarski microscopy. Sections stained with fluorescent secondary antibodies were examined with a fluorescence microscope. Charting was carried out using a camera lucida. Adjacent sections were stained with cresyl violet (Pfalts and Bauer) in order to 1)identify boundaries of laminae, and 2) study sizes and densities of neurons in different laminae of the visual wulst. The densities of cells were estimated based on the number of cells obtained by the method of recursive translation (Rose and Rohrlich, '87). This method uses the size distribution of the soma, and has advantages of insensitivity to section thickness, sample size, and somal morphology. Control conditions for immunohistochemistry were carried out by the omission of the primary antibody incubation. Staining was abolished for all antibodies under this condition. In the followingdescription of immunohistochemical results, however, immunostaining will be referred to as neurotransmitter-, receptor-, or neuropeptide-like immunoreactivity (LI). Although the term "-LI" is sometimes omitted in the text, we always imply neurotransmitter-, receptor-, or neuropeptide-like immunoreactiuity.

RESULTS Before presenting the immunohistochemical results of the present experiments, it is useful to briefly describe some characteristics of the four laminae of Whs: HA, IHA, HIS, and HD (see Fig. 1). 1. The HA contains the largest neurons in Whs (Pettigrew, '79; Watanabe, et al., '83). The somata of these neurons range from 9 to over 20 pm in diameter (m = 14.67, u = 3.02, n = 134). The cells are distributed rather sparsely; i.e., about 30.4 x lo3 cells/mm3. The axons of HA neurons can be traced medially into the tractus septomesencephalicus (TSM), which is the only direct, extratelencephalic efferent pathway of Whs (see Karten et al., '73). The TSM contains efferents of both the anterior (somatosensory/ motor) and posterior (visual) wulst. The TSM descends along the medial telencephalic wall, enters the diencephalon, and terminates in various visual (see Karten et al., '73) and somatosensory/motor (see Karten, '71) areas of the brainstem. 2. The IHA is a thin granular layer, which contains small cells (4-10 km, m = 7.74, u = 1.52, n = 146) with a density of about 50.5 x lo3cells/mm3.The IHA is the major recipient of input from the avian equivalent of the dorsal


350 division of the lateral geniculate nucleus (Karten et al., '73; Streit et al., '80a,b; Watanabe et al., '83). The thalamic input to IHA is organized retinotopically (Perisic et al., '71; Pettigrew and Konishi, '76; Wilson, '80; Denton, '81). 3. The HIS contains many small to medium-size cells (5-15 km, m = 8.27, u = 2.70, n = 155) with a density of about 38.8 x lo3cells/mm3.This layer is separated from the deeper layer (HD) by the lamina frontalis suprema (LFM). 4.The HD contains many cells of variable size (6-19 pn, m = 11.53, u = 3.64, n = 253), and with a high density (about 63.3 x lo3 cells/mm3). The lateral portion of H D receives a projection from the visual nuclei of the dorsal thalamus (Karten et al., '73; Watanabe et al., '83). The medial portion of H D receives a projection from the medial, nonvisual thalamic nuclei (nuclei dorsolateralis pars medialis and dorsomedialis anterior), which may be comparable to the anterior thalamic complex of mammals (Karten et al., '73). The HD appears to send intratelencephalic projections to several areas in the telencephalon (Karten et al., '73; Ritchie, '79;Wild et al., '85; Shimizu et al., '89). The patterns of immunohistochemical labelling in the -1st are illustrated in Figures 3-16. The figures of the left hemisphere show the distribution of cell bodies, while the figures of the right hemisphere show that of the neuropil. The solid lines indicate boundaries between major laminae, and dotted lines represent boundaries between different intensities of immunoreactive staining for various compounds. All subdivisions are not necessarily apparent in figures for each compound. Only the transverse sections are presented here, although the sagittal sections were also analyzed to clarify boundaries. Similarly, the doublelabelling for immunofluorescence was helpful to confirm boundaries, although we do not specify the cases. Thus, the same tissue was processed for one type of immunoreactivity with FITC and also for another type with RITC in order to determine whether overlapping of subdivisions occur or not. The density of stained cells (D) was classified as low (D I 400 cells/mm3), moderate (400 < D I 800 cells/ mm3),or high (D > 800 cells/mm3).The intensity of staining in the neuropil was scaled rather subjectively. In the scaling, we consider high the highest intensity of labelled neuropil observed for any antiserum, and low the lowest intensity. Any intermediate labelling was scaled as moderate. This rating proved to be sufficient in other experiments (Shimizu et al., '87; Britto et al., '89). Samples of low, moderate, and high intensity of neuropil staining are shown in Figure 2. A summary of the relative density of cells and neuropil staining is shown in Table 1.

Classicaltrdttersandreceptors Choline acetyltransferase (Fig. 3 ) and nicotinic acetylcholine receptor (Fig. 4). No C U T - L I cell bodies were found in Whs. The dorsolateral portions of HA, IHA, and the vallecular area contained a moderate density of ChAT-stained fibers. In contrast, only a few scattered ChAT-stained fibers were located in medial portions of HA. A small number of intensely stained nAChR-immunoreactive cells were found, primarily in the dorsal portion of HA, IHA, and HIS. Only a few nAChR-stained cells were found in the ventromedial portion of Whs (Fig. 4). A very low density of nAChR-LI fibers a n d o r punctate structures was Seen throughout whs' without laminar segregation. Tyrosine hYdroXYlase (Fig. 5 ) . No TH-stained cells were seen. A low to moderate density of TH-LI fibers was

Fig. 2. Darkfield photomicrographs showing samples of low (A), moderate (B), and high (C) intensities of immunostained neuropil. All neuropil stained in these photomicrographs were stained with an antiserum against TH, and observed in the basal telencephalon at the same level of the wulst (scale bar = 100 pm).

IMMUNOHISTOCHEMICAL STUDY OF THE AVIAN WULST -

TABLE 1. The Relative Densities of Immunostained Cell Bodies and Neuropil in the Wulst'

Putative neurotransmitters

-

Cells C!hAT nAChR TH 5-HT

GAD-1440 GAD-2 GABAA SP CCK

L.ENK NPY NT SlUF

HAd

HAv

-

-

+ -

++ -

-

++ -

+ -

-

t+

++ -

+

-

IHA

-

+

-

++ -

++ + -

HIS

HDm

HD1

HL

-

-

-

-

+ -

++ -

++ + -

++ -

-

+

+

++

++

-

-

-

+t+

+ +++

-

-

+

-

+

++ +t + + ++ ++

351

least. A moderate density of punctate labelling and a few varicose fibers were observed in the ventromedial portion of HA, IHA, and the most dorsolateral portion of Whs. In particular, IHA, and the area immediately dorsal to the caudal HD contained the highest density of GAD-positive processes. In contrast, HIS and HD contained a low density of GAD-LI staining. GABA,R-LI staining was also found throughout Whs. In particular, the caudal HD appeared to contain a somewhat greater density of GABAARimmunoreactivity.

Peptides

Substance P (Fig. 9). The SP-immunoreactivityexhibited a very distinctive laminar pattern in the wulst. Many CW ++ + + SP-immunoreactive cells were very faintly stained. These WP SP-stained cells were primarily located in HA and IHA, Neuropd CIAT + + ++ + + ++ where the majority of them were located in the region nAChR + + + + + + + immediately mediodorsal and lateral to HD (Fig. 9C,D). TI3 ++ + + + + + + 5-KT +++ +++ ++ ++(+I + + + There were only a few in HD. The HD itself consists of two GAD-1440 + + + t + + + GAD-2 + + ++ + +(t) +(+I ++ divisions, a medial portion (HDm) and a lateral portion (HDl).Within HD, the SP-immunoreactivecells were mostly GABAA t + + + ++ + + SF + ++ ++ + + + CCK + + ++ +++ ++ ++ restricted to HDm. The HA, IHA, HD, and an area near the vallecula (the L-EtiK + + + t + NI'Y + + + + + + + hyperstriaturn laterale, HL) contained SP-LI neuropil which NT + + + ++ + (+I SRIF +(+) +(+) + t + + + appeared rather delicate and punctate. In contrast to the CRF + + + (+) (+) (+) + dorsorostral portion (HAd),the immunoreactivity was parVIP + + ticularly dense in the ventrocaudal portion of HA (HAv) 'Density: -, absent; +, low; + +, moderate, + + +, high.Parentheses indicate a density that is near the ventricle along with a caudal portion of IHA, and slightbless than that indicated. the zone of the highest density was located dorsomedial to HD (an area designated as HD-shell in Figs. 9, 19). This found throughout Whs. Many of these fibers formed pericel- dorsomedial region of high density was gradually displaced lular nests, with the majority localized in HA (Fig. 171, and laterally at more caudal levels through HD (Fig. 9C-E). The similar to even more extensive TH-positive pericellular HDm contained a slightly higher density of SP-stained nests in the caudal neostriatum. In IHA, HIS, and HD, a fibers than HD1 (Fig. 9C). In addition, although the density of SP-staining in Hp is less dense plexus of TH-immunoreactive fibers was also found. In contrast, the LFS contained a high density of not illustrated in Figure 9, APH and Hp contained an fibers which appeared to course in a dorsolateral direction. extremely intense staining of SP-immunoreactive cell bodkn sagittal sections, the LFS appeared to be the path of the ies and neuropil in contrast to a low SP-immunoreactivity in the dorsal portion of HA just adjacent to AF'H (Fig. 2OA). fibers to Whs. Cholecystokinin (Fig. 10). The pattern of CCK-LI Serotonin (Fig. 6). No 5-HT-stained cell bodies were seen. A dense plexus of thin 5-HT-LI fibers was found was in sharp contrast to that of SP-staining. CCK-LI was throughout Whs, with the region of highest density located only occasionally seen in faintly stained cell bodies and fine in HA (Fig. 18). In IHA, 5-HT-stained fibers were directed fibers in HA. The highest intensity of CCK-staining was predominantly radially (perpendicular to the boundary located in HIS and HD (Fig. 20B). Intense CCK-immunorebetween HA and HIS). In addition to the rather fine activity, including CCK-stained neuropil and cell bodies, 5-HT-stained fibers throughout Whs, a small number of was present in HDm and HL. In contrast, HDl and HIS darkly stained fibers was found almost exclusively in HIS showed a somewhat lower density of CCK-staining. In addition, HV, which is located immediately ventral to and HD. These intensely 5-HT-stained fibers appeared to course primarily in a tangential direction (parallel to LFS). HD, also contained a high density of CCK-immunoreactive GLutamic acid decarboxylase (Figs. 7 , 8 ) and the cell bodies and neuropil. Leucine-enkephalin (Fig. 11). A small number of GA33AAreceptor. We used three types of monoclonal antibodies (GAD-1, GAD-2, and GAD-5) and one type of widely scattered L-ENK-stained cell bodies was found antiserum (GAD-1440) that recognize GAD. Patterns of throughout Whs. In particular, an area around IHA conGAD-LI staining were different dependingon which antibod- tained a few, intensely positive L-ENK-stained cells and iedantiserum were used. In sections stained with GAD- fibers. The staining for L-ENK was faint, and L-ENK1440 (Fig. 7 ) , a moderate number of faintly GAD-LI cells positive fibers were sparse in the wulst. A sparse distribuwere sparsely distributed throughout Whs. A faintly stained tion of L-ENK-LI fibers was found in HA, in contrast to the plexus of GAD-immunoreactive fibers and punctate pro- higher density of L-ENK-stained varicose fibers in HDm, cesses were observed throughout Whs. Although no clear HL, and particularly LFS. These L-ENK-immunoreactive pattern of distribution was seen in the wulst, slightly fibers were directed dorsolaterally and tangentially. Neuropeptide Y (Fig. 12). NPY-LI fibers were modergreater intensity of GAD-stainedprocesses was found in the dorsolateral portion of Whs. On the other hand, sections ately dense in LFS where there were also a few, lightly stained with GAD-1, GAD-2, and GAD-5 showed more stained NPY-LI cells. NPY-stained fibers were rarely obdistinct laminar and regional differentiation, with GAD-2 served in the HAd, IHA, HIS, or HD. The caudal portion of (Fig. 8), giving the most intense staining and GAD-5 the Whs contained a small number of NPY-stained fibers, in ++

++ +

-

+

+

+


352

nAChR

ChAT

I

HAd

APHd

I

Figs. 3-16. A series of line drawings arranged from rostra1 to caudal (A-E) schematically illustrate the distribution of immunoreactive cell bodies (dots in the left hemisphere) and/or neuropil (hatch patterns in the right hemisphere) stained with antibodies and antisera to ChAT, nAChR, TH, 5-HT, GAD-1440, GAD-2, SP, CCK, L-ENK, NPY, NT, SRIF, CRF, and VIP, respectively. Composite figures have been used to

I

HAd

APHd

represent the brains of different animals. The sections A-E approximately correspond to A 13.0, A 12.0, A 11.0, A 10.0, and A 9.0 in the coordinates of Karten and Hodos ('67). The density of hatch patterns indicates the intensity of immunoreactive staining in neuropil (scale bar = l m m ) .

IMMUNOHISTOCHEMICAL STUDY OF THE AVIAN WULST

353

5HT

TH

I

HAd

HAd

APHd

Figure 5

APHd

Figure 6


354

GAD-2

GAD- 1440

HAd

HAd

APHd

Figure 7

I

APHd

Figure 8


355

SP

CCK r

I

I

1

r-

HAd

I

I

APHd

Figure 9

HAd

APHd

Figure 10


356

N PY

L-ENK I

I

HAd

I

HAd

1

APHd

Figure 11

I

Figure 12

357


SRIF

NT I

c

-

HAd

1 r

APHd

Figure 13

HAd

APHd

Figure 14


358

CRF

VIP ,

I

I

HAd

I

APHd

Figure 15

HAd

-

APHd

Figure 16


Fig. 17. Photomicrographs of immunohistochemicalstaining for TH-LI, as demonstrated with the ABC staining method: A Darkfield photomicrograph of a moderate number of TH-LI fibers in HA (scale bar = 100 km). B: Brightfield photomicrograph of pericellular nests in HA (scale bar = 50 km). The region of the wulst from which the photomicrographs were taken is outlined in Figure 5-B.

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Fig. 18. Darkfield photomicrograph of dense 5-HT-LI fibers in HA, as demonstrated with the ABC stainingmethod (scalebar = 100 pm). The region of the wulst from which the photomicrograph were taken is outlined in Figure 6C.

contrast to a high density of NPY-immunoreactive fibers and intensely stained NPY-LI cell bodies in the Wrh. Neurotensin (Fig. 13). No NT-stained cells were seen in Whs. Punctate structures of NT-LI were faint in HIS and HA. A moderate density of NT-staining was found in HDm and slightly higher in LFS. In contrast to the low to moderate density of NT-immunoreactivity in Whs, a moderate to high density of NT-LI punctate structures was found in APH. Somatostatin (Fig. 14). A moderate number of SRIF-LI cells were observed in HA and HL. The HA and HL contained a few SRIF-positive varicose fibers and punctate structures. The SRIF-immunoreactivity was somewhat less intense in HIS and HD. Corticotropin-releasing factor (Fig. 15). In HA, HIS, and particularly IHA, there were a moderate number of cells which contained CRF-LI. The pattern of staining of cell bodies was markedly different from that found with other antibodies. Multiple irregularly shaped CRF-LI objects were found within the cytoplasm, and the intervening cytoplasm was unlabelled or only faintly labelled. Examination with Normarski microscopy or counter-staining with cresyl violet showed that the CRF-immunoreactivity appeared to be only inside cell bodies, and thus it is unlikely that the CRF-stained objects were terminal boutons. We therefore interpreted that the CRF-immunoreactive profiles represent the endoplasmic reticulum, although an examination by the electron microscope may be required for definite confirmation. The endoplasmic reticulum staining in cells of HA and HIS was rather intense, in contrast to

small cells with faintly labelled CRF-stained endoplasmic reticulum near the ventricle. The pattern of regional distribution of CRF-stained punctate structures had some similarity to that of SP. Thus, intense CRF-LI was seen in HA, the area immediately dorsal to caudal HD, and APH, whereas a lower intensity of CRF-staining was found in HIS and HD. Vasoactive intestinal polypeptide (Fig. 16). No VIP-stained cells were seen. There was faint VIP-LI in HA, and fainter VIP-staining was seen in HIS and HD. The Whs contained VIP-stained punctate structures and very few fibers, in contrast to more VIP-LI fibers in APH and Hp.

DISCUSSION Generalfindings The major findings of the present experiments are threefold. First, the visual wulst contained immunoreactivity to a wide variety of transmitters, neuropeptides, and receptors. Second, many chemical-specific cells and fibers were distributed in accordance with the cytoarchitectonic laminar organization of Whs. Finally, the distribution of some neuroactive substances indicate the existence of regional variations within each lamina. A variety of substances. The present experiments confirm the existence of immunoreactivity in the wulst for at least fourteen types of transmitter-related chemicals, including C U T , nAChR, GAD, GABA,R, 5-HT, TH, SP, CCK, L-ENK, NPY, NT, SRIF, CRF, and VIP. The hetero-


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Fig. 19. Photomicrographs to illustrate laminar and regional differentiation of the immunohistochemical staining for SP-LI neuropil on transverse sections in Whs, as demonstrated with the ABC staining method: A A section of the posterolateral Whs correspondingto one between drawings D and E of Figure 9; B: A section of the posteromedial Whs corresponding approximately to drawing C of Figure 9. The ventricle is indicated by an asterisk (scale bar = 100 Fm).

geneity of chemical substances in Whs is partially a consequence of the diverse input and output connections of the wulst. The major sources of input to Whs include 1) the archistriatum (Bagnoli and Burkhalter, '83) and APH (Casini et al., '86) in the telencephalon, 2) the dorsolateral thalamic area, including visual (see Karten et al., '73) and somatosensory (see Wild, '87) nuclei, 3) the hypothalamus (Berk and Hawkin, '85), 4) monoamine-containing cell groups in the brainstem, including locus coeruleus, area ventralis of Tsai, n. tegmenti pedunculo-pontinus pars compacta (Bagnoli and Burkhalter, '83; Miceli and Rep&ant,, '85; Kitt and Brauth, '86a,b), griseum centrale, n. linearis caudalis, medial and lateral reticular formation, and n. annularis (Bagnoli and Burkhalter, '83; Miceli and Reperant, '85). Descending pathways from Whs include both intra- and extra-telencephalic projections. There are multiple pathways from the wulst to several areas within the telencephalon (Karten et al., '73; Ritchie, '79; Shimizu et al., '89). Extratelencephalic efferents terminate in several visual (see Karten et al., '73) and somatosensory/motor (see Karten, '71) nuclei in the diencephalon and mesencephalon. Laminar distribution of staining. The majority of immunohistochemical staining was not distributed evenly

throughout the wulst. The pattern of staining for each compound was distributed generally in accordance with the laminar organization. Several types of neurochemically specific cell bodies were found in Whs. Many of them were faintly stained in contrast to the intensely stained cells found either in the basal telencephalon (the avian equivalent of the basal ganglia of mammals) or in the brainstem. The SP-immunoreactive cells occurred more frequently than cells stained for other substances in HA, although the staining for SP was often faint. These SP-positive cells were mainly located in the ventral portion of HA. The HA also contained moderate number of GAD- (GAD-1440) and SRIF-stained cells, as well as a low density of nAChR- and L-ENK-positive cells. The HIS contained a moderate number of GAD- (GAD-1440) and CCK-immunoreactive cell bodies, as well as a few cells expressing nAChR-, SP-, and L-ENK-LI. The HD also contained many CCK-stained cells, with the greatest number of CCK-immunoreactive cells located in the mediodorsal portion of HD. The GAD(GAD-14401, SP-, and L-ENK-stained cell bodies were also found in HD. The HL contained the greatest density of NPY-stained cells in the wulst, although the number of NPY-immunoreactive cells was very small. GAD- (GAD-

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Fig. 20. Photomicrographs to show boundaries between the Whs and adjacent areas: A: The immunohistochemical staining for SP-LI neuropil, illustrating the boundary from the Wrh (APH). (The section corresponds approximately to one between drawings C and D of Fig. 9.

B: The immunohistochemical staining for CCK-LI neuropil, illustrating the boundary from the HV (the section corresponding approximately to drawing C of Fig. 10).The ventricle is indicated by an asterisk (scale bar = 100 bm).

1440) and L-ENK-immunoreactive cells were sparsely distributed throughout Whs. The superficial layer of Whs, HA, contained the highest densities of 5-HT, TH-, GAD- (stained with GAD-1, -2, and 4, SP-, NPY-, SRIF-, CRF-, and VIP-positive neuropil in the wulst, whereas the highest density of GABA,R-, CCK-, NT-staining was found in the deepest layer of Whs, HD. A distinctive pattern of distribution of immunoreactive staining was also found in the most dorsolateral portion of the wulst near the vallecula (HL), which may include portions of the traditionally defined HA, IHA, HIS, and HD. The HL contained the highest densities of C U T - and L-ENKstained fibers in the wulst, as well as substantial densities of 5-HT-, TH-, GAD- (GAD-1,GAD-2, and GAD-51, SP-,and CCK stained neuropil. In contrast to the regional distributions of those immunoreactivities mentioned above, nAChRand GAD-LI (GAD-1440) neuropil were distributed rather uniformly throughout Whs. Regional variation. The present study confirms the significance of immunohistochemistry as a method to def i ~ ethe subtle boundaries of subregions. The boundary between Wrh and Whs is difficult to define based on only cytoarchitecture. However, cells and neuropil in Wrh were intensely stained for SP, L-ENK, NT, and NPY, in contrast

to the low to moderate densities of immunoreactivity for these substances in the adjacent area of Whs. The Whs has traditionally been classified on the basis of four laminae of (HA, IHA, HIS, and HD). The immunoreactive staining, however, appeared to reveal several subregions within each lamina. The layer HA contains at least two such regions: a dorsorostral portion (HAd) and a ventrocaudal portion (HAv). The HAd was characterized by dense TH-immunoreactive staining, and the HAv contained dense SP-staining. The HD also contains at least two divisions: a ventromedial portion (HDm) and a dorsolateral portion (HD1). The HDm was characterized by GABA,R-, SP-, CCK-, L-ENK-, and NT-positive neuropil or cell bodies. In contrast, the HD1 contained little immunoreactivity for these compounds. The dorsolateral portion of the wulst, HL, also showed distinctive distributions of immunoreactivity for several compounds. A caudal portion of HL may correspond to the lateral portion of IHA, which receives bilateral input from the visual thalamic nuclei (Karten et al., '73; Streit et al., '80a,b; Watanabe et al., '831, although combined anterohetrograde transport with immunohistochemical studies are necessary to confirm this possibility. The Whs is considered to include at least three functional regions: 1)the anterior portion, containing the somatosen-


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sory representation of the contralateral side of the body caudal neostriatum and the prefrontal cortex (Wild et al., (Delius and Bennetto, '72; Karten et al., '78; Wild, '87; '90). The LFS may be the entry route of catecholaminergic Funke, '89a,b); 2) the posterior portion, or the visual area innervation to overlying regions. It has been previously suggested that there is norepineph(Karten et al., '73; Bagnoli et al., '82b); and 3) a medial "limbic" portion of HD (Karten et al., '73). The present rine, rather than dopamine, in W h s (Tohyama et a]., '74; study was mainly concerned with the posterior Whs. Find- Bagnoli et al., '83a; Bagnoli and Casini, '85). We were not ing the boundary between the visual portion (posterior able to identify the type of catecholamine of the wulst in the wulst) and the non-visual portion (anterior wulst) was present experiments. Attempts to use antibodies against difficult since there were no clear indications that the two other enzymes of the catecholaminergic biosynthetic pathregions have different patterns of immunoreactivity for any way, including dopamine @-hydroxylaseand phenyl-etanolachemical compounds. The rostral HD, however, was clearly mine-N-nethyl-transferase, failed to reliably demonstrate differentiated from the caudal HD. The distinctive pattern immunoreactive patterns in the avian brain. Such results GAD-2, and GAD-5) and SP- may be attributable to species-specific differences with of intense GAD- (GAD-1, immunoreactivity dorsal to HD (HD-shell in the figures) antigens. Since abundant dopamine D, receptors were was found only in the caudal wulst. found in the wulst (Dietl and Palacios, ,881, further studies Although we used transverse and sagittal planes of are needed to resolve questions about the nature of catesections and the double-labelling technique in order to cholaminergic systems of Whs. delineate subdivisions carefully and thoroughly, it seemed Serotonergic system. There was an absence of 5-HTthat overlapping of the distributions of several immunoreacstained cell bodies, and a moderate density of 5-HTtivities was frequently likely. This indicates, on the basis of subtle differences of chemical anatomy, that there are more immunoreactive fibers, in Whs. This result agrees quite well with those of previous studies (Vischer et al., '82; subdivisions than we described here. Yamada et al., '84; Yamada and Sano, '85; Sako et al., '86; Alesci and Bagnoli, '88; Alesci et al., '89). The avian Comparisonswithpreviousstudies equivalent of n. raphe, n. linearis caudalis, has been sugClassical transmitters gested to be the source of 5-HT innervation of the wulst Cholinergic system. A cholinergic system in Whs has (Vischer et al., '82; Bagnoli and Burkhalter, '83). been previously suggested based on evidence of the existGABAergic system. With GAD-1440, there were rather ence of nAChR (Bradley and Horn, '811, muscarinic recep- faint GAD-immunoreactive cells, varicose fibers, and punctors (Bradley and Horn, '81; Vischer et al., '82; Dietl et al., tate features throughout Whs. In sections stained with '88a), and ChAT activity (Bagnoli et al., '82a; Vischer et al., GAD-1, GAD-2, or GAD-5, variable density of punctate '82) in the wulst. In agreement with the previous studies, labelling was found in different laminae. These results are we found both ChAT-immunoreactive fibers and nAChR- consistent with those described in earlier publications with stained cells in the wulst. In our studies, the ChAT-stained respect to the low to moderate concentrations of GABA fibers and/or nAChR-stained cells were located in the (Vischer et al., '82; Domenici et al., '88) and GAD (Bagnoli clorsolateral portions of IHA, HIS, HD, and HL. The et al., '82a; Vischer et al., '82) in the wulst. On the other distribution of immunoreactivity is consistent with that hand, while we found only low to moderate density of reported by Bagnoli et al. ('82a), who found a high level of GABAAR-stainingthroughout Whs, a high density of GABA ChAT-activity in the dorsolateral portion of the wulst, and receptors has been previously found in the wulst (Vischer et a lower level in the medioventral portion. The dorsolateral al., '82; Dietl et al., '88b). These conflicting results about portion of the wulst, including HL, receives bilateral visual GABA receptors may be due to characteristics of the projections from the dorsal thalamus (Karten et al., '73; Streit et al., '80a,b; Watanabe et al., '831, which is perhaps antibody used. The antibody could have recognized only a one of the sources of the cholinergic system of Whs (Bagnoli subset of the avian GABA,R in the wulst. The GABAAR et al., '81). The ChAT-stained cells were indeed found in the antibody used in the present study was made against visual nuclei of the dorsal thalamus (unpublished observa- mammalian antigen, and the antibody recognizes only about 40% of GABAARof mammals (A. de Blas, personal tion). Catecholaminergic system. No TH-immunoreactive cell communication). Further studies about identity of GABAAR bodies were seen. A low to moderate density of TH-stained in the wulst may clarify this issue. Bagnoli and co-workers ('82a) reported, in the rostral fibers was found in Whs. These results are generally Whs, higher GAD activity in the ventrolateral portion than comparable to previous studies of catecholaminergic innervation in the wulst (Kitt, '81; Takatsuki et al., '81; Bagnoli in the dorsomedial portion. Similarly, in sections stained and Casini, '85). In the present study, many intensely with GAD-1, GAD-2, and GAD-5, we found that the ventroTH-stained fibers were found in LFS, a low to moderate medial portion of HA, IHA, and HL contained more intense density of fibers in HIS and HD, and thin fibers forming GAD-LI labelling than the dorsolateral HA. Moreover, in pericellular nests primarily in HA. A similar pattern of sections stained with GAD-1440, although the staining was dense catecholaminergic (perhaps dopaminergic) innerva- faint, a slightly higher density of GAD-LI was seen in the tion in LFS and pericellular nests in the caudal neostriatum dorsolateral portion of HA than in other areas. The possible was shown by Divac and Mogensen ('85), and a fine extrinsic source(s) of GABA inputs to the wulst has been catecholaminergic plexus in IHA and HIS was found by studied by Bagnoli and co-workers ('83b), who found that Bagnoli and Casini ('85). Divac and Mogensen ('85) sug- injections of tritiated GABA into the wulst resulted in gested that pericellular nests in the caudal neostriatum retrogradely labelled neurons in a visual nucleus of the correspond to those of the prefrontal cortex in rats. The dorsal thalamus. This may be related to the high density of present study shows that pericellular nests are not unique GAD-LI (GAD-1, GAD-2, and GAD-5) labelling in IHA. In to the caudal neostriatum in the avian brain, and thus more addition to the cholinergic system, thus the GABAergic studies are necessary for further comparisons between the afferent to Whs may arise in the visual thalamic nucleus.

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Peptides Substance P. Although there are previous studies on SP-LI in other parts of the avian brain (Reiner et al., '83; Ball et al., '881, SP-LI in the wulst has not been reported before. The SP-immunoreactive cells and punctate structures show distinct patterns in the pigeon wulst. The immunoreactivity was particularly dense in the caudal IHA and the ventral portion of HA near the ventricle, with the zone of the highest density located mediodorsal to HD in the HD-shell. No or only low density SP-staining was found in the deeper layers of the Whs, HIS and HD. This unique pattern of SP-LI in HD-shell is also found in the wulst of zebra finches and penguins (Shimizu and Karten, unpublished observation). Thus, SP-immunoreactivity and its distribution pattern in the wulst of pigeons may be quite common in birds in general. Cholecystokinin. Although Ball and co-workers ('88) studied CCK-LI in the vocal control regions in songbirds, there has been no previous report on CCK-LI in the wulst. There was intense CCK-immunoreactivityin a thalamorecipient area of Whs, namely, HD. The CCK-LI was only occasionally seen in faintly stained cell bodies and neuropil in HA, whereas the highest intensity of CCK-staining was located in HIS and HD. Furthermore, the CCK-stainedcells were observed in the dorsal thalamic nuclei (unpublished observation). These results suggest that one of the sources of CCK input to Whs may be the dorsal thalamus. Enkephalin. A low density of L-ENK-immunoreactivity was observed in Whs. This result agrees generally with previous reports (Bayon et al., '80; Reiner et al., '84b). In the present experiments, however, a small number of widely scattered L-ENK-stained cells were also observed throughout Whs. Reiner et al. ('84b) reported that ENKstained cells were found in HD and APH. The differences of the distribution pattern might be partially related to differences between the species used in the two experiments (pigeons vs. chickens). Neuropeptide Y. A limited number of NPY-immunoreactive fibers were found in Whs, in contrast to the moderate to high numbers of NPY-stained fibers in LFS and APH. The pattern of NPY-immunoreactivitydistribution is similar to that seen with antibodies to the avian pancreatic polypeptide (unpublished observation). The immunoreactive staining with avian pancreatic polypeptide antisera, in fact, has been demonstrated to be due to NPY (Dimaggio et al., '85). Thus, the present report includes only the result of NPYimmunoreactivity. Neurotensin. The distribution of NT-stained punctate structures are generally in agreement with a previous study of NT-binding sites (Brauth et al., '86). Thus, HA and HIS contain the low or moderate levels of NT-immunoreactivity, while HD appears to contain a greater density than those of HA and HIS. In the present experiments, the HDm showed a higher intensity of NT-LI than HD1. Such differential staining in subdivisions of HD has not been reported previously. Somatostatin. The pattern of staining observed for SRIF-immunoreactivity contrasts with the earlier finding of Takatsuki et al. ('81),who noted only sparse numbers of SRIF-stained cells in HA. The result of the present experiRonts showed a moderate number of SRIF-immunoreactive cells and faintly stained SRIF-immunoreactive fibers in the wulst, particularly HA and HL. One reason for the discrepancies with the previous report might be attribut-

T. SHIMIZU AND H.J. KARTEN able to the use of different species as the subjects in these studies (pigeonsvs. parakeets). Corticotropin-releasing factor. The CRF-stained cells showed a peculiar pattern of staining characteristic of the endoplasmic reticulum. These CRF-LI cells were found mainly in HA, HIS, and HL. Bons and co-workers ('88), reported CRF-immunoreactive cells in HA, but did not indicate if the CRF-staining in their study was also restricted to endoplasmic reticulum. Vasoactive intestinal polypeptide. We found faint VIPimmunoreactive punctate structures and a small number of fibers in Whs. Although VIP-immunoreactivity has been found in other areas of the brain in several avian species (Yamada and Mikami, '82; Ball et al., '88; Silver et al., '88), the existence of VIP in the wulst has not been reported previously.

Comparisonswiththemammabnneocortex Classical transmitters In mammals, the neocortex is extensively innervated by subcortical, extrathalamic cell groups: noradrenergic neurons in the locus coeruleus, 5-HT neurons in the raphe nuclei, dopaminergic neurons in the ventral tegmental area, and cholinergic neurons in the nucleus basalis of Meynert (reviewed by Foote and Morrison, '87). Within mammals, there appear to be species differences in the organization of these putative neurotransmitter systems, and the pattern of distribution may vary in different cortical areas even within a single species. Cholinergic system. In rats, CUT-immunoreactive fibers are distributed throughout all cortical layers in the motor cortex, while the somatic sensory area has a low density of fibers in layer IV, and a higher concentration in layer V (Houser et al., '85). On the other hand, in monkeys, layer IV of sensory cortices contains a dense cholinergic innervation, while a lower density is reported in deeper layers (Campbell et al., '87). The area of the cholinergic innervation in Whs, mainly the possible thalamorecipient areas, may be comparable to the cortical layer IV. The cortex of rats also contains CUT-positive cells, which are perhaps intrinsic (Houser et al., '85; Levey et al., '84). CUT-stained cells are not found in the avian wulst, the cortex of cats (De Lima and Singer, '86) or of monkeys (Campbellet al., '87). The distribution of nAChR-LI in the rodent brain was studied by Swanson and co-workers, using the autoradiographic localization of antibodies ('87). In the neocortex nAChR was found generally in layers Ia, IV, and deeper parts of V, while in the cingulate gyms and medial prefrontal cortex, label was located over layers I, 111, and V-VI. On the other hand, whs of birds contained uniform staining throughout layers, although the stained cells were found mainly in the dorsolateral portion of Whs. Catecholaminergic system. In rats, fine noradrenergic fibers innervate all six layers of the cortex (Morrison et al., '78), whereas the primate cortex exhibits greater regional specificity in both density and laminar pattern of innervation (Foote and Morrison, '87). For example, layer IV of the primary visual cortex has very few noradrenergic fibers, while layer IV of other cortical areas displays a more dense innervation. An immunohistochemical study using an antiserum directed against TH (Berger et al., '85) suggests that rats have less dense dopaminergic innervation in the visual cortex than other areas. A similar study with primates (Lewis et al., '87) showed that sensory cortices were all sparsely innervated by dopaminergic fibers-primarily

IMMUNOHISTOCHEMICALSTUDY OF THE A

W WULST

in layer I. The avian wulst contains catecholaminergic innervation, although its type remains obscure. The pattern of TH-immunoreactivity in the wulst is more similar to that of rats than primates in that the laminar differentiation of catecholaminergic innervation is not as clear as in primates. Furthermore, pericellular nests of TH-stained fibers are also found in the prefrontal cortex of rats ( Lindvall and Bjorklund, '78). Serotonergic system. 5-HT innervation of the rat neocortex shows rather uniform density across all layers (Lidov et al., '80). Primate cortices have a tendency to exhibit a greater density of 5-HT-LI fibers in layer N (Foote and Morrison, '87). This tendency is particularly notable in the primary visual cortex (Morrison et al., '82), in which layers 111 and IV have a dense innervation, and layers V and VI have an intermediate innervation. In the wulst, however, 5-HT innervation is not particularly dense in IHA, which corresponds to the granular layer of the mammalian neoRather, the most dense 5-HT innervation cortex (layer N). oEWhs is found in HA, the area containing those cells that provide the major output to extratelencephalic structures. GABAergic system. There are substantial numbers of GABA-containing neurons among all layers in the neocortex, including the visual cortex (Meinecke and Peters, '87), and the accumulated evidence suggests strongly that GABA plays an important role in inhibitory intrinsic circuits in the neocortex (Hendry and Jones, '81; Somogyi et al., '81). A rather uniform distribution of GAD-immunoreactive (GAD1440) cells in Whs is consistent with these mammalian data. Thus, the functions of GABA in the avian wulst might be similar to those in the mammalian cortex. In addition, GABAergic neurons in the hypothalamus project extensively to the neocortex (Vincent et d . , '83).The existence of a comparable projection in birds is unknown. The immunoreactivity of GABAARwas found throughout Whs, without clear laminar segregation. The localization of the GABAARwas studied in rodent brains with the same antibody used in the present experiment (de Blas et al., '88). In the cerebral cortex, the staining of GABAAR-LIwas also present over all layers, particularly layer I.

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et al., '77; Sar et al., '78; Wamsley et al., '80). In brains treated with high doses of colchicine to enhance the visualization of neuronal perikarya, isolated L-ENK-immunoreactive neurons were found in layers I1 and I11 of the many telencephalic regions of rats, including the occipital neocortex (Khachaturian et al., '83).This pattern of distribution contrasts with that of the avian wulst. The L-ENKcontaining neurons were distributed rather uniformly throughout Whs. Neuropeptzde Y. NPY-containing cells and processes show laminar patterns in the mammalian cortex (Hendry et al., '84; Kuljis and Rakic, '89). The Whs, however, contained virtually no NPY-stained cells and fibers, although a moderate intensity of NPY-LI fibers were seen in LFS, and a few were observed in the caudal wulst near APH. In contrast to Whs, the Wrh contained considerable numbers of NPY-LI cells and fibers. Neurotensin. Little or no NT-LI has been reported in the mammalian isocortex (Jennes et al., '82). Similarly, low levels of NT binding sites are found in specific sensory areas of the cortex (Quirion et al., '82). On the other hand, there was a moderate intensity of NT-immunoreactive punctate structures in LFS and HD, and a low density of NT-LI neuropil staining was also present in other areas of the wulst. Somatostatin. There are SRIF-containing cells in cortical layers 11-VI, although there are differences in distribution patterns between rats and monkeys (Campbell et al., '87). In monkeys, layers V and VI are relatively sparsely labelled in the visual, auditory, and visual association areas in contrast to uniform labelling in other areas such as the anterior cingulate area. On the other hand, in rats, the majority of SRIF-containing cells are seen in layers I1 to VI (Johansson et al., '84; McDonald et al., '821, but particularly in the infragranular layers (Campbell et al., '87). The avian wulst contained SRIF-stained cells primarily in HA, which may correspond to the infragranular layers of the neocortex in that cells of these layers provide a major output to the tectum. In general, the SRIF-containing cells are assumed to be related to the intrinsic organization of the mammalian Peptides Substance P. In rats, fine SP-immunoreactive terminal- neocortex, largely based on their nonpyramidal morphology like structures are found in layers I1 and IV throughout the and colocalization with GABA (Hendry et al., '84). Similar cerebral cortex, except the medial prefrontal cortex (Iritani roles might be found in SRIF-stained cells in the avian et al., '891, but no SP-LI cells have been reported (Iritani et wulst. Corticotropin-releasing factor. There are at least a few al., '89; Ljungdahl et al., '78). On the other hand, all regions CRF-positive cells in every major neocortical areas of rats of the monkey cortex contain many SP-containing fibers and cell bodies in layers 11-VI, and layer I contains many (Merchenthaler et al., '82; Swanson et al., '83). The majorgranular, SP-stained structures, possibly terminal buttons ity of such cells are found in layers 11-111 and occasionally in (Iritani et al., '89). As in the rat cortex, the wulst contains the deeper layers. In the wulst, CRF-staining was restricted SP-stained punctate structures, and few fibers, although to endoplasmic reticulum within the cell bodies. However, the wulst contains many SP-immunoreactive cells. Further- no such pattern of staining has been reported in the more, as in the rat cortex, Whs had a clear laminar mammalian neocortex. Furthermore, these immunoreacsegregation of SP-LI distribution, with the highest density tive cells in Whs were distributed partly in the upper layer (HA) and partly in the deeper layer (HIS), but not the found in HAv and the caudal IHA. Cholecystohinin. CCK-positive cells are found in all deepest layer (HD). Vasouctive intestinal polypeptide. VIP-containing cells cortical layers, although the highest concentration is in the layers 1-111 (Demeulemeester et al., '85; Morrison and are found throughout the neocortex, and considered to be Magistretti, '83). Furthermore, the axons of CCK-positive intrinsic to the cortex (Fuxe et al., '77; Morrison and cells appear to course downward, and terminate primarily Magistretti, '83). The present study, however, did not in layer VI (Larsson and Rehfeld, '79). The CCK-stained reveal the presence of VIP-stained cells in the avian wulst. The organization of transmitter-specific lemniscal pathcells in Whs were located predominantly in the deeper ways in mammals is largely unknown, although several layers (HIS and HD). Leucine-enkephalin. Only occasional enkephalin-LI fi- neuropeptides, such as SP in the medial lemniscus (Cuello bers and cells have been found in the neocortex (Simantov and Kanazawa, '84) and CCK in the geniculostriate path-


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way (Fallon and Seroogy, '841, have been found. The presence of C U T and GAD in the thalamo-telencephalic visual pathways in birds (Bagnoli et al., '82a, '83b) is unusual and has not been observed in the visual pathways in mammals.

CONCLUSIONS The majority of transmitter-related compounds mentioned above are distributed differentially in the mammalian neocortex in accordance with the regional cytoarchitectonic and laminar organization. In the avian wulst, similar regional- and laminar-specific distributions of neurotransmitters/peptides were found in previous studies (Bagnoli et al., '82a; Bagnoli and Casini, '85) and in the present study. The results indicate that the laminar configuration of the avian wulst is comparable to that of the mammalian neocortex in terms of differentiation of chemical contents, as well as other features previously described, such as cytoarchitectonics, afferent and efferent connections, and electrophysiology. However, there are marked differences of distribution patterns of various compounds between mammals and birds, suggesting differences in organizations of these putative neurotransmitter system. It is important to note that there are also striking differences of distribution patterns for these compounds even within various mammalian species. The present study showed multiple regional subdivisions within the laminar constituents. Further studies of cytoarchitecture and connections of these areas may reveal additional areas of the wulst. The present study suggests that the wulst consists of multiple regional components some of which are comparable to different cytoarchitectonic areas of the mammalian neocortex. Cytoarchitectonically, the medial boundary of Whs with Wrh has been difficult to define. On the basis of studies with the immunohistochemical method, however, the Wrh is clearly segregated from adjacent HA of Whs. The Wrh contains a moderate to high intensity of immunoreactivity for several compounds, whereas a lower intensity of these immunoreactivity is seen in HA of Whs. Some histochemical features of Whs are different from those of mammalian neocortex. In general, the two structures have similarly extensive innervations of classical transmitters, whereas there are considerable differences in innervation patterns of various peptides. These results contrast with findings on the reptilian and avian equivalent of the basal ganglia. Thus, in the reptilian or avian basal ganglia, both classical transmitters and various peptides are distributed similar to those found in the mammalian basal ganglia (see Reiner et al., '84a, for a review). These studies indicate that the chemical characteristics of neocortex are more likely to be species-specific,in comparison with the rather conservative nature of the basal ganglia. Furthermore, in the neocortex, some histochemical features (classical transmitters) seem phylogenetically conservative, while others (the majority of peptides in the present study) are rather variable. The former might be essential for the functions of the telencephalic neural structures. On the other hand, the latter might be related to the behavioral processes and/or their underlying mechanism unique to the species.

ACKNOWLEDGMENTS This work was supported by NEI grant EY-06890-05, NINCDS grant NS24560, and ONR contract N00014-88-K0504. We thank Drs. T.E. Hughes, K.T. Keyser, L.G. Britto, D.E. Hamassaki, H. Bravo, and J.M. Wild for their advice in the course of this study. We are also grateful to Ms. J.B. Schimke for excellent histological assistance, Ms. C.K. Oldmen for editorial assistance with the manuscript, and Ms. E.B. Watelet for secretarial assistance.

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Projections of the parabrachial nucleus in the pigeon (Columba livia).

The retino-thalamo-hyperstriatal pathway in the pigeon (Columba livia).

Failure of interocular transfer in the pigeon (Columba livia).

A cytoarchitectonic analysis of the spinal cord of the pigeon (Columba livia).

An immunocytochemical analysis of the lateral geniculate complex in the pigeon (Columba livia).

Vasoactive intestinal peptide binding sites and fibers in the brain of the pigeon Columba livia: an autoradiographic and immunohistochemical study.

The visual forebrain and eating in pigeons (Columba livia).

The trigeminal system in the pigeon (Columba livia). I. Projections of the gasserian ganglion.

The complete mitochondrial genome of the ice pigeon (Columba livia breed ice).

The urban pigeon (Columba livia, Forma urbana) - A biomonitor for the lead burden of the environment.

The complete mitochondrial genome of the Jacobin pigeon (Columba livia breed Jacobin).

Estimating the surface area of birds: using the homing pigeon (Columba livia) as a model.

Organization of the cerebellum in the pigeon (Columba livia): I. Corticonuclear and corticovestibular connections.

Studies on the biosynthesis of pancreatic glucagon in the pigeon (Columba livia).

The complete mitochondrial genome of the Fancy Pigeon, Columba livia (Columbiformes: Columbidae).

Efferent and afferent connections of the olfactory bulb and prepiriform cortex in the pigeon (Columba livia).

Steroid-synthesizing cells in the embryonic and adult gonads of the domestic pigeon, Columba livia (Gmelin).

Origin, course and terminations of the rubrospinal tract in the pigeon (Columba livia).

Helminth-bacteria interaction in the gut of domestic pigeon Columba livia domestica.

Visual-discrimination deficits after lesions of the centrifugal visual system in pigeons (Columba livia).

Cholesteryl esters of pigeon (Columba livia) aortas as a function of age.

The distribution of neurotransmitters and neurotransmitter-related enzymes in the dorsomedial telencephalon of the pigeon (Columba livia).

Argon laser photocoagulation of the pecten oculi of the domestic pigeon (Columba livia) using a specially designed contact lens.

The distribution of neuropeptides in the dorsomedial telencephalon of the pigeon (Columba livia): a basis for regional subdivisions.