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

THE JOURNAL OF COMPARATIVE NEUROLOGY 314~721-749 (1991)

An Immunocytochemical Analysis of the Lateral Geniculate Complex in the Pigeon (Columba livia) ONUR GUNTURKUN AND HARVEY J. KARTEN Allgemeine Psychologie, Universitat Konstanz, D-7750 Konstanz, Federal Republic of Germany (O.G.); Department of Neuroscience, School of Medicine, University of California at San Diego, La Jolla, California 92093 (H.J.K.)

ABSTRACT The lateral geniculate complex (GL) of pigeons was investigated with respect to its immunohistochemical characteristics, retinal afferents, and the putative transmitters/ modulators of its neurons. The distributions of serotonin-, choline acetyltransferase-,glutamic acid decarboxylase-, tyrosine hydroxylase-, neuropeptide Y- (NPY), substance P- (SP),neurotensin- (NT), cholecystokinin- (CCK), and leucine-enkephalin- (L-ENK) like immunoreactive perikarya and fibers were mapped. Retinal projections were studied following injections of Rhodamine-B-isothiocyanateinto the vitreous. Transmitter-specific projections onto the visual Wulst and the optic tectum were studied by simultaneous double-labelling of retrograde tracer molecules and immunocytochemical labelling. The GL can be divided into three major subdivisions, the n. geniculatus lateralis, pars dorsalis (GLd; previously designated as the n. opticus principalis thalami, OPT), the n. marginalis tractus optici (nMOT), and the n. geniculatus lateralis, pars ventralis (GLv). All three subdivisions are retinorecipient. The GLd can be further subdivided into at least five components differing in their immunohistochemical characteristics: n. lateralis anterior (LA); n. dorsolateralis anterior thalami, pars lateralis (DLL), n. dorsolateralis anterior thalami, pars magnocellularis (DLAmc);n. lateralis dorsalis nuclei optici principalis thalami (LdOPT);and n. suprarotundus (SpRt). The LdOPT consists of an area of dense CCK-like and NT-like terminals of probable retinal origin. Three subnuclei (DLL, DLAmc, SpRt) were shown to project to the visual Wulst. Cholinergic and cholecystokinergic relay neurons participated in this projection. The nMOT occupies a position between the GLd and GLv and encircles the rostra1 pole of n. rotundus and the LA. It is characterized mainly by medium sized NPY-like perikarya which were shown to project onto the ipsilateral optic tectum. Bands of NPY-like fibers in the tectal layers 2, 4, and 7 could at least in part be due to this projection of the nMOT. Most of the antisera used revealed transmitter/modulator-specific fiber systems in the GLv which often showed a layer-specific distribution. Perikaryal labelling was only obtained with glutamic acid decarboxylase. On the basis of its chemoarchitectonics, topography, and connectional pattern, the GLd complex of pigeons is most directly equivalent to the mammalian GLd. However, although the different subdivisions of the avian GLd may represent functionally different channels within the thalamofugal pathway similar to the lamina-specific differentiation within the mammalian geniculostriate projection, direct comparison of subnuclei of birds and mammals is not justified at this time. The nMOT appears similar to the intergeniculate leaflet (IGL) and the avian GLv clearly corresponds in many features to the mammalian GLv. Key words: serotonin, neuropeptides, choline acetyltransferase, tyrosine hydroxylase, glutamic acid decarboxylase, visual projections, transmitter-specific projections

Accepted September 12, 1991. Address reprint requests to Onur Gdnturkiin, Allgemeine Psychologie, Universitat Konstanz, D-7750 Konstanz, Federal Republic of Germany. O

1991 WILEY-LISS, INC.

0. GUNTURKUN AND H.J. KARTEN

722 The avian optic tract and primary visual projections to the thalamus were the subject of several early studies of the vertebrate visual system. Bellonci (18881, using normal material, was the first to describe retinal endings in a discrete area of the ventrolateral thalamus of pigeons. He labelled this retinorecipient structure "corpus geniculatum thalamicum." From his drawings it is evident that he recognized only the ventral portion of the avian lateral geniculate complex, the n. geniculatus lateralis, pars ventralis (GLv). Bellonci also recognized fibers in the dorsolateral thalamic area as being of retinal origin but was not sure whether they ended there or passed through to terminate at midbrain levels. Perlia (1889) was the first to trace degenerating optic fibers, after enucleation, to the contralateral brain stem, and thus verified the previously postulated complete decussation at the optic chiasm in birds (Carus and Meckel, 1826). However, Perlia (1889) failed to find degeneration in either the ventral or the dorsal portion of the lateral geniculate complex. Edinger and Wallenberg (1899) reported that after enucleation degenerating terminals were detected both in the GLv and in the dorsal part of the lateral geniculate complex, the n. geniculatus lateralis, pars dorsalis (GLd),designated by Karten et al. ('73) as the n. principalis opticus thalami (OPT). Several studies in the first decades of this century replicated these findings (e.g. Huber and Crosby, '29) and were also confirmed by more recent experiments (Cowan et al., '61; Karten and Nauta, '68). Hirschberger ('67) demonstrated that the GLd did not consist of a single homogeneous cell group, but could be subdivided into two retinorecipient nuclei, which correspond, according to the present terminology, to the n. lateralis anterior (LA) and the n. dorsolateralis anterior thalami, pars lateralis (DLL). The studies of Reperant ('73) in pigeons and Ehrlich and Mark ('84) in chicks provide the most recent detailed reports on avian retinal projections. They subdivided the main division of the lateral geniculate complex into a dorsal (GLd) and a ventral (GLv) component and further noted that the dorsal part can be subdivided

into at least five subdivisions based on cytoarchitectonic properties and the distribution of retinal terminals. The avian GLv is a trilaminar structure in the ventrolatera1 thalamus (Guiloff et al., '87). In addition to retinal afferents, GLv receives inputs from the tectum and the Wulst of both hemispheres (Karten et al., '73; Crossland and Uchwat, '79; Miceli et al., '87). Projections of the GLv can be followed to the ipsilateral tedum and the area pretectalis (Crossland and Uchwat, '79; Gamlin et al., '84). Electrophysiological studies have demonstrated many movement-sensitive (Pateromichelakis, '79) and color-opponent (Maturana and Varela, '82) units in this nucleus. The avian GLv seems thus to be comparable to the mammalian ventral lateral geniculate nucleus (GLv) which also is known to receive aKerents from the retina (Cajal, 'll),the superior colliculus (Harting et al., '73), and the visual cortex (Hughes and Chi, '81) and projects onto the superior colliculus and to a part of the pretectal nuclei (Swanson et al., '74). However, in contrast to its avian counterpart, color-coded (10%)and movement-sensitive (18%)units only make up a minority of cells of the GLv of cats (Hughes and Ater, '77; Hughes and Chi, '83). After the retinal projections to the dorsal part of the avian lateral geniculate complex were established, studies of the last two decades mainly concentrated on the ascending projections of these neurons. Karten and Nauta ('68) were the first to demonstrate that the GLd projects to the telencephalon, and thus might be directly comparable to the n. geniculatus lateralis, pars dorsalis (GLd), of mammals. These findings were replicated and extended in a large number of different studies using different bird species (pigeons: Hunt and Webster, '72; Karten et al., '73; Meier et al., '74; Mihailovic et al., '74; Miceli et al., '75; Streit et al., '80; Miceli and Reperant, '82, '85; Bagnoli and Burkhalter, '83; zebra finches: Nixdorf and Bischof, '82; falcons: Bravo and Inzunza, '83; quails: Watanabe et al., '83; chicks: Ehrlich and Stuchberry, '86; owls: Karten et al., '73; Bravo and Pettigrew, '81). These experiments demonstrate that

Abbreviations ABC ACh AL CCK ChAT DIVA DIAmc DLL DLLl DLLm DMA FG FITC FPL GAD GLd GLv HD HIS HV IGL IHA LA LdOPT le L-ENK li LMmc ne

avidin-biotin-conjugate acetylcholine ansa lenticularis cholecystokinin choline acetyl transferase n. dorsalis interniedius ventralis anterior n. dorsolateralis anterior thalami, pars magnocellularis n. dorsolateralis anterior thalami, pars lateralis n. dorsolateralis anterior thalami, pars laterolateralis n. dorsolateralis anterior thalami, pars lateromedialis n. dorsomedialis anterior thalami Fluoro-Gold fluorescein isothiocyanate fasciculus prosencephali lateralis glutamic acid decarboxylase n. geniculatus lateralis, pars dorsalis n.geniculatus lateralis, pars ventralis hyperstriatum dorsale hyperstriatum intercalatus superior hyperstriatum ventrale intergeniculate leaflet n. intercalatus hyperstriati accessorii n. lateralis anterior n. lateralis dorsalis nuclei optici principalis thalami lamina externa of the GLv leucine-enkephalin lamina interna of the GLv n. lentiformis mesencephali, pars magnocellularis neuropil layer of the GLv

nMOT nMOTcv nMOTrd

NPY NT OM OPT ov

PB PPC QF Rclb RlTC Rt S

SCN SP SPC SPCd SPCv SpRt SRt T TH TI0 TrBOR TrO TSM TO

n. marginalis tractus optici n. marginalis tractus optici (nMOT), pars caudoventralis n. marginalis tractus optici (nMOT),pars rostrodorsalis neuropeptide Y neurotensin tractus occipitomesencephalicus n. principalis opticus thalami n.ovoidalis phosphate buffer n. principalis precommissuralis tractus quintofrontalis Rhodamine-coupled latex beads Rhodamine-B isothiocyanate n. rotundus serotonin n. suprachiasmaticus substance P n. superficialis parvocellularis n. superficialis parocellularis, pars dorsalis n. superficialis parvocellularis, pars ventralis n. suprarotundus n. subrotundus n. triangularis tyrosine hydroxylase tractus isthmo opticus tractus basalis opticus radici tractus opticus tractus septomesencephalicus tectum opticum

IMMUNOHISTOCHEMISTRY OF THE LATERAL GENICULATE COMPLEX part of the GLd subdivisions project to a specific area in the frontodorsal forebrain, the visual Wulst. The Wulst is a multilayered portion of the rostral pallial roof of the telencephalon. It is macroscopically visible as a bulge that extends from a lateral groove, the vallecula, to the midline. Electrophysiological studies demonstrated important similarities between the visual Wulst of birds and the striate cortex of mammals. As in most mammals studied, in the visual Wulst of owls, falcons, and kestrels most neurons are primarily concerned with binocular visual processing and many cells are selectively tuned to stereoscopic depth cues (Pettigrew and Konishi, '76a; Pettigrew, '78, '79). The frontal binocular field seems to be highly represented in the Wulst of all raptors studied, as it is in cats and monkeys (Pettigrew, '79). Both the avian and the mammalian thalamofugal systems are sensitive to visual experience during the neonatal period (Pettigrew and Konishi, '76b; Hubel, '78). As in the geniculostriate system of mammals, many units in the avian thalamofugal system also have small receptive fields. In the avian GLd, the smallest receptive fields have diameters of 2"-4" in pigeons (Britto et al., '75; JassikGerschenfeld et al., '76, '79; Britten, '87) and 3" in chicks (Pateromichelakis, '81). This is larger than in macaques (0.5"; Wiesel and Hubel, '661, but smaller than in hooded rats (5"; Hale et al., '79). In the Wulst, the size of the smallest receptive fields is in the range of 1" in owls (Pettigrew, '79), 2" in pigeons (Revzin, '691, but 10"-20" in chicks (Wilson, '80b). In monkeys and cats the smallest receptive fields of "simple" cells are less than 1"in monkeys (Hubel and Wiesel, '74) and 0.3"-3" in cats (Lee et al., '77). These similarities are still present but less evident in granivorous birds, such as pigeons and chicks. One important difference between these avian species and frontal eyed mammals, such as monkeys and cats, is the poor representation of the frontal binocular field. The "red field" is an area in the dorsotemporal retina of pigeons which is binocularly stimulated by objects in the inferofrontal visual field (Hayes et al., '87; Nalbach et al., '90). Remy and Giinturkiin ('91) demonstrated that the red field only has an extremely limited representation in the GLd. In agreement with their anatomical study, a single unit electrophysiological study of the Wulst did not reveal the presence of visual units with receptive fields in the frontal binocular field (Miceli et al., '79). The frontal field seems to be represented in the visual Wulst of chicks, but the number of binocularly driven units is less than 1%(Wilson, '80a,b; Denton, '81). The hodological and physiological data on the avian lateral geniculate complex indicate many similarities to the GLd of mammals. A more detailed analysis of the avian GLd complex has been hampered by several problems. Foremost amongst them is the lack of information regarding the boundaries separating individual subnuclei of the GLd. Consistent boundaries of individual cell groups are difficult to discern on the basis of either cytoarchitectonics or afferentation. Cytoarchitectural differences between subdivisions are minimal, and in view of the small size of the subnuclei of the pigeon and chicken GLd, difficult to identify with any consistency. Retinal input to the avian dorsal thalamus is totally crossed, thus no obvious lamination consequent to alternating ipsilateral and contralateral visual afferents is seen, and retinal inputs do not delineate nuclear subdivisions as distinct laminae. Although retinal fibers to individual subnuclei differ in size, density, and trajectory as they enter each subdivision, these differences

723

are rather subtle, and have proven extremely difficult to codify with any consistency even between animals of the same species. Attempts to compare subdivisions of the GLd between pigeons, chickens, and owls have not been successful. Similarly, electrophysiological studies have not provided sufficiently detailed information regarding the physiological differences between subdivisions. A covert lamination of the GLd is partially indicated by differential output of subnuclear components to the ipsi- and the contralateral Wulst (Meier et al., '74; Miceli and Reperant, '82). We do not know if each subdivision has differential projections upon various constituents of the visual Wulst. Although different authors have proposed various subdivisions within the GLd on the basis of differing topological positions, cytology, volume, inputs, and outputs (Karten et al., '73; Reperant, '73; Raffin, '74; Ehrlich and Mark, '84), there is little certainty regarding the validity of the current classification of the GLd complex. Thus a classic neuroanatomical analysis based predominantly upon Nissl-stained sections has not provided adequate criteria for distinguishing distinct subdivisions of the avian GLd. In the present study we have attempted to determine whether immunocytochemical methods, when applied to seemingly undifferentiated areas, can help delineate subdivisions of cell groups according to their biochemical content. In order to correlate the distribution of putative transmitters/neuromodulators with various subdivisions of the geniculate complex, we investigated the chemoarchitectonics of the geniculate complex of the pigeon in relation to the pattern of direct retinal afferentation.

MATERIALS AND METHODS All experiments were carried out on white Carneaux or homing pigeons (Columba liuia). Three separate studies were conducted: 1) The distributions of various peptides, enzymes, and transmitters in the pigeon lateral geniculate complex were determined with antibodies directed against cholecystokinin (CCK), neurotensin (NT), substance P (SP), leucine-enkephalin (L-ENK), neuropeptide Y (NPY), glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (S).2) The distributions of efferent fibers and terminals of retinal ganglion cells within the geniculate complex were determined with injections of Rhodamine-B-isothiocyanate (RITC) into the vitreous. 3) The projections of neurotransmitter/modulator-specific geniculate cell groups onto the visual Wulst or optic tectum were determined by injections of various fluorescent retrograde markers into the Wulst or the tectum. The geniculate complex was then examined for the presence of tracer molecules in immunocytochemically labelled cells.

Immunocytochemistry For the immunocytochemical experiments, 12 animals were injected 20 minutes before perfusion with 1,000 IU heparin. Pigeons were then anesthetized with Ketamine (Parke-Davis; 1ccikg) and perfused through the left ventricle with 6% dextran in 0.1 M phosphate buffer (PB; 40"C, pH 7.2) followed by a fixative consisting of 4% paraformaldehyde in 0.1 M PB (4"C, pH 7.2). The brains were removed and stored for 4 hours in the same fixative, transferred to a fresh solution of 30% sucrose in 0.1 M PB, and stored overnight at 4°C. The brains were cut in the frontal plane in

724 steps of 25 krn on a freezing microtome and the sections collected in a solution of 0.1% sodium azide in 0.1 M PB. Free floating sections were incubated in one of the primary antisera in 0.3% Triton X-100 for 24 hours at 4°C in microcentrifuge tubes. The dilutions of the primary antisera are outlined below. Sections were washed three times in PB for a total of 30 minutes and incubated for an additional 30 minutes at room temperature in 10% serum of the same species of animal in which the secondary antibody was raised. The sections were washed for 10 minutes in PB and processed either for the indirect immunofluorescence or the avidin-biotin-conjugate (ABC)technique. For indirect immunofluorescence, a fluorescein isothiocyanate (F1TC)-coupled secondary antibody diluted 1:100 in 0.3% Triton X-100 was applied to the tissue for 1 hour at room temperature. The sections were then washed three times in PB for a total of 30 minutes, mounted, and coverslipped with a glycerine-carbonate buffer. For the ABC technique, following incubation in normal serum and subsequent washing in PB, the sections were incubated in biotinylated secondary antibody diluted 1:200 in 0.3%Triton X-100 for 1 hour at room temperature. The tissue was then washed in three changes of PB for 10 minutes each and incubated for 1 hour in avidin-coupled peroxidase diluted 1 : l O O in Triton X-100 at room temperature. Following three washes in PB for 10 minutes each, the sections were incubated with 0.05%diaminobenzidine (DAB) in PB for 15 minutes. Hydrogen peroxidase was added to the incubation medium to make a final concentration of 0.01%, and the dish was gently shaken as the reaction proceeded. After 20 minutes the reaction was stopped with several washes of PB. Sections were mounted on gelatincoated slides and dried. Afterwards the DAB reaction product was intensified by immersing the sections in 0.1% osmium tetroxide in PB for 30 seconds. After a final buffer wash, the sections were cleared and coverslipped. One out of six sections was stained with cresyl violet to allow correlation of immunohistochemical findings with traditional cytoarchitectonic subdivisions. Eleven different antisera were used for the present study. Antisera directed against cholecystokinin, substance P, neurotensin, leucine-enkephalin, and serotonin were purchased from Immunonuclear Corporation and were used at working dilutions of 1:1,000. Antisera directed against tyrosine hydroxylase were purchased from three different sources (Eugene Tech., 1:25; Boehringer Mannheim, 1:40; Chemicon, 1:250). Two different antisera directed against choline acetyltransferase were used; one was purchased from Chemicon (working dilution 1:500) and the other was kindly provided by Miles Epstein of the University of Wisconsin (working dilution 1:1,000). GAD-2, an antiserum directed against glutamic acid decarboxylase, kindly provided by David Gottlieb of Washington University in St. Louis, Missouri, was used at a working dilution of 1:2,000. Two different controls were conducted to demonstrate the specificity of the immunocytochemical staining pattern. As a first control, a complete staining sequence was run without prior incubation in any primary antibody. In this case staining was completely abolished. As more specific M of controls, primary antisera were incubated with the appropriate antigens, when available, for 12 hours at 4°C before application onto the sections. In these cases the sections were either completely blank or displayed only an extremely light brown and completely undifferentiated background. Since neither tyrosine hydroxylase nor glu-

0. GUNTURKUN AND H.J. KARTEN tamic acid decarboxylase could be purchased as antigens, this type of control could not be performed for these enzymes. However, to avoid misleading statements about the transmitters/modulators used by different neurons, all positive immunocytochemical results were described with the suffix “-like immunoreactivity” (e.g., serotonin-like immunoreactivity, S-li). The distribution of the various antibodies was mapped onto drawings of the geniculate complex. For this purpose, first some of the cresyl violet stained reference sections were projected onto a white paper with a final magnification of x30. The outlines of the thalamus and of clearly definable structures such as the optic tract, the GLv, the n. rotundus, or the n. ovoidalis were drawn at intervals of 250 pm onto these charts. Sections from the same animal stained for the visualization of different antigens were then projected onto these preliminary maps. Clearly visible cell groups and fiber tracts were drawn. In a third step, fine details of the immunocytochemical staining were added with microscopic inspections of the sections. In some instances it was important to differentiate between immunocytochemically defined terminal areas of retinal and nonretinal origin. For this purpose three animals were unilaterally enucleated under deep anesthesia. In one of these pigeons the complete eyeball was removed. In the two other pigeons the frontal chamber of one of the eyes was opened and the retina was removed with cotton swabs. After a survival time of 11-14 days the anesthetized animals were perfused and the brains were processed as outlined above.

Tracing of retinal fibers In five pigeons the projections of retinal fibers were studied with the aid of Rhodamine-B-isothiocyanate (RITC; Sigma) as an anterograde tracer. Twenty microliters of a solution consisting of 1%RITC in 1%DMSO dissolved in distilled water was injected under anesthesia into the vitreous. Eight days later the animals were perfused as outlined above, the brains sectioned at 25 km, and individual sections mounted on slides. Retinal fibers could be visualized without additional treatment of the sections, with a fluorescence microscope with an excitation wavelength of 550 nm. Ganglion cell projections were photographed and drawn onto the maps of the geniculate complex which were prepared as previously described. In order to visualize simultaneously the distribution of different antisera and the RITC labelled retinal fibers, sections from the thalamus of animals which had received an RITC injection into the vitreous were stained with FITC, the indirect immunofluorescence method. By changing the epifluorescence filter set of the microscope, FITC and RITC labelling could be visualized within a single section.

Double-labelling experiments To demonstrate neurotransmitterimodulator-specific projection systems of the subnuclei of the lateral geniculate complex, 11 animals received injections of retrograde fluorescent tracers either into the visual Wulst or the optic tectum. The tracers used were either Fast Blue (Sigma), Fluoro-Gold (Fluorochrome Inc.), or Rhodamine-coupled latex beads (Luma Fluor). Fast Blue and Fluoro-Gold were dissolved at 2% in distilled water. Rhodamine-coupled latex beads were diluted 1:5 in 0.1 M PB. The animals were anesthetized, their heads were placed in a stereotaxic holder, the skin over the skull was incised, and a small

IMMUNOHISTOCHEMISTRY OF THE LATERAL GENICULATE COMPLEX

fiber density values

o=O

[3=1

-2

725

m=3 m = 4

m=5

Fig. 1. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex at the anteroposterior coordinate A7.75 of the pigeon brain atlas tKarten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of

density and location. The left side of the section for serotonin ( S )is used as a key since no labelled perikarya expressing this antigen were detected within the geniculate complex. The grey levels at the right side of each figure indicate different fiber densities. The key for these grey values is given at the bottom of the figure. For abbreviations refer to the list of abbreviations.

portion of the skull was removed with a dental burr. One of ) then pressure injected with a the tracers (0.05 ~ 1 was Hamilton microsyringe. Since the three tracers used could be clearly differentiated with fluorescence microscopy, in most animals two or three injections with different tracers were performed. With this procedure differential projections onto various portions of the tectum or the Wulst could be simultaneously visualized. In several animals, tracers were placed in the tectum without a microsyringe. The bone overlying the tectum was removed and a small cut was made with a surgical microblade. Tracer crystals then were directly applied to the incision. This procedure generally yielded a significantly higher number of retrogradely labelled neurons in the afferent areas. After the injections were completed, the tip of the microsyringe was withdrawn, and the skin was pulled over the skull and sutured. After 24-48 hours the subjects were perfused as described above. Free floating sections were processed for indirect FITC or ABC immunocytochemistry using antisera against CCK, ChAT, GAD, or NPY. The sections were checked for the simultaneous presence of one of the tracers together with FITC or ABC immunoreactivity. The combined colocalization of Fast Blue or FluoroGold with FITC was generally less conclusive, since light emitted from both tracers overlapped slightly into the wavelength range in which FITC labelled cells were visualized. Better results were obtained with injections of Rhodamine coupled latex beads and subsequent ABC immunocytochemistry. However, for this technique, processed tissue sections were not dehydrated and cleared, but rather were coverslipped with glycerine-carbonate buffer. Contrary to the instructions given by Luma Fluor the latex beads were not dissolved by this procedure.

RESULTS On the basis of the immunocytochemical data the lateral geniculate complex of pigeons consists of three major subdivisions: the n. geniculatus lateralis, pars dorsalis (GLd), the n. marginalis tractus optici (nMOT), and the n. geniculatus lateralis, pars ventralis (GLv). These three components will be described separately. Additionally the GLd can be further subdivided into five components. In the following section the distribution of the various antibodies used will first be described. Then the distribution of retinal fibers in the different subcomponents of the lateral geniculate complex will be analysed. Finally, the results of the double-labelling experiments which revealed neurotransmitterimodulator-specific projections of the lateral geniculate complex onto the optic tectum and the visual Wulst will be described.

Immunocytochemistry N. geniculatus lateralis, pars dorsalis (GLd) N . lateralis anterior (LA). The LA is situated at the rostra1 pole of the GLd and is completely encircled by the n. marginalis tractus optici (nMOT). It extends from A7.75 to A7.25 in the atlas of the pigeon brain (Karten and Hodos, '67). In its anterior half it has an ovoid shape with the long axis oriented along the dorsolateral to ventromedial dimension. In its caudal part it assumes a spherical appearence with a location close to the dorsolateral edge of the optic nerve. Antibodies directed against serotonin demonstrated a dense network of serotonin-like (S-li)fibers which permeate the entire nucleus (Figs. 1-3). Most of these fibers possessed large numbers of swellings along their path through


726

A 7.50

I I

@

NPY

\

R

Fig. 2. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex at the anteroposterior coordinate A7.50 of the pigeon brain atlas (Karten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by hlack dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of

density and location. The left side of the section for serotonin (S)is used as a key since no labelled perikarya expressing this antigen were detected within the geniculate complex. The grey levels at the right side of each figure indicate different fiber densities. The key for these grey values is given only in Figure 1 but applies to all sections. For abbreviations refer to the list of abbreviations.

A7.25

L-ENK

Fig. 3. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex at the anteroposterior coordinate A 7 2 5 of the pigeon brain atlas (Karten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of

density and location. The left side ofthe section for serotonin (S)is used as a key since no labelled perikarya expressing this antigen were detected within the geniculate complex. The grey levels at the right side of each figure indicate different fiber densities. The key for these grey values is given only in Figure 1 but applies to all sections. For abbreviations refer to the list of abbreviations.


727

Fig. 4. Schematic mapping of the distribution o f the antigens studied in the lateral geniculate complex at the anteroposterior coordinate A7.00 o f the pigeon brain atlas (Karten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate o f

density and location. The left side of the section for serotonin ( S ) is used as a key since no labelled perikarya expressing this antigen were detected within the geniculate complex. The grey levels at the right side of each figure indicate different fiber densities. The key for these grey values is given only in Figure 1 but applies to all sections. For abbreviations refer to the list of abbreviations.

the nucleus resembling boutons-en-passant.Choline acetyltransferase- (ChAT-li), tyrosine hydroxylase- (TH-li), substance P- (SP-li), and leucine-enkephalin-like (L-ENK-11) fibers were visible within the LA but did not reach high densities. Antibodies directed against glutamic acid decarboxylase (GAD) demonstrated a large number of GAD-like fibers. In additional to the neuropil staining, a low number of GAD-11 small perikarya were present within this nucleus. Neuropeptide Y-like (NPY-11)fibers were present in the LA, but had a lower density than in the other subnuclei of the GLd. Most of these NPY-li fibers exhibited numerous swellings along their paths and traversed the LA in a ventrodorsal direction. N. dorsolateralis anterior thalami, pars magnocellularis (DLAmc). The DLAmc is an oval shaped nucleus with its long axis oriented in the dorsomedial to ventrolateral dimension. In frontal sections it first appears at A7.25 and reaches its maximal size at A7.00. It disappears between A7.00 and A6.75. S-li fibers were present throughout the whole nucleus (Figs. 3 , 4).Their density and appearance were equal to those described for the LA. Antibodies directed against choline acetyltransferase demonstrated scarce neuropil labelling but consistently a large number of ChAT-liperikarya. GAD-11 fibers were detected in low density throughout the whole extent of the nucleus. GAD-li medium sized perikarya were also present in the DLAmc but their number was considerably lower than that of CUT-11 somata. Antibodies directed against tyrosine hydroxylase revealed a fiber network of very low density without perikaryal staining in this

nucleus. The DLAmc was additionally characterized by a low density of NPY-, SP-,CCK-, and L-ENK-li fibers. N, dorsolateralis anterior thalami, pars lateralis (DLL). The DLL is by far the largest subnucleus within the GLd. It extends from A7.25 to A6.00 where it defines the caudal end of the GLd. Only a very small portion of the DLL is visible in its most frontal part where it borders the dorsolateral edge of the thalamus. At more caudal levels the DLL enlarges, especially medially at the border of the n. dorsomedialis anterior thalami (DMA). S-li fibers were homogeneously distributed throughout the DLL but their density never reached that of the LA or the DLAmc (Figs. 3-8). Antibodies directed against choline acetyltransferase labelled a large number of perikarya within the DLL. The staining intensity of the cells was low, but since this labelling pattern occurred consistently and was always above background, it was accepted to represent a population of cholinergic neurons. The distribution of ChAT-li somata revealed a clear distinction between a lateral (DLL1) and a medial (DLLm) component of the DLL. Perikarya in the lateral part were more numerous and consistently more intensely labelled than those of the medial half. As depicted in Figures 3, 4 and 11, DLLl occupies the whole rostral part of the DLL (A7.25-A7.00). Addi- tionally, from A6.75 to A6.00 it encompasses the lateral third of this nucleus with an elongation along the border with the n. superficialis parvocellularis (SPC). ChAT-li fibers had the same distribution as CUT-li somata with a higher density in the DLLl than in the DLLm. The distinction between DLLl and DLLm was also evident in the distribution of

0. GUNTURKUN AND HJ. KARTEN

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A 6.75

Fig. 5. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex at the anteroposterior coordinateA6.75 of the pigeon brain atlas (Karten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of

density and location. The left side of the section for serotonin (S) is used as a key since no labelled perikarya expressing this antigen were detected within the geniculate complex. The grey levels at the right side of each figure indicate different fiber densities. The key for these grey values is given only in Figure 1 but applies to all sections. For abbreviations refer to the list of abbreviations.

GAD-li labellings. DLLl was characterized by a large number of GAD-11 fibers and perikarya, while in DLLm both types of labellings were relatively scarce. As in the DLAmc, TH-li fibers were distributed with low density throughout DLL. Antibodies directed against neuropeptide Y also revealed heterogeneity within the DLL with high density fiber patches in the lateral DLLl and lateral DLLm. CCK-li fibers could not be detected within the DLL, while CCK-Ii perikarya were visible throughout the whole nucleus (Figs. 3-8). While a few L-ENK-11 fibers were present in the DLL, no SP-li or NT-11 labelling patterns were detected. N. superficialis parvocellularis (SPC). The SPC is located dorsally to DLL and is clearly divisible into a dorsal portion (SPCd) with small cells, and a ventral portion (SPCv) with medium sized neurons. The tractus septomesencephalicus (TSM) passes through SPCv such that most neurons within this area are scattered between the bundles of the TSM. Rostrally the TSM moves slightly dorsal so that the SPC flattens out, and is no longer evident at A6.75. S-li fibers are uniformly distributed both in the dorsal and the ventral portion of the SPC (Figs. 5-81, ChAT-li fibers could be detected in SPCd, while their density in SPCv was only slightly higher than background level. ChAT-li perikaryal staining was mainly visible in SPCd but a few somata were also labelled in SPCv. A weak GAD4 and TH-li fiber staining was present in SPCd but not SPCv. Antibodies directed against neuropeptide Y clearly differen-

tiated between the dorsal and the ventral portion of the SPC. While NPY-li stained neuropil was densely labelled in SPCv, only few scattered fibers could be detected in SPCd. Antibodies directed against substance P labelled fibers and a large number of somata in SPCd. Especially at more caudal levels (A6.25-A6.00) the SP-li stained cells made up a major portion of this subnucleus and reached medially up to the vicinity of the stria medullaris. In SPCv only a small number of SP-11 fibers could be recognized. The labelling pattern for CCK was very similar to that for SP: in SPCd both neuropil and perikarya were stained while SPCv was free of labelling. L-ENK-li fibers were homogeneously distributed throughout the whole SPC without concomitant perikaryal staining. N. lateralis dorsalis nuclei optici principalis thalami (LdOPT). The LdOPT is the smallest substructure within the GLd. Even in its maximal extent it never exceeds a diameter of 600 Fm. In Nissl-stained material it is only visible as a small, cell-poor area in the lateral part of the DLL (Fig. 9a,b). As demonstrated later, the LdOPT becomes clearly visible when retinal fibers are labelled, which heavily terminate within this area. S-li neuropil labelling was visible within the LdOPT without staining of S-li perikarya (Figs. 5-7). Antibodies directed against glutamic acid decarboxylase demonstrated a heavily labelled GAD-li neuropil encompassing an area slightly larger than the LdOPT, so that parts of the


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A6.50

Fig. 6. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex at the anteroposterior coordinate A 6 5 0 of the pigeon brain atlas (Karten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots, The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of


surrounding DLL were also involved (Fig. Sj). A large number of GAD-11 perikarya were located at the outer border of the LdOPT and processes of some of these neurons could be followed into this structure. The only perikaryal population within the LdOPT were a small number of SP-li neurons (Fig. 91). Although their somata were located within this structure, their processes could be partly followed out of the LdOPT to penetrate the surrounding DLL. TH-li and ChAT-li fibers or somata were not observed within the LdOPT (Fig. Sc). Antibodies directed against neuropeptide Y defined the outer border of the LdOPT in a negative way: NPY-li fibers encircled the LdOPT without penetrating it (Fig. 9k). NPY-li perikarya were absent while a few L-ENK-lifibers could be detected in the LdOPT. Antibodies directed against cholecystokinin and neurotensin labelled structures within the LdOPT which could not be easily defined as either perikarya or terminal endings and which demonstrated extreme variabilities in size (Fig. 9f,i).They reached from small dots with diameters of about 1km to large structures with diameters of up to 9.7 bm and surfaces of up to 43 km2.Since typical perikaryal characteristics of a nucleus and surrounding cytoplasm could not be detected, it was assumed that these structures represent very large terminals of unknown origin. Immunohistochemical stainings of unilaterally enucleated animals demonstrated that these NT- and CCK-11 structures disappeared contralateral to the enucleated side. The GAD-11 neuropil was unaltered in the same animals. These results suggest

the possibility that the NT- and CCK-11 structures represented very large transmitter-specific retinal terminals. However, we could not exclude the possibility that the disappearance of the peptidergic labelling was due to secondary effects of the enucleation. Therefore double-labelling experiments were performed to simultaneously demonstrate retinal terminals and NT- or CCK-li structures. Three animals received 20 p1, RITC injections into the vitreous of one eye. Eight days later the animals were perfused and processed either for NT-FITC or CCK-FITC immunohistochemistry. Fluorescence microscopy revealed large RITC labelled retinal terminals of very high density within the LdOPT. Many of these terminals appeared simultaneously positive for NT-FITC (Fig. Sg,h). On adjacent sections of the same animals it was similarly shown that some RITC labelled synapses were also CCK-FITC positive (Figs. 9d,e, 12e,f). Since the antibodies directed against NT and CCK were derived from the same species, it was not possible to simultaneously stain for NT and CCK. Thus it could not be decided whether NT and CCK were colocalized or were labelling adjacent terminals. N. suprarotundus (SpRt). The SpRt is a small nucleus in the caudal GLd. It consists of a thin sheet of neurons overlying the n. rotundus (Rt) and extends from A650 to A6.00. In Nissl-stained material it is visible as a narrow layer of small, tightly packed neurons. S-li fibers heavily innervate the SpRt, so that the lateral aspects of this nucleus were clearly differentiated from the


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A 6.25

Fig. 7. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex a t the anteroposterior coordinate A625 of the pigeon brain atlas (Karten and Hodos, ’67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of

density and location. The left side of the section for serotonin (S) is used as a key since no labelled perikarya expressing this antigen were detected within the geniculate complex. The grey levels at the right side of each figure indicate different fiber densities. The key for these grey values is given only in Figure 1 but applies to all sections. For abbreviations refer to the list of abbreviations.

overlying DLL and the underlying Rt (Figs. 6-8). More medially the SpRt became difficult to delineate, since the ventrally located n. triangularis (T) and parts of the dorsomedial Rt were also covered with a dense network of S-li fibers. Antibodies directed against glutamic acid decarboxylase revealed a dense network of GAD4 fibers and a number of GAD-li perikarya within the SpRt. TH- and NPY-11 fibers also had a higher density in SpRt than in the more dorsally situated DLL. Antibodies directed against CCK labelled the majority of SpRt neurons. Some of them exhibited a dark, Golgi-like labelling with processes ramifying within the flat sheet of this nucleus. Additionally CCK-Ii fibers were present throughout the whole extent of the SpRt. The same was true for L-ENK-11 fibers but with a considerably lower density than for CCK. N. mnrginalis tractus optici (nMOT). The nMOT is located in the anterolateral thalamus and extends from a level slightly caudal to A8.00 up to A6.00. It is thus one of the most rostrally located thalamic nuclei and nevertheless reaches up to the caudal levels of the geniculate complex. Rostrally it completely encircles LA and extends along the dorso- and ventrolateral border of the lateral forebrain bundle. Proceeding caudalward, it also encircles the Rt. From A650 to A6.00 the SpRt replaces the nMOT along the dorsal border of the Rt so that the nMOT now appears like a large “U” bridging the gap between the dorsal and the ventral part of the lateral geniculate complex.

S-li fibers were present throughout the complete extent of the nMOT, without S-li perikaryal labelling (Figs. 1-8). Antibodies directed against glutamic acid decarboxylase labelled a small number of neurons and a low density fiber network in the nMOT. The number of fibers increased in the lateral and especially dorsolateral part of this nucleus. The most outstanding labelling pattern was revealed with antibodies directed against neuropeptide Y. A large number of darkly stained NPY-li neurons and fibers could be detected throughout the whole nMOT so that this antibody could be used as a marker for the location and extent of this nucleus (Fig. 10a,b).Although NPY labelled a large number of nMOT neurons, consistently about half of the cells within this nucleus were completely unstained as could be seen using Nomarski optics. NPY-li fibers were generally more densely distributed in the most dorsolateral part of the nMOT. This heterogeneity within the nMOT was even more obvious using other antibodies. For example, SP-I1 perikarya and fibers were only detected in the dorsolateral part of the caudal (A7.00-A6.00) nMOT. More rostrally this SP positive portion of the nMOT increased in size to occupy the complete extent of the nucleus from A7.75 to A7.25. Thus the nMOT could be partitioned into a rostrodorsal (nMOTrd) SP positive and a caudoventral (nMOTcv) SP negative component. This distinction could be verified using antibodies directed against cholecystokinin and tyrosine hydroxylase. CCK-11 perikarya and fibers were only


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A 6.00

Fig. 8. Schematic mapping of the distribution of the antigens studied in the lateral geniculate complex at the anteroposterior coordinate A6.00 of the pigeon brain atlas (Karten and Hodos, '67). The labels at the top left of each section designate the abbreviation of the antigen depicted. To the left of each frontal section, labelled perikarya are symbolized by black dots. The number of dots does not indicate the exact number of labelled neurons but provides only an estimate of


present in the nMOTrd, while the nMOTcv was virtually free of labelling. Generally CCK-li perikarya were more numerous than those labelled with SP. A dense TH-li fiber pattern was observed in nMOTrd while few TH-li fibers were observed in nMOTcv. A weak L-ENK-11 fiber network was present in both components of the nMOT without labelling of perikarya. N. geniculatus lateralis, pars ventralis (GLv). The GLv is a prominent feature of the avian thalamus. In frontal sections it has a lens-like shape with the convex side completely embedded into the ventrally located optic tract. It is first recognizable at A7.75. From A750 caudally, it assumes its typical three-layered appearance with an outer parvocellular lamina externa (le), a cell-poor core with neuropil (ne),and an internal magnocellular lamina interna (li). While there is a smooth transition zone between le and ne, the internal layer li is a readily distinguishable entity. Proceeding caudalward the GLv shifts slightly medial to disappear at A5.25. S-li fibers were loosely distributed within the whole GLv without a layer-specificdifferentiation (Figs. 1-8). A CUT-li neuropil was present throughout the nucleus, but definite fibers were visible only in the le layer. In the ne and li layers, antibodies against ChAT produced a light brown labelling which could be differentiated from the white background. GAD4 fibers were especially pronounced in li,

although a GAD-li neuropil was also present in the other two layers. Additionally GAD-liperikarya could be observed throughout the whole extent of the GLv. A ranking of the perikaryal density labelling showed highest densities in li, intermediate in le, and lowest in ne. NPY-11 fibers were observed in all layers of the GLv but with a higher density in the two outer layers, le and ne. NT-li labelling patterns were not observed within the GLv. Four antibodies revealed differences in the labelling pattern between the most medial 100 pm of the GLv and the rest of the nucleus. This was the case for TH, SP, CCK, and L-ENK. The lateral portion of the GLv has an extent of approximately 1.5 mm and thus makes up the largest part of this nucleus. In this portion TH-li fibers were present in all layers of the GLv, but with a higher density in layer li where TH-11 fibers could be observed to encircle single perikarya with a large number of boutons. SP-Ii fibers were distributed only within layer li of the GLv while no CCK- or L-ENK-11 labelling patterns were observed. The most medial 100 pm of the GLv demonstrated several important differences. First of all, TH-, SP-, CCK-, and L-ENK-Ii fibers were present in a high density. Additionally, only in the medial GLv were TH-, SP-, and L-ENK-li perikarya observed. The immunocytochemically divergent medial part of the GLv undergoes a number of important topological changes in the rostrocaudal dimension. These can be


732

Figure 9


733

exemplified by a detailed description of the TH-11 staining DLAmc was a consistent result in all animals. To control for pattern. The same is essentially also the case for SP, CCK, incomplete ocular injections, the optic tectum was always and L-ENK but it is simply less obvious than for TH. From examined for complete labelling of the retinorecipient tectal A6.75 to A5.25 TH-li somata and a high density of TH-11 layers. Since this was always the case, it can be assumed fibers were present in the medial 100 km of the GLv. that the RITC injections labelled all aspects of the retinal Rostra1 to A6.75 this medial area shifted to an even more projection. Thus the partial innervation of the DLAmc medial position and parted from the GLv. Proceeding seems to be a characteristic feature of this nucleus. rostralward this medial part could be followed as an entity Between A7.00 and A6.75 the SCN moved laterally and separate from the GLv up to A850 while the GLv itself had joined the medial aspect of the GLv. From this level on, to disappeared at A7.75. These observations are consistent the caudal border of the GLv, the medial 100 Fm of this with the suggestion that the immunohistochemically distin- nucleus always contained a higher density of retinal fibers guishable medial extent of the GLv is a separate nucleus (Figs. 15, 16). Not all retinal axons which entered the GLv which only parly overlaps in position with the GLv (Gamlin terminated there. A large number of them proceeded et al., '82). dorsalward to innervate the nMOT. Consistently few fibers were observed to transverse the Rt to innervate the overlyRetinal projections upon the geniculate ing SpRt (Figs. 15, 16). No bouton-like swellings were complex detected on these fibers, although the resolution of light Intraocular RITC injections produced intense labelling of microscopy is of course not sufficient to exclude this the contralateral optic nerve. The fibers could be followed to possibility. The SPC received only a minority of retinal axons. All of the optic chiasm where they interdigitated with the axons from the other eye. Approximately at that level a small these optic fibers innervated SPCd, where they course number of fibers were observed to return to the ipsilateral medially to the vicinity of the stria medullaris (Fig. 12b). side where they joined the fibers of the other optic nerve SPCv was consistently free of labelled axons. At A6.75 the LdOPT was evident, with the previously (Fig. 12a). These ipsilateral fibers were concentrated at the dorsal aspect of the chiasm. Most of them coursed within described large terminals (Figs. 9d,g, 12c,e,fJ.The surroundthe tractus nuclei basalis opticus radici to innervate the n. ing DLL was, at that level, no longer uniformly covered basalis opticus radici. An even smaller number could be with retinal fibers, but assumed the heterogeneity defollowed to the ipsilateral LA, DLL, and SPC. No ipsilateral scribed in the section on immunohistochemistry. While the dorsal, lateral, and ventral DLL were heavily innervated, a fibers were observed to enter the optic tectum. The vast majority of optic fibers proceeded to the con- core portion and the medial DLL were only sparsely innertralateral side. Between A8.00 and A7.75 a fiber bundle left vated (Figs. 15, 16). More caudally, the dorsal aspect of the the optic nerve to innervate the most rostral part of the DLL became the primary region of optic afferentiation. In nMOT (Figs. 13-16). At A7.75 the LA and the GLv were this area the retinal fibers formed a large number of innervated by a large number of RITC labelled fibers. basket-like structures which were located within an approxMedial to the GLv, a small nucleus could be distinguished imately 300 Fm wide band oriented parallel to the SPC overlying the optic tract. Due to its topology and its (Figs. 12c,d, 15, 16). At A6.00, the caudal border of the retinorecipient status it probably corresponds to the n. DLL, retinal axons were observed only at the lateral and suprachiasmaticus (SCN) of other birds. Proceeding cau- laterodorsal aspect of the nucleus. The other parts of this dally, the GLv assumed its typical laminated appearance nucleus were virtually free of optic fibers. To test the possibility that retinal baskets and DLL relay with very large dot-like retinal boutons in the li and small fibers in the le and ne (Fig. 12j). In the LA a dorsoventral neurons have common locations, the distribution of both split could be observed which consisted of a virtually DLL relay neurons and retinal axons were simultaneously fiber-free area (Figs. 13, 14). This split was still visible at visualized in one experiment. This animal received an A7.25 where the LA disappeared. At approximately that intraocular RITC injection and, 8 days later, an injection of level, the most rostral apects of the DLL and the DLAmc the same tracer into the visual Wulst. With an excitation came into sight. While the DLL was full of retinal fibers, wavelength of 550 nm retrogradely labelled DLL relay only the dorsal aspect of the DLAmc was covered with RITC neurons and anterogradely labelled retinal fibers were labelled axons (Fig. 14). This partial innervation of the visualized. As depicted in Figure 12c,g, a number of retrogradely labelled relay neurons were located within the baskets and seemed to be covered with endings (Fig. 12k). Since these baskets could only be visualized with intraocuFig. 9. Frontal sections of the pigeon thalamus at about A6.50 show lar RITC injections and never occurred after Wulst injecthe LdOPT. The bar in a equals 100 )*mand applies to all sections. For tions, this result suggests within the resolution of light abbreviations refer to the list of abbreviations. Dorsal is upward and microscopy a direct retinal input to DLL relay neurons, in medial is to the right. Kluver-Barrera (a)and cresyl violet (b)staining of the LdOPT. Note the paucity of perikarya in the area of the LdOPT. agreement with the electron microscopic results of Watanabe ('87) in quail. Photomicrographs c, f, and i-I are taken from immunocytochemical staining experiments performed with the ABC method. Note the intense GAD-li labelling pattern in j , but the lack of NPY-li staining in k. In 1 only a few SP-li perikarya were labelled. Photomicrographs b, d, and e are taken from the same section and depict a double-labelling experiment with intraocular RITC injection and subsequent CCKFITC immunocytochemistry. Note the occurrence of the same structures in d and e. After the photographs were taken the section was stained for cresyl violet to obtain the picture given in b. The same applies for g and h which represent another double-labelling experiment with intraocular RITC injection and subsequent NT-FITC immunocytochemistry.

Double-labellingexperiments The aim of the double-labelling experiments was to demonstrate neurotransmitterimodulator-specific projections of the lateral geniculate complex. In the first series of experiments the peptide-specific projection of the nMOT onto the optic tectum was investigated. In the second series we investigated the transmitteripeptide-specific projection of the GLd subnuclei onto the visual Wulst.

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Fig. 10. Frontal sections of the thalamus of the pigeon taken at the same magnification factor depict NPY-li staining patterns. The borders of the nMOT are indicated by broken lines. The bar equals 200 km. The

sections correspond to the frontal levels A6.75 (a)and A7.25 (b).For abbreviations refer to the list of abbreviations. Dorsal is upward and medial is to the right.

nMOTINPY-li projections upon the optic tecturn. Previous unpublished experiments had demonstrated a projection from the nMOT to the optic tectum (Karten, Gamlin, and Reiner, unpublished observations). Immunocytochemical experiments had revealed the presence of a moderately dense plexus of coarse NPY-li axons in layers 2, 4,and 7 of the optic tectum, with an extremely dense plexus of very fine dustlike NPY-11 staining in layer 5. In order to determine the possible contribution of nMOT neurons to the NPY-Ii staining in the tectum, Fast Blue or Fluoro-Gold injections were performed into various areas of the optic tectum. These experiments always resulted in a large

number of retrogradely labelled neurons in the nMOT. NPY-FITC staining of the same sections simultaneously labelled NPY-li perikarya. Alternating filters for FITC and RITC revealed the simultaneous presence of tracer molecules and immunocytochemical staining patterns within

Fig. 11. Mapping of the various subdivisions of the pigeons's lateral geniculate complex as derived from the present experiments. The numbers at the top left refer to the planes of frontal sections taken from the atlas of Karten and Hodos 1'67). For abbreviations refer to the list of abbreviations. Dorsal is upward and medial is to the right.

A7.75

T% LA -nMO rd

A 7.00

A 7.2 5

n

A 6.7

A 6.

\

I

Figure 11

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IMMUNOHISTOCHEMISTRY OF THE LATERAL GENICULATE COMPLEX single neurons of nMOT. Virtually all retrogradely labelled neurons in nMOT were also NPY-FITC positive (Fig. 17a,b). Only few neurons were found to be tracer positive but NPY negative. As expected, a large number of NPYFITC positive neurons demonstrated no uptake of tracer molecules. Thus, the tracer applications had invaded only an area around the injection needle and consequently labelled only a small portion of the nMOT neurons. Due to the intensity of NPY-FITC staining, fluorescent breakthrough into the range of the retrograde tracers was a common occurrence. Therefore, to confirm the projection of nMOT to tectum, an alternate tracer was employed. In one animal, an injection of Rhodamine coupled latex beads (Rclb) was placed in the optic tectum. Subsequently the sections were processed for NPY-ABC. With this procedure NPY-11 neurons could be visualized in transmitted light microscopy while retrogradely labelled neurons were demonstrated with fluorescence microscopy. This single experiment gave the same results as the previously described NPY-FITC cases. In order to determine if the NPY-li perikarya of the nMOT were directly contacted by retinal fibers, selected sections from the retinal tracing experiments containing nMOT were labelled with NPY-FITC. Alternately illuminating sections for RITC and FITC demonstrated apposition of anterogradely labelled retinal fibers and NPY-li perikarya. In all cases, the NPY-11 somata were “wrapped” in a meshwork of retinal fibers. Within the technical limits of light microscopy these data suggest a direct retinal input upon NPY-li neurons of nMOT. Projections of chemically specified OPT neurons upon the Wulst. Using the same technical approach, the projection of the various subnuclei of GLd upon the visual Wulst was investigated. Two enzymes and one peptide were included in the analysis: ChAT, GAD, and CCK. Both the FITC labelled antibody plus fluorescent tracer technique and the ABC plus Rclb technique were used with all three antibodies. However Fluoro-Gold was not used as a retrograde tracer, as it produced bright fluorescingneurons with substantial breakthrough of the FITC filter and could

Fig. 12. a: RITC labelled retinal axons in the optic chiasm taking a turn (arrow) to the ipsilateral side. Both fibers course at the dorsal aspect of the optic nerve close to the overlying thalamus (dark region). b: Single RITC labelled retinal axon in the SPCd. c: Anterogradely labelled retinal axons after an intraocular RITC injection and retrogradely labelled relay neurons after an injection into the ipsilateral visual Wulst with the same tracer. Picture is taken from a frontal section through the lateral aspect of the GLd. Note that a band of retinal fibers is made up of basket-like structures (open arrows). Intermingled are relay neurons projecting to the ipsilateral Wulst (white arrows). d: A single basket made up of RITC labelled retinal axons in DLLI. e: High magnification of retinal terminals labelled with RITC in the LdOPT. f: Same section as e visualizes with another epifluorescence filter set the occurrence of CCK-li structures. g: Higher magnification from another section of the same experiment depicted in c, shows the overlap or close apposition of retinal axonal baskets and DLLl relay neurons (arrows). h: RITC labelled retinal terminals in LdOPT. i: Fluoro-Gold (FG) labelled relay neuron (arrow) of the same section as in h. Note the labelled dendrites (open arrow) partly penetrating the LdOPT. j: RITC labelled retinal axons in the GLv. k DLLl relay neuron retrogradely labelled after RITC injection into the ipsilateral Wulst. This cell is covered with bright dots probably representing RITC labelled retinal ganglion cell terminals. For abbreviations refer to the list of abbreviations. With the exception of d and e, where the bars equal 10 pm, all bars give a length of 100 pm. Dorsal is always upward and medial is to the right.

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therefore not be adequately discriminated from the less intensely stained ChAT- and CCK-FITC positive cells. ChAT-li labelled neurons in DLL and the DLAmc were found to be simultaneously labelled with retrograde tracers placed in the visual Wulst (Fig. 17c,d). However a large number of retrogradely labelled neurons were Dbserved to be ChAT-negative. This may indicate that a substantial proportion of the GLd projection onto the Wulst is noncholinergic. In the DLL and the SpRt, CCK-li neurons were found to be double-labelled, indicating a cholecystokininergic projection onto the Wulst (Fig. 17e,D. In the DLL, most of the retrogradely labelled neurons were found to be CCK negative. This was not the case for the SpRt. In this nucleus virtually all tracer labelled neurons were observed to be CCK-11. No GAD-li neurons were observed to be retrogradely labelled in any nucleus examined. The results of these experiments are schematically summarized in Figure 18. Most of the injections into the visual Wulst were performed at A1l.OO, between Lateral 1.00 and 2.00 and at a depth of approximately 1.5 mm below the surface. However, in eight injections, the location was varied between A9.00 and A13.00 and Lateral 0.50 and 4.00. Thus, a large part of the visual Wulst was explored for afferents from the lateral geniculate complex. In none of these experiments were retrogradely labelled neurons found in nMOT or GLv. But even within the GLd, not all substructures were found to project to the visual Wulst. Retrogradely labelled neurons were consistently observed in DLL, DLAmc, SPC, and SpRt, but not in LA. Several injections yielded retrogradely labelled cells not within but in close proximity to LdOPT, with their dendrites penetrating this structure (Fig. 12h,i). Due to the small size of this structure we presently cannot exclude the prospect that also a set of cells within LdOPT may project onto the forebrain.

DISCUSSION The avian lateral geniculate complex The lateral geniculate can be subdivided from dorsal to ventral into three major components: the n. geniculatus lateralis, pars dorsalis (GLd), the n. marginalis tractus optici (nMOT),and the n. geniculatus lateralis, pars ventralis (GLv) (Fig. 11).The GLd can be further subdivided into smaller subdivisions which may represent parallel channels within the ascending thalarnofugal pathway. The results of the present study demonstrate that a large number of neurotransmitter/modulator-specificneuronal systems interact in the avian lateral geniculate complex (Table 1).The present study has demonstrated that cholinergic and cholecystokininergic GLd neurons project to the visual Wulst. Within the DLL, where both systems occur, it is not clear whether ChAT and CCK are colocalized in single neurons. The dorsal portion of the avian lateral geniculate complex has been the subject of controversy with respect to its afferents, efferents, boundaries, nomenclature, and extent of similarity to the mammalian cell group of the same name. Previous attempts to subdivide the GLd into smaller entities relied upon cytoarchitectonic criteria andlor results from tracing experiments. We suggest that the chemoarchitectonic characteristics provide additional, and complementary, criteria for defining subdivisions of the GLd. This approach results in a somewhat different delineation of this structure than previously proposed. The different GLd subdivisions will be discussed below.

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1 Fig. 13. Drawings of the distribution of retinal projections to the thalamus as derived from anterograde tracing experiments with intraocular RITC injections. The numbers at the top give the position of the drawing according to the frontal sections in the atias of Karten and Hodos ('67). The numbers at the bottom and to the sides give the length in millimeters for mediolateral and ventrodorsal dimensions, respectively. The zero point of these coordinates equals that in the pigeon brain atlas. For abbreviations refer to the list of abbreviations.

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Fig. 14. Drawings of the distribution of retinal projections to the thalamus as derived from anterograde tracing experiments with intraocular RITC injections. The numbers at the top give the position of the drawing according to the frontal sections in the atlas of Karten and Hodos ('67). The numbers at the bottom and to the sides give the length in millimeters for mediolateral and ventrodorsal dimensions, respectively. The zero point of these coordinates equals that in the pigeon brain atlas. For abbreviations refer to the list of abbreviations.


N. lateralis anterior (LA), n. lateralis dorsalis nuclei optici principalis thalami (LdOPT), and n. suprarotundus (SpRt). The LA is the most easily defined substructure of the GLd. There is no disagreement as to its location, retinorecipient status, and nomenclature (pigeons: Edinger and Wallenberg, 1899; Huber and Crosby, '29; Cowan et al., '61; Karten and Nauta, '68; Reperant, '73; Meier et al., '74; Mihailovic et al., '74; Miceli et al., '75; Gamlin and Cohen, '88; chicks: Raffin, '74; Ehrlich and Mark, '84; quail: Watanabe, '87; Norgren and Silver, '89; owls: Hirschberger, '67; Karten and Nauta, '68). The variety of neurotransmitter-specific fiber systems within the LA contrasts with the fact that the retina is the only confirmed source of afferents to LA. However, it is possible that different transmitterspecific retinal ganglion cells project to the LA and thus contribute at least partly to the heterogeneity of neuroactive substances within this nucleus (Karten et al., '90). The LdOPT was first described by Hirschberger ('67) who suggested that it was a caudolateral continuation of the LA. According to his observations in the unilaterally enucleated screech owl, this area is characterized by extremely large axonal endings with diameters of 8-14 Fm. Most of these terminals were interspersed between perikarya but some were observed to entirely cover one half of the neuronal somata. According to the present study, these structures represent endings from CCK- and NT-11 retinal ganglion cells. This accords with several studies which provided evidence for the existence of CCK- and NTcontaining ganglion cells (Kuljis and Karten, '83; Kuljis et al., '84; Reiner, '86; Eldred et al., '881, and an electron microscopic study of Watanabe ('87) who demonstrated retinal terminals with dense cored vesicles in a part of the quail's GLd, which probably corresponds to the pigeon's LdOPT. A large number of these terminals in quail contacted dendrites of relay neurons which were shown to project to the Wulst. The SpRt contains both cholecystokininergic and GABAergic neurons. The CCK-li cells were shown to project to the Wulst while a similar projection pattern could not be revealed for the GAD-li neurons. Most authors defined the SpRt as the n. dorsolateralis anterior thalami, pars lateralis, pars ventroventralis (DLLw), and recognized an exclusively ipsilateral projection from this structure to the Wulst (Hunt and Webster, '72; Meier et al., '74; Miceli et al., '75, '79; Nixdorf and Bischof, '82). Bagnoli et al. ('83)labelled a very small number of presumably SpRt neurons after injections of [3H]-GABAinto the visual Wulst. This latter result may indicate that a small population of GABAergic cells with projections to the forebrain are localized in the SpRt but were not detected in the double-labelling experiments of the present study. N. dorsolateralis anterior thalami,pars magnocellularis (DLAmc), and n. dorsolateralis anterior thalami, pars lateralis (DLL). The DLAmc and the DLL are the two main retinorecipient subnuclei of the avian dorsolateral thalamus which project onto the visual Wulst. They represent the two largest and, with respect to the number and boundaries of their subdivisions, most controversial parts of the avian GLd. The mapping and nomenclature of the pigeon visual system provided by Reperant ('73) in his fundamental work dominated a large number of subsequent studies by different authors. Reperant ('73) divided this area of the GLd into four subcomponents: a magnocellular (DLAlm), a medial (DLAm), a lateral (DLAlp), and a laterorostral part (DLAlr). D W m appears equivalent to

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the DLAmc of the present study. It thus constitutes an independent thalamic entity with respect to its immunocytochemical characteristics, cytoarchitectonics, and efferents. The DLAm is located medial to the DLL. It occupies a position outside of the location of ChAT-li neurons, receives no retinal innervation, and does not project to the visual Wulst. Thus it does not seem to be a part of the avian lateral geniculate complex. According to the present study, the last two components, DLAlp and DLAlr, do not seem to represent separate entities, but are part of the DLL. Due to its more lateral position several authors mislabelled the LdOPT of the present study as the D W r according to Reperant (Ehrlich and Mark, '84; Watanabe, '87; Norgren and Silver, '89). The retinorecipient status of the DLAmc and the DLL has long been established (e.g., Edinger and Wallenberg, 1899). However, most authors describe a retinal innervation pattern which covers only parts of these nuclei. For the DLAmc this is the lateral (Meier et al., '74; Norgren and Silver, '89) or lateral plus dorsal part (Reperant, '73; Miceli et al., '75). This is in close correspondence to the results of the present study, where RITC labelled retinal fibers were observed to course only through the lateral and dorsal aspect of the DLAmc. The RITC technique demonstrated that this pattern of partial retinal innervation seems also to apply to the DLL: at more caudal levels the central and medial aspect of this nucleus was virtually free of labelled fibers. Since in the same animals the retinorecipient tectal layers were always completely labelled throughout their dorsoventral extent, it can be assumed that the RITC injections labelled all aspects of the retinal projection. The partial retinal innervation has been described until now for three avian species and could thus represent a general pattern of the GLd (pigeon: Meier et al., '74; Miceli et al., '75; Gamlin and Cohen, '88; chick: Ehrlich and Mark, '84; quail: Watanabe, '87). Tracing experiments demonstrated reciprocal connections of DLAmc and DLL with the visual Wulst (Adamo, '67; Karten et al., '73; Meier et al., '74; Miceli et al., '75, '80, '83, '87; Nixdorf and Bischof, '82; Bagnoli and Burkhalter, '83; Miceli and Reperant, '85; Watanabe, '87). According to the present study cholinergic neurons seem to participate in this projection while GAl3Aergic neurons were not found to project to the Wulst. Additionally cholecystokininergic neurons of the DLL also project to the Wulst. The presence of GAD-li immunoreactive neurons in DLAmc and DLL accords with the immunocytochemical study of Domenici et al. ('88).Bagnoli et al. ('83) failed to label DLAmc or DLL cells by injections of ['HI-GABA into the Wulst, providing further evidence that GABAergic neurons in the GLd lack direct projections to the visual forebrain. The relatively high muscimol binding and the low GAD activity in these nuclei further suggest that intrinsic and/or afferent GAl3Aergic neurons are more important than efferent neurons (Vischer et al., '82). Several studies provide direct and indirect evidence to confirm the present findings of cholinergic neurons in DLAmc and DLL. Aprison et al. ('64) and Aprison and Takahashi ('65) described high levels of ChAT and Ach in the pigeon thalamus. In a more specific study Vischer et al. ('82) described a dense acetylcholinesterase staining pattern and high ChAT activity levels in the area of the pigeon DLAmc and DLL. Bagnoli et al. ('81), who used radioactively labelled choline for identification of afferent cholinergic neurons by selective retrograde transport, labelled neurons in the ipsilateral DLAmc after injection of ['HI-

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Fig. 15. Drawings of the distribution of retinal projections to the thalamus as derived from anterograde tracing experiments with intraocular RITC injections. The numbers at the top give the position of the drawing according to the frontal sections in the atlas of Karten and

Hodos ('67). The numbers at the bottom and to the sides give the length in millimeters for mediolateral and ventrodorsal dimensions, respectively. The zero point of these coordinates equals that in the pigeon brain atlas. For abbreviations refer to the list of abbreviations.

choline into the Wulst. Shimizu and Karten ('90) detected ChAT immunoreactive fibers in the dorsolateral portion of the Wulst, which receives a projection from the GLd. An additional argument for the presence of a cholinergic innervation of the visual Wulst are the high ChAT activity levels of this forebrain structure (Bagnoli et al., '82; Vischer et al., '82); which decrease by about 40% following electrolytic lesions of the pigeon GLd (Vischer et al., '80).Wachtler ('85) and Wachtler and Ebinger ('89) demonstrated high concentrations of muscarinic acetylcholine receptors in pigeon and goose hyperstriatum dorsale (HD), hyperstriatum intercalatus superior (HIS), and nucleus intercalatus hyperstriati accessorii (IHA), the input layers of the visual Wulst. Together with the present results, these experiments provide reasonable evidence of possible cholinergic projections from the DLAmc and the DLL to the visual Wulst.

of them is the serotonergic projection from the raphe nuclei (Pasquier and Villar, '82; De Lima and Singer, '87b). The activity of serotonergic raphe neurons is modulated according to the sleep-wake cycles with high activity levels during waking states and decreased activity during slow-wave sleep (Jouvet, '72). The serotonergic fibers are evenly and diffusely distributed throughout all laminae of the primate GLd (Pasik et al., '88). In cats, the innervation density varies with low values in A, A l , and C, higher densities in the laminae Cl-C3, and highest values in GLv (De Lima and Singer, '87b; Mize and Payne, '87). At the ultrastructural level the serotonergic fibers in the GLd make only few conventional synapses and the serotonergic effects seem to be at least in part due to nonsynaptic release (De Lima and Singer, '87a; Pasik et al., '88; Wilson and Hendrickson, '88). This release leads to an inhibition of spontaneous and evoked activity of GLd relay neurons (Kayama et al., '89). Pape and McCormick ('89)demonstrated that the serotonininduced hyperpolarization of GLd neurons enhances a CAMP-mediated sodium/potassium ion current. This enhancement reduces the ability of GLd neurons to generate rhythmic burst firing which is characteristic for slow-wave sleep. It thus promotes a state of excitability during periods of waking. Similar to the situation in mammals the present study demonstrates also an innervation of the avian GLd with S-li fibers. Like in cats this innervation density varies with higher densities in the GLv and differences in densities between the GLd subnuclei. Within the avian GLd the S-li

Comparative considerations The avian lateral geniculate complex, on the basis of its topology, afferents, efferents, and synaptic organization, is thought to be equivalent to the mammalian lateral geniculate complex (Nauta and Karten, '70). The present study provides evidence that these similarities also may pertain to chemoarchitectonic features. 11. geniculatus lateralis pars dorsalis (GM). The mammalian GLd receives at least three distinct transmitterspecific brain stem afferents (Sherman and Koch, '86). One

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Fig, 16. Drawings of the distribution of retinal projections to the thalamus as derived from anterograde tracing experiments with intraocular RITC injections. The numbers at the top give the position of the drawing according to the frontal sections in the atlas of Karten and

Hodos ('67). The numbers at the bottom and to the sides give the length in millimeters for mediolateral and ventrodorsal dimensions, respectively. The zero point of these coordinates equals that in the pigeon brain atlas. For abbreviations refer to the list of abbreviations.

fibers demonstrate the same pattern of diffuse distribution as described for mammals (De Lima and Singer, '87a). It is not clear whether these resemblances between pigeons and cats hold to the ultrastructural and electrophysiological level but the similarities in the general outline of the system open the possibility that the serotonergic innervation may also alter the state-dependent level of excitability of neurons in the avian lateral geniculate complex. The second source of transmitter-specific brain stem afferents to the mammalian GLd is the cholinergic projection from the pontomesencephalic tegmental field (Mesulam et al., '83; De Lima and Singer, '87b). In the present study cholinergic elements were demonstrated by visualization of choline acetyltransferase (ChAT). ChAT can be assumed to occur exclusively within cholinergic cells and thus ChAT immunocytochemistry is considered to be the most selective method available to assess the distribution of cholinergic systems (Levey et al., 84). Acetylcholine directly excites GLd relay neurons due to a nicotinic receptormediated increase in cation conductance followed by a muscarinic M, receptor-mediated decrease in potassium conductance. GABAergic GLd interneurons and perigeniculate neurons are inhibited through an increase of potassium conductance via muscarinic M, receptors (Francesconi et al., '88; McCormick and Pape, '88; McCormick, '89). McCor-

mick ('89) suggests that the transition from synchronized thalamic activity during slow-wave sleep to desynchronized activity during arousal is associated with the activation of the cholinergic brain stem system. De Lima et al. ('85) identified cholinergic synapses primarily in the synaptic glomeruli of the GLd where they contacted both GABAergic interneuronal as well as relay neuronal processes. Synaptic glomeruli were observed by Watanabe ('87) in an area of the quail GLd which is comparable to parts of the DLLl of the present study. The pigeon's DLLl is characterized by a high density of cholinergic and GABAergic elements. Thus, we are inclined to believe that at least parts of the cholinergic processes in the pigeon GLd represent fibers from cholinergic tegmental neurons which could subserve similar functions in the avian and mammalian thalamus. The pigeon's GLd is characterized not only by a presence of cholinergic axons but also of ChAT-li perikarya. As also previously shown by Bagnoli et al. ('81) these cells represent relay neurons with projections to the visual Wulst. This is in sharp contrast to the situation in mammals. To our knowledge in none of the mammalian species studies so far have cholinergic perikarya been identified in the GLd. According to the present study this seems to be a sole but important difference between the chemoarchitectonics of birds and mammals.

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Fig. 17. Photomicrographs of the double-labelling experiments demonstrate transmitter-specific projections of the pigeon’s geniculate complex. a: NPY-FITC labelled cells in nMOT. b: Fluoro-Gold labelled nMOT neuron (arrow) in the same section as in a. The cell was retrogradely labelled with the FG injection into the ipsilateral optic tectum. c: ChAT-FITC labelled cells in the DLAmc. d: FB labelled


neurons of the same section as in c. The neurons were retrogradely labelled after a Wulst injection. e: CCK-ABC labelled cell in the DLL. f: Rhodamine-latex beads in the same neuron as in e after an injection of beads into the ipsilateral Wulst. Bars in a and b give a length of 100 km, all others a length of 15 krn. For abbreviations refer to the list of abbreviations.


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responsible for the excitatory effects of noradrenaline (Balthazart et al., '89). The mammalian lateral geniculate complex is characterized by a large number of GABAergic neurons. In virtually all mammalian species studied the GLv has a much higher number of GABAergic elements than the GLd (Hendrickson et al., '83; Penny et al., '84; Rinvik et al., '87). This is in perfect agreement with the results of the present study in pigeons. Like in mammals, the pigeon's GLd demonstrates also different densities of GABAergic neurons in distinct subdivisions (Montero and Zempel, '86; Rinvik et al., '87). Most of the GABAergic input in the mammalian GLd is based on GABA, receptors which increases the chloride conductance and thus creates a shunting inhibition (Sherman and Koch, '86). This intrageniculate inhibition is thought to play a role in the adjustment of the receptive field properties and seems to be mediated by interneurons and GABAergic elements of the perigeniculate nucleus (Creutzfeldt et al., '79). GABAergic relay neurons from the mammalian GLd seem to be absent (Montero and Zempel, '85; Montero, '89). This is also in agreement with the present study where GAD-li relay neurons could not be detected (but see Bagnoli et al., '83). The undecapeptide substance P (SP) is a member of the family of tachikinin peptides and has a wide distribution in the nervous system (Karten and Brecha, '80; Hokfelt et al., '87). Several studies demonstrated SP-li fibers in the mammalian GLd while SP-11perikarya were not observed (Cuello and Kanazawa, '78; Ljungdahl et al., '78; Mantyh and Kemp, '83).This is similar to the results of the present study where SP-li fibers were observed in the whole GLd while SP-li perikarya were virtually absent in most subnuclei. Cholecystokinin (CCK) is probably the most abundant CNS neuropeptide (Beinfeld et al., '81). It can be released by a calcium-dependent mechanism (Emson et al., '80) and Fig. 18. Schema showing the transmitter-specific projections of the has potent excitatory effects when applied iontophoretically avian lateral geniculate complex as revealed by the present experi- (Dodd and Kelly, '81). Fallon and Seroogy ('84) were ments. For abbreviations refer to the list of abbreviations. successful in demonstrating the existence of a large number of CCK-li GLd relay neurons with projections onto V1 in The third major mammalian transmitter-specific brain the rat. According to their data approximately 25% of the stem area with projections onto the GLd is the noradrener- GLd relay neurons were CCK-positive. Although no quantigic locus coeruleus (De Lima and Singer, '87b). In the tative evaluations were performed in the present study, the present study noradrenergic fibers were demonstrated by double-labelling experiments demonstrated nonetheless an visualization of tyrosine hydroxylase (TH). TH catalyzes abundance of CCK-li relay neurons in the pigeon's GLd. the first step in the biosynthesis of catecholamines and is Thus, CCK has to be considered as an important neurotransthus a specific marker common to dopaminergic, noradren- mitterimodulator in the vertebrate visual geniculo-telenergic, and adrenergic neurons (Cooper et al., '86). Applica- cephalic pathway. Leucine-enkephalin (L-ENK) is an opioid peptide which tion of noradrenaline or stimulation of the locus coeruleus increases the excitability of GLd relay neurons (Kayama et generally exerts an inhibitory effect on neuronal activity al., '82; McCormick and Prince, '88). This facilitatory effect either via blockade of the sodium channel or through decrease in potas- increased potassium ion conductance that secondarily supis due to an alpha-1-adrenoceptor-coupled sium conductance. This generates a slow depolarization presses calcium-dependent spike activity (Emson and Hunt, which brings the membrane potential closer to single spike '82). The presence of L-ENK-li fibers but not perikarya in firing threshold (McCormick, '89). The present study dem- the pigeon's GLd conforms with observations in rats (Manonstrated in agreement with the study of Bagnoli and tyh and Kemp, '83). Reiner et al. ('89) studied the distribuCasini ('85) a dense TH-li fiber network within the different tion of opiate receptors in the pigeon brain and found subareas of the pigeon lateral geniculate. Tohyama et al. medium concentrations of delta receptors in the whole ('74) described the avian locus coeruleus according to the lateral geniculate nucleus. Delta receptors are known to presence of catecholaminergic and probably noradrenergic have high affinities to enkephalin pentapeptides (Zukin and cells. Kitt and Brauth ('86)demonstrated a projection from Zukin, '84). the pigeon's locus coeruleus to the GLd and GLv. Similar to Both the avian and the mammalian GLd are subdivided mammals (Jones et al., '85) the avian GLd is also character- into several areas with diverse morphological and immunoized by a high density of alpha-1-receptors which are cytochemical characteristics. In mammals all of these subdi-

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TABLE 1. Distribution of Retinal Projections and Nine Different Antigens in the Various Subdivisions of the Pigeon's Lateral Geniculate Complex. + + High Density; + = Low Density; - = Absent; P = Perikaiya; F = Fibers. The Characteristics of the Medial Portion of the Glv Are Not Given.

visions are retinorecipient and contain relay neurons with projections onto V1 and/or V2 (Jones, '85). The present study demonstrates that this is not the case for all subdivisions of the pigeon's GLd. As demonstrated by various previous authors and also by present results, the LA is a retinorecipient thalamic structure which lacks projections onto the forebrain (Hunt and Webster, '72; Meier et al., '74; Miceli et al., '75, '79; Nixdorf and Bischof, '82; Bagnoli and Burkhalter, '83; Miceli and Reperant, '85; Ehrlich and Stuchberry, '86). This is a situation different from mammals and casts doubt on the assumption of the LA being a part of the avian GLd, at least according to a definition of the GLd adopted from mammals. However the situation in pigeons is similar to that in reptiles. In most reptiles studied the dorsal lateral geniculate complex consists of several retinorecipient subdivisions (Kosareva, '67; Reperant, '72, '75, '78; Kunzle and Schnyder, '83; Ulinski and Nautiyal, '88).The forebrain projections of these subnuclei are subject to controversies which seem to be related to species differences within the class of reptiles (Reperant, et al., '90). In Pseudemys scripta elegans and Chrysemys picta belli (Testudines) the entire dorsal lateral geniculate complex including its two retinorecipient subdivisions projects onto the forebrain (Rainey and Ulinski, '86). However in the saurian Tupinambis nigropunctatus (Lohman and van Woerden-Verkley, '78) as well as Iguana iguana and Gecko gecko (Bruce and Butler, '84) only one subdivision of the retinorecipient dorsal lateral geniculate complex projects to the telencephalon. Thus, the differences in the thalamofugal pathway within the class of reptiles seem to reflect also differences between birds and mammals. Only according to a definition reflecting the situation in mammals has the LA to be excluded from the lateral geniculate complex.

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Another substructure of the avian GLd which does not easily fit into the framework of the mammalian GLd is the SPC. Previous experiments (Reperant, '73) as well as the present study demonstrated that only few retinal fibers reach the dorsal aspect of this structure while the ventral SPC is completely devoid of direct visual afferents. Wild ('89b) demonstrated a somatosensory projection to an area which, according to pictures given by this author, is probably the ventral SPC. Additionally it has been demonstrated that the SPC projects not only to the visual but also to the somatosensory Wulst (Funke, '89a,b) and the area parahippocampalis (Casini et al., '86). Thus, the SPC is not a specific visual structure, neither in its afferents nor in its efferents. Due to its position and its connections it resembles the n. dorsolateralis anterior (DL) in several species of reptiles (Reperant et al., '90). The DL receives sparse retinal afferents in turtles (Kosareva, '67) and lizards (Kenigfest et al., '86) and projects onto the possible reptilian equivalent of the hippocampal formation and adjacent pallial areas (Lohman and van Woerden-Verkley, '78; Bruce and Butler, '84; Desan, '88). Desan ('88) and Reperant et al. ('90) speculate, although along slightly different lines of evidence, that the projection over the reptilian DL could be equivalent to a thalamo-hippocampal (Herkenham, '78) or even retino-thalamo-hippocampal pathway in mammals (Reperant et al., '87). Surely the comparison of the avian SPC with possibly related structures in reptiles and mammals has to await further experiments before sound conclusions can be drawn. But according to the present state of evidence the SPC does not seem to be a subdivision of the avian lateral geniculate complex. N. marginalis tractus optici (nMOT). The nMOT is situated between GLd and GLv and is characterized by the

IMMUNOHISTOCHEMISTRY OF THE LATERAL GENICULATE COMPLEX presence of NPY-11perikarya. It thus resembles the mammalian intergeniculate leaflet (IGL), a thin neuronal layer situated between GLd and GLv with a retinal input separate from its neighbouring structures (Hickey and Spear, '76). The IGL can be observed in a variety of mammals including hamsters, rats, and humans and consists mainly of NPY-11 and methionine-enkephalin-li (M-ENK-1i) neurons (Moore, '89). The M-ENK-li cells comprise a commissural projections to the contralateral IGL, while the NPY-li neurons were observed to project to the ipsilateral n. suprachiasmaticus (Card and Moore, '89). The NPYergic projection from the IGL to the SCN seems to play a role in the entrainment of the circadian rhythm (Moore and Card, '89). The IGL is not known to project to the superior colliculus, unlike its seeming avian counterpart, the nMOT (Edwards et al., '79). Since the projection of the nMOT onto the tectum is probably confined to the superficial layers, specifically upon layers 4 and 7 (Karten, Gamlin, and Reiner, unpublished observations), this indirect pathway could modulate the activity of the direct pathway from the retina onto the contralateral tectum. Wild ('89a) demonstrated a projection from the n. lentiformis mesencephali (LM) onto the ipsilateral GLd. According to his drawings a part of this projection terminates in the area of the nMOT. The LM is particularly concerned with horizontal optokinetic nystagmus and contains neurons sensitive to slow temporal-to-nasal motion (Gioanni et al., '83; Winterson and Brauth, '85). These data open the possibility that the projection over the nMOT might modulate the tectal activity patterns during locomotion-induced head saccades. Hamassaki and Britto ('90) additionally demonstrated a projection of NPY-li nMOT neurons onto the ipsilateral nucleus of the basal optic root and suggested a role of these cells in eye-stabilizing mechanisms. Thus, the avian nMOT and the mammalian IGL show a number of similarities with respect to their topology, immunocytochemistry, and their retinorecipient status. At least according to the present state of knowledge, their projections seem to differ. N. geniculatus lateralis, pars ventralis (GLv). The immunocytochemical results on the avian GLv are virtually identical to the results of authors studying the mammalian GLv with the same approach (S:Wilson and Hendrickson, '88; ChAT: Steriade et al., '88; TH Kromer and Moore, '80; GAD: Ohara et al., '83; SP: H kfelt et a]., '87; NPY: Chronwall et al., '85). Thus, the present study reveals a remarkable similarity in the chemoarchitectonics of the avian and the mammalian GLv. Tracing studies demonstrate that these similarities can also be confirmed at the connectional level. Both the avian and the mammalian GLv are retinorecipient (Hickey and Spear, '76; Crossland and Uchwat, '79; Peduzzi and Crossland, '831, have reciprocal connections with the optic tectum (Crossland and Uchwat, '79; Nakamura and Kawamura, '88),receive afferents from the visual forebrain (Hughes and Chi, '81; Miceli et al., '87), and project to a part of the pretectal nuclei (Swanson et al., '74; Gamlin et al., '84). We are thus inclined to believe that the avian and the mammalian GLv represent equivalent entities of the vertebrate thalamus. With the RITC technique we could demonstrate that the retinal termination pattern is strikingly different between the GLv laminae. The lamina externa (le) and the ventral neuropil (ne) were predominated by thin fibers while the lamina interna (li) was mostly covered by large terminal swellings. This result suggests that different classes of retinal ganglion cells could participate in a layer-specific

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projection onto the GLv. This assumption is supported by the results of Britto et al. ('89) who showed that the GLv receives d e r e n t s from TH- and SP-li retinal ganglion cells. In the present study TH-li fibers were distributed in all layers but with a higher density in layer li where they formed terminals similar to those observed with RITC. SP-li fibers had a different pattern of distribution and were only observed to ramify within layer li. Thus, the SP-11 retinal ganglion cells seem to project selectively to layer li of the GLv. Part of the TH-li fibers within GLv probably represent the noradrenergic projection from the locus coeruleus (Kitt and Brauth, '86). Since Balthazart et al. ('89) and Ball et al. ('89) could not demonstrate a remarkable density of alpha-1 and alpha-2-receptors in the quail GLv, a noradrenergic projection to the GLv might exert its effects via betaadrenergic receptors. Pape and McCormick ('89) showed that noradrenaline binding to beta-adrenergic receptors leads to an increase of intracellular CAMP enhancing a hyperpolarization-activated current. According to the same authors this effect reduces the responsiveness to large hyperpolarizing inputs and could thus mediate an increased efficacy of information transfer during periods of increased attentiveness. The medial GLv differs from the rest of this nucleus with regard to the presence and distribution of TH-, SP-, CCK-, and L-ENK-11 elements. The same area receives a strong retinal input. This partly obscures the lamination pattern of the GLv. Rostra1 to A6.75 this retinorecipient area moves slightly medial and separates from the remainder of the GLv. The same changes occur in the TH-, SP-, CCK-, and L-ENK-li staining patterns. The structure now positioned medial to GLv is probably the main portion of the n. suprachiasmaticus (SCN). This is reflected in its retinorecipient status (Reperant, '73; Ehrlich and Mark, '84; Cassone and Moore, '87; Norgren and Silver, '89) and its immunocytochemical characteristics (Cassone and Moore, '87). The present results thus indicate that the pigeon's SCN, or at least the lateral avian hypothalamic retinorecipient nucleus (Norgren and Silver, '901, is partly contiguous with the GLv proper. Since this nuclear colocalization can be observed extending to the most caudal level of the GLv a t A5.25, the avian SCN would have a considerably larger size then previously assumed.

ACKNOWLEDGMENTS Special thanks are in order to Thom Hughes, Kent T. Keyser, and Christine Laverack for expert advice during the conduct of this research. This study was supported by the German Research Council through grants Gu 22711-1 to O.G. and ONR N 00014-88-K-0504, NEI EY-06890, and NINDS NS-24560 to H.J.K.

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

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

Visual discrimination performance after lesions of the ventral lateral geniculate nucleus in pigeons (Columba livia).

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

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

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

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

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 ice pigeon (Columba livia breed ice).

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

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).

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.

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

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

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

Perception of complex motion in humans and pigeons (Columba livia).

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

Detection and discrimination of complex sounds by pigeons (Columba livia).

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

Responses of sympathetic postganglionic neurons to peripheral nerve stimulation in pigeon (Columba livia).