Distribution and relative abundance of neurons in the pigeon forebrain containing somatostatin, neuropeptide Y, or both.

THE JOURNAL OF C0MPARA"E NEUROLOGY 299:261-282 (1990)

Distributionand RelativeAbundance of Neuronsin thePigmnForebrain ContainingSomatosta%in, Nempeptide Y, or Both KEITH D. ANDERSON AND ANTON REINER Department of Anatomy and Neurobiology, University of Tennessee H.S.C., Memphis, Tennessee 38163

ABSTRACT Immunohistochemical studies in several mammalian species and in red-eared turtles have shown that somatostatin (SS)and neuropeptide Y (NPY) co-occur in a substantial proportion of the telencephalic neurons containing either. To explore further the possibility that telencephalic neurons co-containing SS and NPY may be evolutionarily conserved among amniotes, we determined the distribution and co-occurrence of SS and NPY in forebrain neurons in pigeons. Single-label immunohistochemical studies revealed the presence of overlapping populations of SS+ neurons and NPY+ neurons in most of the major subdivisions of the telencephalon. Double-label immunofluorescence studies revealed that in subdivisions of the telencephalon that are comparable to mammalian cortex (i.e., those dorsal and lateral to the basal ganglia), the vast majority of NPY+ neurons were also SS+,whereas a major and regionally variable percentage of the SS+ neurons were not NPY+. In contrast, within the basal telencephalon (including the basal ganglia and several other structures) neurons labeled only for NPY or only SS were more abundant than those containing both neuropeptides. Outside the telencephalon, the only forebrain cell group containing neurons in which SS and NPY were co-localized was in the lateral hypothalamus. A series of double- and triple-label immunohistochemical studies was undertaken to determine the extent of co-occurrence of SS and NPY in striatal neurons and the relationship of these neurons to striatal neurons containing other neuropeptides. In addition, immunohistochemical single- and double-label techniques were employed in conjunction with retrogradelabeling by fluorogold to determine the projections of SS+ and NPY+ striatal neurons. The results indicate that: 1) a population of striatal interneurons containing both SS and NPY exists in pigeons and constitutes approximately the same fraction of all striatal neurons as reported in mammals, 2 ) neurons containing NPY (but not SS)form a second, larger population of striatal interneurons, 3) neurons containing SS (but not NPY) form a third population of striatal interneurons that is approximately half as abundant as the NPY+ interneuron population, and 4)one-third of the substance P-containing striatonigral projection neurons also contain SS. The existence in pigeons of a major population of neurons containing both SS and NPY throughout the telencephalon, the existence of a population of neurons containing only SS in cortex-equivalent parts of the telencephalon, and the existence of a population of interneurons containing only NPY in the striatum is consistent with findings in mammals and turtles. The existence of these peptide-specific populations in members of each class of amniotes favors the view that these populations were present in the last common ancestor of amniotes and were conserved during amniote evolution. Thus, they most likely play fundamentally important roles in telencephalic circuits. However, the avian striaturn contains a substantial population of SS+ interneurons and a population of SS+/SP + striatonigral projection neurons. SS+ projection neurons have also been reported in the guinea pig, but not in other mammals. Thus, the presence of some types of SS-containing striatal neurons may vary among species. Key words: basal ganglia, telencephalon, striatum, substantia nigra, neuropeptide co-occurrence

Accepted June 9,1990

o 1990 WILEY-LISS, INC.

K.D. ANDERSON AND A. REINER

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Immunohistochemical studies in representatives of most striatal interneurons (DiFiglia and Aronin, '82, '84; Takagi classes of vertebrates have identified populations of neu- et al., '83; Vincent and Johansson, '83; Aoki and Pickel, '88; rons containing somatostatin ( S S )and populations contain- Vuillet et al., '89a). Studies combining retrograde labeling ing neuropeptide Y (NPY) throughout the brain (Bennett- of striatal projection neurons with immunohistochemistry Clarke et al., '80; Goossens et al., '80; Vandesande and in cats and monkeys indicate that SS+ or NPY+ striatal Dierickx, '80; Finley et al., '81; Inagaki et al., '81; 01- neurons do not project outside the striatum, providing schowka et al., '81; Takatsuki et al., '81; Allen et al., '83a; direct evidence that these striatal neurons are striatal local Bear and Ebner, '83; Nozaki and Gorbman, '83; Johansson circuit neurons (Chesselet and Graybiel, '86; Smith and et al., '84; Weindl et al., '84; Chronwall et al., '85; Danger et Parent, '86). However, in the guinea pig, a population of al., '85; Smith et al., '85; Vincent et al., '85; Bennett-Clarke medium-sized SS+ striatal neurons that do not contain and Joseph, '86; Chan-Palay et al., '86; De Quidt and NPY and that have a morphology similar to that of spiny Emson, '86; Wright, '86; Reiner and Northcutt, '87; Davila projection neurons has also been observed (Vincent and von et al., '88; Vallarino et al., '88; Kuljis and Rakic, '89a; Krosigk, '88). Thus, a population of SS+ striatal projection Laquerriere et al., '89; Noe et al., '89; Pontet et al., '89). neurons may be present in some mammalian species. Double-label studies in several groups of mammals, includ- Electrophysiological and neurochemical studies have shown ing primates and rats (Vincent et al., '82a,b; Chronwall et that SS plays a neuroactive role in the striatum (Chesselet al., '84; Hendry et al., '84a; Smith and Parent, '86; Gaspar and Reisine, '83; Beal and Martin, '84a; Arneric and Reis, et al., '8'71, and in a reptilian species, the red-eared turtle '86; Beal et al., '86; Meyer et al., '89). The similar distribution of neurons containing SS, NPY, (Reiner and Oliver, '871, have shown that a substantial percentage of telencephalic neurons containing either of or both neuropeptides throughout the telencephalon in all these neuropeptides contain both of them. Somatostatin- species examined, including members of two of the three immunoreactive (SS+)/NPY-immunoreactive(NPY+) neu- classes of amniotes (i.e., mammals and reptiles), indicates rons are abundant in all parts of the cerebral cortex in that these peptide-specific populations may be a common monkeys (Hendry et al., '84a), and in all parts of the feature of telencephalic organization in modern-day amequivalent telencephalic areas in turtles (the dorsal ventric- niotes. Thus, these findings raise the possibility that these ular ridge and cortedpallial thickening complex) (Reiner populations were also present in the last common ancestor and Oliver, '87). It appears that in the cerebral cortex of of amniotes and were inherited by modern-day amniotJes mammals, nearly all NPY+ neurons are SS+ and the from this ancient reptilian ancestor. To further examine majority of the SS+ neurons are NPY+ (Chronwall et al., this possibility, we studied the forebrain in the third major '84; Jones and Hendry, '86). Similarly, in reptiles virtually group of amniotes (i.e., birds) to determine if it contains all NPY+ cortical neurons are SS+,while a number of SS+ SS+,NPY+, and SS+/NPY+ neuronal populations distribneurons are not NPY+ (Reiner and Oliver, '87). In mon- uted similarly to those in mammals and reptiles. Single-, keys, the typically non-pyramidal morphology of these double-, and triple-label immunohistochemical techniques, neurons indicates they are intracortical local circuit neu- alone or in conjunction with retrograde labeling by fluorons (Hendry et al., '84a), although both pyramidal and rogold, were used to determine the distributions, projecnonpyramidal SS+/NPY+ neurons have been observed in tions, and relative abundances of SS+,NPY+, and SS+/ the rat cortex (Chronwall et al., '84). The role of these NPY + neuronal populations in the forebrain of pigeons. We neurons requires further study, but a transmitter role for found that in most telencephalic regions outside the basal cortical somatostatin is suggested by electrophysiological ganglia (i.e.,the telencephalic areas comparable to mamniastudies (Ioffe et al., '78; Phillis and Kirkpatrick, '80; Delfs lian neocortex, piriform cortex, hippocampus, and amygdala) our results in pigeons were similar to those previously and Dichter, '83). In the striatum of mammals and turtles, the co- described in other amniotes. In the basal ganglia, two occurrence of SS and NPY in neurons is also extensive; few populations of interneurons that have previously been neurons label for only one of these neuropeptides (Smith found in other amniotes were observed, one whose neurons and Parent, '86; Reiner and Oliver, '87). In mammals, SS+ contain both SS and NPY and one whose neurons contain neurons and NPY+ neurons in the striatum are typically only NPY. Thus these various telencephalic populations medium in size with aspiny dendrites, implying they are appear to be phylogenetically conserved among amniotes.

Abbreviations

AA Ac Ad AL APH Av Bas CA

cnrJ co E FPL HA

HD HIS

HV INP LPO

anterior archistriatum nucleus accumbens archistriatum, pars dorsalis ansa lenticularis area parahippocampalis archistriatum, pars ventralis nucleus basalis anterior commissure area corticoidea dorsolateralis optic chiasm ectostriatiim fasciculus prosencephali lateralis hyperstriatum accessorium hyperstriatum dorsale hyperstriatum intercalatus superior hyperstriatum ventrale intrapeduncular nucleus lobus parolfactorius

N NF NI NC nST OB PA PP PVM QF S SL SM TPO TSM TuOI VP

neostriatum neostriatum frontale neostriatum intermedium neostriatum caudale bed nucleus of the stria terminalis olfactory bulb paleostriatum augmentatum paleostriatum primitivum magnocellular periventricular nucleus tractus quintofrontalis septum lateral septum medial septum area temporo-parieto-occipitalis tractus septomesencephalicus olfactory tubercle ventral pallidum

SS AND NPY NEURONS IN THE PIGEON FOREBRAIN However, we also found a population of striatal interneuTons containing only SS and a large population of striatonigral projection neurons containing both SS and SP. These types of SS+ striatal neurons may be more phylogenetically variable, appearing in some but not all amniotes.

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Immunohistochemical techniques:single-label P-

One of two immunohistochemical procedures were used to label neurons for SS,NPY, and/or SP. In studies to map the distributions of SS+ neurons and NPY+ neurons, the peroxidase anti-peroxidase (PAP) technique was used (as described previously in Reiner, '87a,c), and in studies to examine the MA~SANDMETHODS co-occurrenceof these neuropeptides in individualneurons and Animal pretreatment in fluorogold-labeled(FG+) neurons, an indirect immunofluoWhite Carneaux pigeons (Columba liuia) between 6 and rescence technique was used that allows simultaneous labeling 12 months of age were used. All surgeries were performed of two or three different antigens in a single section (Erichsenet on pigeons deeply anesthetized with ketamine hydrochlo- al., '82; Reiner et al., '85;Wessendorfand Elde, '85; Reiner, '86, ride (Ketaset; 67 mg/kg, i.p.1 and xylazine (Gemini; 6.6 '87a,b; Reiner and Oliver, '87; Wessendorfet al., '87; Anderson mgkg, i.p.1. To enhance immunohistochemical labeling of and Reiner, '90). In the immunoperoxidase studies, a monoclonal mouse perikarya, the axonal transport inhibitor colchicine (Sigma; anti-SS-14 antibody was used at a dilution of 1:500 (gener315 pg in 7 pl distilled water), was injected at a rate of 1 pl every 10 minutes into the lateral ventricle approximately ously provided by J.C. Brown and S.R. Vincent), and a 36 hours prior to sacrifice. Pigeons used in the immuno- polyclonal rabbit anti-NPY antiserum was used at a dilufluorescence studies also received twice daily injections of tion of 1:500 (generously provided by J.M. Polak). Previous apomorphine (Lilly; 3.6 mg in 0.6 ml of 0.75% saline, i.p.) studies have demonstrated the specificity of these antibodfor 7 days prior to injection of colchicine. Apomorphine ies for their respective antigens (Allen et al.,'83a,b; Vincent et al., '85). The structure of SS has been shown to be highly pretreatment substantially elevates striatal levels of subsimilar if not identical among different classes of vertestance P in pigeons and makes neurons containing SP more brates, including birds (King and Millar, '79). Evidence immunohistochemically detectable (Anderson and Reiner, from various species suggest that NPY is a widespread and '90). This treatment did not, however, appear to have an phylogenetically conserved peptide among vertebrates that effect on immunohistochemically detectable SS or NPY in has been present in vertebrate brains since at least the the present studies. To label striatonigral projection neu- divergence of cartilaginous fish and bony fish (Danger et al., rons retrogradely, three of the four apomorphine-treated '85; Noe et al.,'89; Pontet et al., '89; Vallarino et al., '88; pigeons were stereotaxically injected with the fluorescent Reiner and Northcutt, '87, '88; Reiner and Oliver, '87). tracer fluorogold (Schmued and Fallon, '861, into the Sections processed according to the PAP procedure were substantia nigra (coordinates from the atlas of Karten and incubated in primary antisera diluted in a solution of 0.3% Hodos, '67) 7-12 days prior to injection of colchicine. Each Triton-X 100/0.01% sodium azide in 0.1 M PB at 4°C for 3 pigeon received three separate injections of fluorogold, so days, Sections were then incubated in goat anti-rabbit IgG that a total of 0.5 cl.1 of a 5% fluorogold in distilled water or goat anti-mouse IgG (as appropriate) diluted 150 in solution produced an overall injection site that encom- Triton-X lOO/sodium azide PB, followed by incubation with passed most or all of the substantia nigra and ventral the appropriate PAP complex (rabbit or mouse) diluted tegmental area. 1 : l O O in Triton-X 100/sodium azide PB. Incubations in secondary antisera and PAP were carried out at room temperature for 1hour. All incubations were carried out in Fixation microcentrifuge tubes on a rotator. Sections were rinsed Pigeons were placed deeply under chloral hydrate anesthe- between incubations in three 5-minute washes of PB, and sia and perfused transcardially, first with 50-75 ml of 6% were rinsed with three 10-minute washes in distilled water dextran in 0.1 M phosphate buffer (PB) (pH 7.2, room before staining. Sections were stained by incubating them temperature) and then with 250 ml of one of two fixatives at in 50 ml of 0.05 M imidazole/0.05 M cacodylate buffer (pH room temperature. Pigeons whose brains were used for the 7.2) containing 50 mg diaminobenzidine HC1 (DAB) for 10 immunoperoxidase studies were perfused with 4% paraform- minutes, and then adding 200 pl of 3%hydrogen peroxide aldehyde/O.l M lysinei0.01 M sodium periodate (meta) in for 10 more minutes, with continuous agitation through0.1 M PB (PLP fixative, pH 7.2), and pigeons whose brains out. Sections were then washed in distilled water, placed in were used for the immunofluorescence studies were per- PB, mounted onto gelatin-coated slides, dried, dehydrated, fused with 4% paraformaldehyde/O. 1% glutaraldehyde in and cover-slipped with Permount. Blocked controls were 0.1 M PB (pH 7.2). This latter fixative was used to enhance performed using the procedures described above with SS or immunohistochemical labeling of leucine-enkephalin for a NPY added at a concentration of 100 pM to the primary study not reported here; use of a fixative containing glutaral- antiserum incubation. dehyde did not appear to affect labeling for any of the antisera used in this study. Brains were removed and those Immunohistmhemical techniques:double and perfused with PLP fixative were immediately placed into triplelabel procedures 20% sucrose/lO% glycero1/0.02%sodium azide in 0.1 M PB Sections processed according to the simultaneous immuat 4°C for at least 24 hours before sectioning. Brains perfused with the paraformaldehyde/glutaraldehyde fixa- nofluorescence procedure were incubated in a mixture of tive were placed overnight in 4% paraformaldehyde in 0.1 M two or three of the following primary antisera (each raised PB (pH 10.4) at 4"C, and then transferred to the sucrose/ in a different species) in Triton-X lOO/sodium azide PB for 3 glycerol solution at 4°C. Brains were cut frozen on a sliding days at 4°C at the indicated dilutions: polyclonal rabbit microtome into 40-pm-thick transverse sections. Sections anti-SS-14 (Incstar), 1:500; polyclonal sheep anti-NPY (generously provided by J.R. Oliver and W.W. Blessing), 1500; were stored in 0.1 M PB with 0.02% sodium azide at 4°C.


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monoclonal rat anti-SP (Accurate Chemical and Scientific Co.), 1:1,000. The specificity of the anti-NPY and anti-SP for their respective antigens has been demonstrated previously (Cuello et al., '79; Blessing et al., '86). The lack of crossreactivity of sheep anti-NPY for SS was demonstrated in a previous double-label study in turtles (Reiner and Oliver, '87). Moreover, the lack of crossreactivity of these antisera for each others' antigen was evident in the present studies, since neurons labeled (sometimes very intensely) for only one antigen were observed in tissue in which double-labeled neurons were also abundant. For example, throughout most of the diencephalon numerous neurons containing only SS or only NPY were observed, while in one specific site in the hypothalamus numerous neurons containing both were oktserved (Fig. 5). The presence of an SP-like peptide in the brain of pigeons has been shown in a number of previous biochemical and immunohistochemical studies (Reubi and Jessel, '79; Reiner et al., '83; White et al., '85; Reiner, '86; Davis et al., '88). After incubation in primary antiserum, the sections were given three 5-minute washes in PB. The sections were next incubated for 1hour at room temperature in a mixture of appropriate secondary antisera, each conjugated to a different fluorophore: donkey anti-rabbit IgG conjugated to dichlorotriazinylaminofluorescein (DTAF) or tetramethylrhodamine isothiocyanate (TRITC),donkey anti-sheep IgGDTAF, mouse anti-rat IgG conjugated to 7-amino-4methylcoumarin-3-acetic acid (AMCA) (all from Jackson ImmunoResearch, West Grove, PA) and goat anti-ratTRITC (Cappel, West Chester, PA). The secondary antisera were used at dilutions of 1:50. Sections were then given three 5-minute washes in PB, mounted onto gelatin-coated slides, dried, rinsed in distilled water, dried, and coverslipped with 9:l glycero1:carbonate buffer (pH 10.5). Control procedures to demonstrate lack of primary and secondary antiserum crossreactivity or of fluorophore crossemission were carried out as described previously (Erichsen et al., '82; Wessendorf and Elde, '85; Reiner, '86; Reiner and Oliver, '87). Our filter system, as described below, prevented fluorophore cross-emission.

Data collection The distributions of SS+ neurons and NPY+ neurons in the forebrain were mapped from a series of immunoperoxidase-labeled sections with an Olympus microscope with drawing tube. In the analysis of immunofluorescence doublelabeled tissue, the relative percentages of SS+, NPY+, and SS+lNPY+ neurons in the various subdivisions and nuclei of the telencephalon were determined by counting all labeled neurons of each type in every section of a rostral to caudal series of sections through the forebrain (in which each section was separated from the next adjacent section by 440 Fm). I:llustrations of the relative distributions of SS+,NPY+, and SS+iNPY+ neurons and of the relative distributions of SS+ and SS+/SP+ neurons in the basal ganglia portion of the forebrain were made using an overhead projector to trace section outlines and prominent blood vessels. These landmarks were then used to draw the locations of labeled neurons freehand while viewing the sections through the microscope with fluorescence epiillumination. An Olympus BH-2 fluorescence epi-illumination microscope with a mercury light source and three different sets of standard Olympus excitation and dichroic filters was used. One set of filters was used to view DTAF, a second set was used to view TRITC, and a third set was

used to view AMCA or FG. The sets of filters were located in a custom-made three-position slider, so that different fluorophores could be viewed in the same section quickly and easily by moving the slider from one position to the next. We found that several supplementary filters were useful in eliminating any and all cross-emission among fluorophores. A 530-nm long pass excitation filter eliminated crossemission of DTAF fluorescence through the filter system used for TRITC, a 540-nm short pass barrier (emission)filter eliminated cross-emissionof TRITC through the filter systems used for DTAF or AMCMG, and a 500 nm short pass barrier filter eliminated cross-emission of both DTAF and TRI'rC through the AMCMG filter system. Use of the standard filter systems along with these supplementary filters provided for unequivocal identification of fluorophore labeling in tissue labeled with more than one fluorophore. To determine the percentages of each of the various neuropeptide-specific types of neurons identified in the medial striatum relative to all medial striatal neurons, we first determined the percentage of all medial striatal neurons that were SP+. This was calculated by determining the number of SP+ neurons in 14 400-pm2 sample areas (taken from a series of immunoperoxidase-labeled sections) and dividing this value by the total number of striatal neurons in these sample areas (asdetermined in Nissl-stained sections). The percentages of all striatal neurons that were SS+,NPY+, SS+iSP+, or SS+/NPY+ was then calcu1:ited from the ratio of SS+ neurons to SP+ neurons determind in the immunofluorescence double-labelstudies.

RESULTS single-labelstudies Somatostatin-immunoreactive (SS+)and neuropeptide Y-immunoreactive (NPY +) neurons were abundant throughout the cortex-equivalent parts of the telencephalon, including the Wulst [which comprises the hyperstriatum accessorium (HA), the hyperstriatum intercalatus superior (HIS), and the hyperstriatum dorsale (HD)],the hippocampal complex [which comprises the hippocampus (Hp) and the area parahippocampalis (APH)I, much of the dorsal ventricular ridge (DVR) [which comprises the hyperstriatum ventrale (HV), the neostriatum (N), field L (L), and the archistriaturn (A)], the dorsal and lateral pallium [which comprises the temporo-parieto-occipital region (TPO), the area corticoidea dorsolateralis (CDL), and the piriform cortex], as well as the glomerular layer of the olfactory bulbs, and much of the basal telencephalon [induding the medial striatum (lobus parolfactorius, LPO), the olfactory tubercle (TuOl), the accumbens region (Ac), and the bed nucleus of the stria terminalis (nST)]. However, neurons containing SS or NPY were relatively rare in the uentrolateral DVR [including nucleus basalis (Bas) and the ectostriatum (El] and parts of the basal telencephalon [including the lateral striatum (paleostriatum augmentatum, PA), the pallidum (paleostriatum primitivum, PP), the intrapeduncular nucleus (INP), the ventral pallidum (VP), and the septum (S)1(Fig. 1).The distributions of SS+ neurons and NPY+ neurons overlapped one another completely, but SS+ neurons were generally two to three times more numerous than NPY+ neurons. Overall, both types of neurons were most abundant near the lateral ventricle, and were less abundant away from the ventricle. Virtually all neurons were medium-sized (10-20 pm), and appeared to be multipolar or bipolar with round or ovoid perikarya.

SS AND NPY NEURONS IN THE PIGEON FOREBRAIN

A

265

B

Figure 1 (see overleaf 1


266

/

Fig. 1. A rostra1 to caudal series of transverse sections through the telencephalon schematically illustrating the locations of SS-labeled neurons (A, C, E, G , I, K, M, 0)and NPY-labeled neurons (B,D, F, H, J, L, N, PI. Each dot represents ten labeled neurons. Field L is located

in medial neostriatum at the levels shown in K-P. Piriform corlex is located along the ventrolateral surface of thc brain (ventral to 'I'PO, ventral and lateral to paleostriatum and archistriaturn) at the levels shown in G-N.

Doublelabel studies

the DVR) were also SS + , whereas many SS+ neurons 'were not NPY+ (Figs. 2 , 3 ) . Thus, two major populations were found in each region. Neurons in one population contained SS but not NPY and neurons in the second contained both of these neuropeptides. SS+ neurons were 2-5 times more abundant than SS+/NPY+ neurons in the Wulst (relative percentages of SS+,SS+/NPY+, and NPY + neurons were:

Cortex-equivalent telencephalon a n d olfactory bulbs. Double-label immunofluorescence studies revealed that the vast majority of NPY+ neurons in regions of the telencephalon dorsal and lateral to the dorsal medullary lamina (LMD, the boundary separating the striatum from

K

L

N

N

0

P

Figure 1 continued

268

Fig. 2. Pairs of photomicrographs illustrating the co-occurrence of SS and NPY in neurons in the telencephalon. a and b: A section through the neostriatum labeled for SS and for NPY by siniultaneous immunofluorescence, showing SS+ neurons labeled by rhodamine (a), and, in the same field of view, NPY+ neurons labeled by fluorescein (b). Arrows in a and h indicate an example of a neuron labeled for both SS


and NPY. The lateral ventricle is at top in this field. c and d A section through the hippocampal area labeled for SS and for NPY by siniultaneous immunofluorescence, showingSS+ neurons labeled by rhodamine (c), and, in the same field of view, N P Y i neurons labeled by fluorescein (d). Arrows in c and d indicate examples of neurons labeled for both SS and NPY. Scale bar = 50 pm (a,b); 100 bm cc,d).


269

Fig. 3. Pairs of photomicrographs illustrating the co-occurrence of SS and NPY in neurons in the telencephalon. a and b: A section through the archistriaturn labeled for SS and for NPY by simultaneous immunofluorescence, showing SS+ neurons labeled by rhodamine la), and, in the same field ofview, NPY+ neurons labeled by fluorescein (b). Arrows in a and b indicate examples of neurons labeled for both SS and

NPY. c and d: A section through the hyperstriatum accessorium labeled for SS and for NPY by simultaneous immunofluorescence, showing SS+ neurons labeled with rhodamine (c),and, in the same field of view, NPY+ neurons labeled with fluorescein (d). Solid arrows in c and d indicate a neuron labeled for both SS and NPY, and open arrows indicate neurons labeled only for SS. Scale bar = 50 km.

HA: 64%, 36%, 0%; HIS/HD: 61%, 38%, l%), in the dorsal ventricular ridge (HV: 77%, 23%,0%; N: 84%, 15%,1%;A: 76%, 24%, O%), and in the archistriaturn (76%, 24%, 0%) (Table 1). In contrast, SS+/NPY+ neurons were much more abundant than SS+ neurons in the hippocampal complex (30%, 70%, 0%) and piriform cortex (18%, 82%, 0%). Finally, these two types of neurons were relatively equal in number in dorsal pallial regions (TPO: 51%, 47%, 2%;CDL: 60%, 40%,0%). The olfactory bulbs were the only telencephalic region where neurons double-labeled for SS and NPY were not observed. There were no obvious differ-

ences in the morphology among SS+,SS+INPY+, and NPY+ neurons in any region (Figs. 2,3).However, neurons close to the lateral ventricle tended to have more extensive dendritic labeling (Fig. 2a,b). This feature may be attributable to high concentrations of neuropeptides in the dendrites of neurons in close proximity to the ventricle, perhaps as a result of higher exposure to colchicine than more lateral neurons. Basal telencephalon. In parts of the telencephalon ventral to the LMD, the abundance of SS+/NPY+ neurons relative to single-labeled neurons was generally lower, and


2 70

striatonigralprojection neurons in pigeons (Kittand Brauth, '81), and because many of these neurons had morphologies similar to striatonigral projection neurons, we combined ss+ SS+/NPY+ NPY -I Brain area retrograde labeling and immunohistochemistry to see if these neurons were projection neurons or interneurons. Medial, dorsal and lateral telencephalon 36 0 64 Hyperstriatum accessoriu'm Intranigral injection of fluorogold (FG) retrogradely labeled Hyprstriatum intercaiatus sup* numerous neurons in the basal ganglia portion of the 1 38 61 n o r h p r s t r i a t u r n doriale 0 23 77 Hyperstnatum ventrale telencephalon. FG+ neurons were most abundant in the 1 15 a4 Nrostriatum medial striatum (LPO), nucleus accumbens, ventral palli24 0 76 Archistriatum 0 dum, and pallidum, whereas FG+ neurons were never 70 30 Hippocampal complex 2 47 51 Temporo-pxieto-occipitd region observed in the lateral striatum (PA). These results are 40 0 60 Arra cortirnidea dnrsdattralis largely consistent with those described previously in retro(1 1H 82 Piriform cort~x Ventral telrrimphalon grade labeling studies with HRP (Kitt and Brauth, '81). Basal ganglia (stnatum,pallidum, These injections also labeled many neurons in the nS'r, ventral palliduiii, n. a u x m b n s , 4 34 62 olfactmy tubrrclel presumably by labeling fibers-of-passage destined for the 4 38 58' Striaturn and palhdum only nucleus of the solitary tract (Berk, '87). Using the simulta7 38 55 Red nucleus of stria terminalis 11 22 neous immunofluorescence double-label technique we deter67 Seuturn mined that 30-40% of FG+ neurons in the striatum were 'Ofthe 5X'k or neuronb conta~mngSS but not NPY, 20V were observed to contmn only SS and also SS+ (which represented only a fraction of all SS+ 38'8~a r i i t ~ ~ nSP wl neurons) (Fig. 6), whereas none of the FG+ neurons in the striatum were SS+/NPY + or NPY +. Since previous studies the relative abundance of NPY + neurons was higher, than have shown that the vast majority (i.e., 85-95%') of striatoin the more dorsal telencephalon. In the basal ganglia, P (SP)(Anderwhich includes the medial (LPO) and lateral (PA) striatum, nigral projection neurons contain substance son and Reiner, ' 8 7 ) , we next used the simultaneous the pallidum (PP, which contained only occasional SS+ and/or NPY+ neurons), the ventral pallidum, the nucleus immunofluorescence technique to determine the extent to SS+/FG+ neurons were SP+. This study revealed accumbens, and the olfactory tubercle, the SS+, SS+I which NPY+, and NPV+ populations were present in the follow- that virtually all (96-98%) of the FG+/SS+ striatal neuSP+, but this population represented onky a ing proportion: SS+,62%; SS+/NPY+, 4%; NPY+, 34% rons were also (Table 1). In the striatum and pallidum alone, these minority (39%)of all FG+/SP+ neurons. A small number of percentages were: 58%,4%, and 38%. In the bed nucleus of SS+/SP+ neurons were observed that were not FG+, but SS that were not the stria terminalis, the relative percentages of SS+,SS+/ the vast majority of neurons containing SP. The retrogradely labeled with FG did not contain NPY+, and NFY+ neurons were, respectively, 5596, 7%, and 38%, in the septum they were 67%, ll%, and 22%,and results of these studies thus revealed that SP+ striatoniin the ventral telencephalic region surrounding the anterior gral projection neurons consist of two subpopulations, one commissure (a region including part of the nST, but with containing SS and SP, and a second, larger one containing poorly defined boundaries), they were 61%, 17%, and 22%. SP (but not SS).On the other hand, of those neurons Thus, the basal telencephalon differed from dorsal and containing either SS or NPY, three peptide-specific populalateral telencephalon in that there were substantial num- tions appear to exist that do not project to the substantia bers of NPY+ neurons and these NPY+ neurons and the nigra. One of these contains SS (but not SP), the second SS+ neurons were much more abundant than were the contains SS and NPY, and the third contains NPY. It is SS+/NPY+ neurons. Nevertheless, the basal telencephalon possible that a very small population of SS+iSP+ neurons contained numerous SS+/NPY+ neurons. In the medial also may not project to the nigra, but since we cannot be striatum, the overall abundance of SS+/NPY+ neurons certain that all striatonigral projection neurons were retrowas calculated to be 1.3% of the total striatal neuron gradely labeled by the intranigral FG injections (despite our population. The distributions of SS+,SS+INPY+, and apparent success at encompassing the entire nigra with our NPY + neurons are illustrated in Fig. 4. The medium-sized injections), we cannot rule out the possibility that the few bipolar and multipolar morphologies of these different observed SS+/SP+ neurons not retrogradely labeled with peptide-specific neuron types were largely similar through- FG are nevertheless striatonigral projection neurons. In other parts of the basal ganglia (i.e.,PP, VP, Ac, and TuOl), out the ventral telencephalon (Fig. 7). Neurons single-labeled for SS or NPY were also abundant and in the nST, FG+ neurons were never found to be in the diencephalon, but neurons labeled for both SS and labeled for either SS or NPY. NPY were detected in only one location, the dorsal part of Triple-labelstudies the lateral hypothalamus at the level of rostra1 nucleus rotundus. Here, intensely labeled SS+ neurons were abunThe results of the retrograde labeling studies suggested dant, and the majority did not contain NPY. Few if any SS+ striatal neurons containing SP were distinct from 3S+ neurons were NPY+ only. However, virtually all neurons in striatal neurons containing NPY. To examine this possibilthe dorsolateral one third of this group of neurons labeled ity directly, we triple-labeled tissue for SP (using an relatively intensely for both SS and NPY (Fig. 5 ) . The AMCA-conjugated secondary antiserum), SS (using a medium-sized bipolar and multipolar SS+ neurons were TRITC-conjugated secondary antiserum), and NPY (using morphologically indistinguishable from the SS+/NPY+ an DTAF-conjugated secondary antiserum). The tripleneurons. label studies confirmed that many SS+ striatal neurons contained SP (virtually all of which presumably project to Retrograde labelingstudies the nigra) and that these neurons did not contain NPY. Because SS+,NPY+, and SS+/NPY+ neurons were Moreover, NPY+ neurons, SS+INPY + neurons, and many abundant in medial striatum, which is the location of SS+ neurons did not contain SP (all of which, based on the TABLE 1. Percentages of Neurons in Major Subdivisions of the Telencephalon Labeling for Only SS, for SS and NPY, and for Only NPY Relative to All Neurons in a Given Subdivision Labeling for a t Least One of the Neuropeptides

~~~

~

SS AND NPY NEURONS IN THE PIGEON FOREBRAIN retrograde labeling results, are apparently local circuit neurons) (Fig. 7). Since we found that the AMCAconjugated antiserum was somewhat less sensitive than either DTAF- or TRITC-conjugated antisera (which were equally sensitive), we used tissue double-labeledfor SS and SP by DTAF- and TRITC-conjugated secondary antisera to map the distributions of SS+ISP+ neurons (Fig. 8) and to determine the relative proportion of SS+ neurons that were SP+. Of all Ss+striatal neurons, 67% percent were found to also be SP+ (representing 30% of all SP+ neurons). By normalizing the relative number of all SS+ neurons in the SSiSP double-label study with the relative number of all SS+ neurons in the SS/NPY double-label study described above, we were able to combine the data from these two studies to calculate the relative numbers of neurons in the four different peptide-specificpopulations of medial striatal (LPO) neurons containing SS, NPY or both. These values were then translated into a percentage relative t o all medial striatal neurons by determining the percentage of all medial striatal neurons that are SP+ (from immunoperoxidase-labeled and Nissl-stained sections) and using this value to normalize our relative values. The percentages of all medial striatal neurons making up these four peptide-specific populations were: SS+, 6.6%; SS+/SP+, 12.5%;SS+/NPY+, 1.3%;NPY+, 12.5%.

DISCUSSION Basic conclusions The present results indicate that SS+ and NPY+ neurons are abundant and distributed throughout the major subdivisions of the telencephalon in pigeons. Neurons in the medial, dorsal, and lateral telencephalon (i.e., cortexequivalent parts of the telencephalon) that contained either SS or NPY comprised two major peptide-specific populations, one containing SS and NPY and the other containing SS. SS + neurons and SS+INPY+ neurons were also present in the basal (or ventral) telencephalon, and, in addition, a substantial number of NPY + neurons were present in this region. SS+/NPY+ neurons and NPY+ neurons in the striatum are most likely local circuit neurons, since we found no evidence that they project to the substantia nigra or the pallidum. The SS+ neurons were divisible into two populations according t o their projection targets. Neurons in one of these populations appeared to be striatal interneurons, whereas neurons in the second group were also found to contain substance P (SP) and to project to the substantia nigra. Many of the features observed in the pigeon in the present study appear to be common among amniotes. The presence of SS+ neurons in many parts of the forebrain has been reported in one other avian species (Takatsuki et al., '81), and immunohistochemical studies have shown that SS + neurons and NPY + neurons are abundant throughout the forebrain in mammals and reptiles (Bennett-Clarke et al., '80; Goossens et al., '80; Finley et al., '81; Olschowka et al., '81; Allen et al., '83b; Bear and Ebner, '83;Johansson et al., '84; Weindl et al., '84; Chronwall et al., '85; Smith et al., '85; Vincent et al., '85; Bennett-Clarke and Joseph, '86; Chan-Palay et al., '86; De Quidt and Emson, '86; Davila et al., '88; Kuljis and Rakic, '89a,b). In mammals and turtles, most of the telencephalic neurons containing either of these neuropeptides contain both of them (Vincent et al., '82a,b; Chronwall et al., '84; Hendry et al., '84a; Jones and Hendry, '86; Smith and Parent, '86; Gaspar et al., '87; Reiner and

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Oliver, '87). In pigeons, too, the percentage of neurons in which these neuropeptides co-occurred was quite high, but was generally somewhat lower than in other amniotes. In the striatum, neurons containing both SS and NPY or only NPY appear to be local circuit neurons in mammals (Takagi et al., '83; DiFiglia and Aronin, '82, '84; Vincent and Johansson, '83; Chesselet and Graybiel, '86; Smith and Parent, '86; Aoki and Pickel, '881, as also indicated to be the case in pigeons by the present studies. In contrast to the present results in pigeons, SS+ striatonigral projection neurons have not been reported in cats or monkeys (Chesselet and Graybiel, '86; Smith and Parent, '86). However, SS+ striatal projection neurons may be present in at least some mammals. A recent study has shown that SS+ striatal neurons similar in size and morphology to striatal projection neurons are present in the guinea pig striatum (Vincent and von Krosigk, '881, although the projection target(s) of such neurons is uncertain. Thus, SS+ striatal projection neurons may not be unique to the avian basal ganglia, but rather may be a variable feature of basal ganglia organization among amniotes. Finally, the pigeon is the only amniote so far examined in which a substantial population of SS+ striatal interneurons has been reported.

Single-labelstudiesand comparisonswith other species Neurons containing somatostatin. The abundance of the SS + neurons observed in some regions of the pigeon telencephalon was similar to that described in another avian species, the warbling grass parakeet (Takatsuki et al., '81). In both pigeons and parakeets, relatively dense SS+ populations were found in the hippocampal complex (HA and APH), TPO, CDL, and medial striatum (LPO), while only scattered neurons were present in the PA, PP, and S. However, SS+ neurons were found to be far more widely distributed in the pigeon telencephalon than was observed in parakeets. Thus, whereas few SS+ neurons were found in the archistriatum, neostriatum, and the different subdivisions of the hyperstriatum in parakeets, these subdivisions all contained moderate to dense populations of SS+ neurons in pigeons. These differences may reflect a species variation, but the possibility also exists that they are simply the result of technical differences between the two studies. We also examined several parts of the pigeon telencephalon for which the relative number of SS+ neurons has not been previously reported in birds, including the glomerular layer of the olfactory bulbs, the nST, the ventral striatum (Ac), the VP, and the TuOl (all of which contained a moderate to high density of neurons) and the E, the Bas, and the INP (all of which contained few or no SS+ neurons). Somatostatin is a phylogenetically conserved peptide that has been shown to be present in the brains of representative species of all vertebrate classes (King and Millar, '79). Jmmunohistochemical studies have shown that SS+ neuronal populations are present throughout the telencephalon in several species of mammals (Bennett-Clarke et al., '80; Finley et al., '81; Vincent et al., '82a,b; Chronwall et al., '84; Hendry et al., '84a; Johansson et al., '84; Vincent et al., '85; Smith and Parent, '86; Bennett-Clarke and Joseph, '86; Gaspar et al., '87) and in reptiles (Goossens et al., '80; Bear and Ebner, '83; Weindl et al., '84; Reiner and Oliver, '87; Davila et al., '88). Regions shown to possess SS+ neurons in the pigeon telencephalon include regions possibly homologous, on the bases of topography, connectivity,

K.D.ANDERSON AND A. REINER

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A

Fig. 4. Line drawings of transverse sections through two levels of the ventral telencephalon showing the distributions of neurons immunohistochemically labeled only for SS, only for NPY, or for both SS and NPY. For clarity, the neurons at each level are divided between two drawings of the same section. A and B: Sections through a rostra1 level

(A) and a caudal level (B) showing NPY+ neurons (open circles) and SS+/NPY+ neurons (closed triangles). C and D: The same sections as in A and B, showing SS+ neurons (closed circles). Each symbol represents one neuron. Labeled neurons in the septum are not shown.

function, and/or histochemical features, to regions possessing SS+ neurons in the telencephalon of reptiles and mammals. Thus, SS+ neuronal populations in such regions as the olfactory bulbs, the olfactory tubercle, the piriform cortex, the striatum, the ventral striatum, the septum, and the hippocampal complex may be homologous among amniotes. The similar morphology of SS+ neurons (i.e., me-

dium-sized multipolar) in these various telencephalic subdivisions also supports the notion that many of the telencephalic SS+ neuronal populations are homologous among amniotes. The present study also revealed that SS+ neurons in pigeons are prevalent in parts of the lateral and dorsal telencephalon, for example the Wulst, field I,, and the archistriatum, that are involved in visual, auditory, and


2 73

C

Figure 4 continued

motor functions, respectively. SS+ neurons are also present in the portions of the telencephalon in mammals and reptiles that are comparable to Wulst, field L, and archistriatum in their connections and functions. SS+ neurons have been reported in parts of the frog telencephalon apparently homologous to parts of the telencephalon in amniotes, including the olfactory bulbs, dorsal pallium, medial pallium, septum, striatal complex, and ventrobasal telencephalon (Vandesande and Dierickx, '80; Inagaki et al., '81; Laquerriere et al., '89). Thus, the involvement of SS+ neurons in various telencephalic neu-

ronal circuits in modern tetrapod vertebrates may be inherited from ancestral amphibians. In agnathans, SS+ neurons have not been reported in the telencephalon (Nozaki and Gorbman, '83; Wright, '86). The distributions of SS+ neurons in the brains of other fish species have not been reported. Neurons containing neuropeptide Y. NPY also appears to be an evolutionarily conserved neuropeptide in the brains of vertebrates. The human and porcine versions of this 36-amino acid peptide differ by only a single amino acid (Corder et al., '84). In the brain of a frog species (Danger et

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Fig. 5. A pair of photomicrographs illustrating the co-occurrence of SS and NPY in neurons in the dorsal part of the lateral hypothalamus. A single section labeled for SS and for NPY by simultaneous immunofluorescence shows SS+ neurons labeled by rhodamine (a),and, in the same field of view, NPY+ neurons labeled by fluorescein (b).Arrowheads in a and b indicate examples of neurons labeled for both SS and

NPY; open arrows in a indicate examples of neurons labeled only for SS, the locations of which are also indicated by identical arrows .n b. Midline is just to the right of this field of view and dorsal is toward the top. Note that all of the neurons in the dorsolateral one third of the group are labeled for both SS and NPY, whereas the rest are labeled for only SS. Scale bar = 100 Km.

al., ' 8 5 ) ,goldfish (Pontet et al., '89), anglerfish (Noe et al., '89), and a galeomorph shark species (Vallarino et al., '88), an NPY-like peptide has been detected whose retention time in HPLC is similar to that of human or porcine NPY. Although similar data are lacking in birds and reptiles, such results suggest that NPY is a phylogenetically ancient and conserved neu ropeptide. The distribution of NPY+ neurons in the forebrain has not previously been determined in an avian species, but immunohistochemical studies have revealed NPY + neurons in the forebrain in several mammalian species (01schowka et al., '81; Vincent et al., '82a,b; Allen et al., '83b; Chronwall et al., '84, '85; Hendry et al., '84a; Smith et al., '85; Chan-Palay et al., '86; De Quidt and Emson, '86; Smith and Parent, '86; Gaspar et al., '87; Kuljis and Rakic, '89a,b), turtles (Reiner and Oliver, '871, frogs (Danger et al., '85), lungfish (Reiner and Northcutt, '871, ray-finned bony fish (Reiner and Northcutt, '88; Noe et al., '89; Pontet et al., '89), and galeomorph sharks (Vallarino et al., '88). Studies in mammals and turtles have shown that NPY+ neurons are present in all parts of the telencephalon in which SS+ neurons are found, consistent with the present findings in pigeons. Thus, as discussed for SS+ neurons above, many of the NPY+ neuronal populations in the telencephalon may be homologous among amniotes. NPY + neurons have been described in frogs in telencephalic structures that are likely homologous to the hippocampal complex (medial pallium) and to parts of the neocortex/DVR/piriform cortex (dorsal and lateral pallium) in amniotes (Danger et al., '85), and in lungfish in the telencephalic structure likely homologous to the hippocampal complex (medial pallium) in amniotes (Reiner and Northcutt, '87), thereby suggesting that NPY+ neurons may have been involved in the functions of some telencephalic neuronal circuits since the time

of primitive amphibians or before. Although homologies are not as readily deduced between telencephalic structures in ray-finned bony fish and those in other vertebrates, NPY+ perikarya are present in goldfish in the olfactory bulbs and the apparent homolog of the olfactory tubercle (Pontet et al., '89) and in Polypterus in the olfactory bulbs (Reiner and Northcutt, '88).

Multiple-labl

compe with

other species

Cortex-equivalent regions of the telencepha lon. Neurons double-labeled for SS and NPY were present throughout the Wulst, the hippocampal complex, the IIVR, TPO, CDL, and the piriform cortex. Immunohistocheinical studies in several mammalian species and in turtles have likewise shown that SS+/NPY+ neurons are present in virtually all medial, dorsal, and lateral telencephalic regions where either SS+ or NPY+ neurons are located (Vincent et al., '82a,b; Chronwall et al., '84; Hendry et al., '84a; Jones and Hendry, '86; Kohler et al., '86; Smith and Parent, '86; Gaspar et al., '87; Reiner and Oliver, '87). In those mammals examined quantitatively, in turtles, and in pigeons (as shown in the present study) nearly all NPY+ neurorrs are SS+,but a small percentage of SS+ neurons do not contain NPY. Thus, the presence of two major peptide-specific types of neurons, one containing both SS and NPY and the other containing SS, appears to be a common feature in the dorsal, medial, and lateral telencephalon (i.e., cortex or cortex-equivalent parts) of amniotes. Neurons in these populations in pigeons were bipolar or multipolar in shape and medium in size (up to 20 pm in the medial hippocampal complex). Previous studies in pigeons have indicated that neurons in these parts of the telencephalon, with the


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Fig. 6. Photomicrographs of striatonigral projection neurons containing both SS and SP. Sections through the striatum containing neurons retrogradely labeled by an injection of FG into the substantia nigra were labeled for SS and SP by simultaneous immunofluorescence. a, b, and c: The same field of view in a single section showing (a) SS labeling (fluorescein),(b) FG labeling, and (c) SP labeling (rhodamine). Solid arrows identically placed in all three micrographs show the same

neuron labeled by all three markers. A neuron labeled for FG and SP, but not SS (open arrows) and a neuron labeled for only SS (arrowheads) are also indicated. d, e, and f A single field of view from one section showing (d) SS labeling (fluorescein), (el FG labeling, and (D SP labeling (rhodamine). Arrows in d, e, and f indicate examples of neurons labeled by all three markers. Scale bars = 50 bm (a+); 30 Km (d-0.

exception of the Wulst and the archistriatum, do not project to extratelencephalic targets (Nauta and Karten, '701, which implies that most if not all of the SS+ andior NPY+ neurons do not have extratelencephalic projection targets. Similarly in mammals and reptiles, most or all neurons containing SS or NPY in cortical portions of the telencephalon have the non-pyramidal morphology of neocortical local circuit neurons (Bennett-Clarke et al., '80; Goossens et al., '80; Finley et al., '81; Olschowka et al., '81; Takatsuki et al., '81; Allen et al., '83b; Bear and Ebner, '83; Johansson et al., '84; Weindl et al., '84; Chronwall et al., '85; Smith et al., '85; Vincent et al., '85; Bennett-Clarke and Joseph, '86; De Quidt and Emson, '86; Davila et al., '88; Kuljis and Rakic, '89a,b). Observations in monkeys that neurons labeled for NPY, SS, or both send processes into superficial layers of cortex, where they form dense plexuses containing SS+ and NPY+ synaptic terminals (Hendry et al., '84a; Kuljis and Raluc, '89b), are consistent with the notion that these are local circuit neurons. In the hippocampus too, NPY+ neurons have the morphology and distribution of local circuit neurons (Kohler et al., '86; Chan-Palay et al., '86).

Basal forebrain. The basal forebrain contains the basal ganglia, the bed nucleus of the stria terminalis and the septum. SS+/NPY+ neurons were present throughout these parts of the forebrain in pigeons. In the septum, the abundance of SS+/NPY+ neurons relative to single-labeled neurons was less than that reported in humans, whereas in the bed nucleus of the stria terminalis it was greater in pigeons than that in humans (Gaspar et al., '87). In the striatum of pigeons, neurons containing only SS or only NPY outnumbered neurons containing both of these neuropeptides, unlike in mammals. Thus, the percentage of SS+/NPY+ neurons relative to all neurons labeled for at least one of these peptides (4%)was far less than in turtles and monkeys. In turtles and monkeys, the vast majority (85-90%) of striatal neurons containing SS or NPY are reported to contain typically both of these neuropeptides, and those containing only one of these neuropeptides tend to be NPY+ (Smith and Parent, '86; Reiner and Oliver, '87). It is possible, as in any immunohistochemical study, that the number of neurons containing both SS and NPY is greater than that indicated in the present studies, and that some of the neurons we observed labeled only for SS

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the striatum. A rmgle section labeled for NPY, SS, and SP by simultaneous immunofluorescence shows NPY + neurons laheled by fluorescein (a),and, in the same field of view, S S i neurons labeled by rhodamine (b)and, also in the same field of view, SP+ neurons labeled by AMCA ( c ) .An open white arrow in a and b indicates an example of a neuron labeled for both NPY and SS;solid white arrows in b and c

indicate examples of neurons laheled for SS and SP; a white arrowhead in a indicates an example of a neuron labeled only for NPY, the locai.ion of which is also indicated by identical arrows in b and c; a pair of wbite arrows in b indicates an example of a neuron labeled only for SS, the location of which is also indicated by identical symbols in a and c; a black arrowhead in c indicates an example of a neuron labeled only for SP, the location of which is also indicated by identical arrows in a and b. Scale bar = 50 bm.

or only for NPY contained the other of these two neuropeptides at a level below that detectable by immunohistochemistry. However, when the percentage of striatal neurons containing SS and NPY was calculated relative to all striatal neurons in pigeons, the abundance of neurons of this type (1.3%;1was found to be comparable to the 1-2% value calculated for this type of neuron in the striatum of rats (Vincent arid Johansson, '83). This similarity suggests that the relative abundance of SS+/NPY+ neurons may, in fact, be a phylogenetically conserved feature of the striatum among amniotes. Thus, the difference observed between pigeons and other amniotes in the abundance of SS-only and NPY-only neurons may be due to an increase in pigeons in the number of striatal neurons containing only SS and only NPY rath,er than to a reduction in the number of neurons Containing both neuropeptides. Retrograde labeling of striatonigral projection neurons in combination with immunohistochemistry indicated that striatal neurons containing NPY (some of which are SS+/ NPY + and some of' which are NPY + ) do not project to the substantia nigr:s in pigeons. This result is consistent with the possibility that they are striatal interneurons. Although it is possible tha.t some of these neurons project to the other major target area of the striatum, the pallidum, most of the NPY+ neurons are located in the medial striatum, which does not conta:in striatopallidal projection neurons (Kitt and Brauth, '81). Moreover, neither SS+ nor NPY+ fibers or terminals are evident in the pallidum. In mammals, striatal neurons containing SS and NPY are reported to

have a medium-sized aspiny morphology (Takagi et al., '83; DiFiglia and Aronin, '82, '84; Vincent and Johansson, '83; Aoki and Pickel, '88; Vuillet et al., '89a). Neurons of this type do not appear to project outside the striatum, but instead have short axons synapsing within the striatum. Studies in cats and monkeys combining retrograde labeling with immunohistochemical labeling have directly demonstrated that SS+ or NPY+ neurons do not project to the major striatal projection targets, indicating that virtuadly all such neurons are local circuit neurons in these species (Chesselet and Graybiel, '86; Smith and Parent, '86). However, in guinea pigs, in addition to a population of SS+/NPY+ neurons with interneuronal morphology, SS+ striatal neurons are present that do not label for NPY and that possess the morphology of projection neurons (Vincent and von Krosigk, '88). In the present investigation, we similarly found SS+ neurons that did not label for KPY (but did label for SP), which we directly demonstrated to be striatonigral projection neurons. Thus, studies to date indicate that all SS+/NPY+ and all NPY+ neurons in the striatum of mammals and pigeons make intrastriatal connections, whereas at least some neurons containing SS (but not NPY) are present and appear to be projection neurons in at least some avian and mammalian species. Finally, numerous SS+ neurons that were neither NPY+ nor SP+ were observed in the triple-label immunohistochemical study. Since virtually all SS+ projection neurons were also S P + , these SS+ neurons appear to be an additional population of interneurons. A substantial population of SS-only

Fig. 7. A trio of photomicrographs illustrating the co-occurrence of

SS and NPY and the co-occurrence of SS and SP in different neurons in


277

Fig. 8. Transverse sections through a rostra1 (A) and a caudal (B) level of the ventral telencephalon, schematically illustrating the distributions of SS-only neurons (open dots) and SS+/SP+ neurons (closed

triangles) in the ventral telencephalon. Each symbol represents one neuron. Note that SS+/SP+ neurons are largely confined to the striatum.

striatal interneurons has not been reported in other amniotes.

including CA1 hippocampal neurons in rats (Colmers et al., '87; Martel et al., '86, '87; Tatemoto et al., '82; Allen et al., '82, '83a; Lundberg et al., '84).The physiological significance of the co-occurrence of SS and NPY in cortex is unknown. Virtually all SS+ or NPY+ neurons in many cortical areas in cats and monkeys have been shown to label also for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD),suggesting that the influence of these neurons on their target neurons may involve an interaction of the effects of these neuropeptides and GABA (Hendry et al., '84b). Role of S S and NPY in the striaturn. The role of neurons containing SS and/or NPY in the mammalian

Functional considerations Role of SS and NPY in cortex. SS exerts predominantly excitatory effects on cortical neurons, including corticospinal neurons, which is consistent with a role for SS as a neurotransmitter in the cortex (Ioffe et al., '78; Olpe et al., '80; Phillis and Kirkpatrick, '80; Delfs and Dichter, '83). Although little data are available on the influence of NPY on cortical neurons, receptors for NPY are present in both cortex and striatum. NPY is known to have generally inhibitory effects on target structures to which it is applied,

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striatum has been examined in a number of studies. Neuronal type(!;) postsynaptic to NPY+ and SS+ striatal neurons appear to include striatal spiny projection neurons (Takagi et al., '83; DiFiglia and Aronin, '82, '84; Aoki and Pickel, '88). Consistent with this observation are data showing that S9 influences the release from striatal brain slices of the transmitter contained in the vast majority of striatal spiny projection neurons, GABA (Meyer et al., '89). Moreover, SS and/or NPY stimulate dopamine (DA) release from striatal dopaminergic terminals (Chesselet and Reisine, '83; Beal and Martin, '84a; Arneric and Reis, '86; Beal et al., '86'1 and the release produced by SS and NPY acting together is greater than that produced by either alone (Beal et al., '86). Thus, SS+/NPY+ neurons may also influence striatal neurons by modulating DA release. Since DA, in turn, can influence a number of different neuroactive substances present in striatal neurons, includingacetylcholine (Arneric and Reis, '86), GABA (Scheel-Kruger, '86; Samuel et al., '88; Bernath and Zigmond, '89), SP, dynorphin, and enkephalin (Young et al., '86), it is clear that SS+/NPY+ striatal interneurons may influence a number of different types of striatal neurons. SS/NPY neurons within the mammalian striatum may play a role in the transmission of signals between the two major subdivisions of mammalian striatum, termed patches (striosomes) and matrix (Graybiel, '84; Gerfen, '84). Striatal projection neurons in patches are segregated from those in matrix, with those of each compartment having different connections with cortex and substantia nigra, and presumably mediating distinct functions. SS+ neurons are located predominantly in the matrix, but are often located near patch-matrix borders where they send dendritic processes into the patches (Gerfen, '84, '85; Chesselet and Graybiel, '86; Gerfen et al., '87). Such neurons may thus be involved in transmitting signals from patches to spiny projection neurons in matrix (Gerfen, '84, '85; Chesselet and Graybiel, '86). In pigeons, macroscopic subdivisions such as patches and matrix are not present, and thus more detailed connectivity data are required to ascertain whether these neurons might play roles in intrastriatal signaling in pigeons similar to those hypothesized in mammals. Inputs influencing striatal SSINPY neurons. In mammals, NPY+ neurons (most of which also contain SS, as discussed above) have been found to receive direct input from GABAergic neurons and glutamatergic neurons. In the nucleus accumbens (or ventral striatum), ultrastructural immunohistochemical investigations have shown that GAD+ terminals (possibly from GABAergic interneurons or axon collaterals of spiny projection neurons) make direct contact with N P Y + neurons (Massari et al., '88).Ultrastructural studies also show that SS/NPY neurons receive direct input from ghtamatergic corticostriatal projection neurons (Vuillet et al., '89b). Moreover, injection of a glutamate receptor agonist, such as quinolinic acid or kainic acid, into the striatum causes destruction of SSiNPY neurons (Beal et al., '89). The sensitivity of these neurons to such excitotoxins indicates that these neurons have receptors for glutamate, which implies that they receive glutamatergic input. The major source of glutamatergic input in the striatum is believed to come from corticostriatal projection neurons (Penney and Young, '86). Ultrastructural immunohistochemical evidence in mammals indicates that NPY+ (presumably mostly also SS+) striatal interneurons receive only slight direct input from dopaminergic neurons (Aoki and Pickel, '88; Vuillet et al.,

K.D. ANDERSON AND A. REINER '89b). Despite the seemingly slight direct dopaminel*gic input, there is some, albeit contradictory, evidence that SS+/NPY+ striatal neurons are influenced by dopamiriergic striatal input. Destruction of dopaminergic neurons in the substantia nigra leads to an increase in NPY levels in striatal neurons that is reversed by subsequent treatment with apomorphine (Kerkerian et al., '86, '88). However, nigral lesions have been reported to have no effect on SS levels (Beal and Martin, '83; Radke et a]., '87) or an effect that varies with time (Ogawa et al., '87). Neuroleptic blockade of dopaminergic neurotransmission induces a reduction in both striatal SS levels (Beal and Martin, 'H4b; Radke et al., '88) and NPY levels (Kerkerian et al., '88). Recently, Weiss and Chesselet ('89) reported that systemic injection of a D, receptor antagonist yields a reduction in preprosomatostatin mRNA in the lateral but not medial striatum in mice, as determined by in situ hybridization, suggesting a topographical heterogeneity in the regulation of SSsynthesis (and thus possibly the level of this neuropeptide) by DA in the striatum. In the present study, althciugh we did not rigorously assess the influence of doparnine agonists in the regulation of the levels of SS or NPY, neither the number nor the labeling intensity of SS+ neurons and NPY+ neurons was obviously influenced by systemic injections of apomorphine for 6 days. This is consistent with the results of studies showing that apomorphine treatment does not affect NPY levels in intact rats (Beal and Martin, '84b; Kerkerian et al., '88). Peptide-specific populations of striatonigral projection neurons. We have previously shown in pigeons that the vast majority of striatonigral projection neurons contain SP (Anderson and Reiner, '871, that virtually all ;3P+ striatal neurons contain dynorphin (DYN) (Reiner, '86; Anderson and Reiner, 'go), and that many of the SP+ striatal neurons also appear to contain neurokinin A (NKA) (Reiner and Anderson, unpublished observations). Evidence in pigeons and rats indicates that most if not all of these neurons also contain GABA (Reiner and Anderson, '87; Kita and Kitai, '88). In the present study we found that virtually all SS+ striatonigral projection neurons also contained SP (which is consistent with our observation that the vast majority of striatonigral projection neurons contain SP), but not all SP+ striatonigral projection neurons were also SS+.Thus, our present results define two major neuropeptideheurotransmitter-specificpopulations of striatonigral projection neurons in pigeons. One populat'ion contains the tachykinins, DYN, and GABA, and the second contains SS in addition to all of these substances. We have also found previously that an additional population of neurons containingenkephalin and (probably)GABA makes up less than 5% of the total population of striatonigral projection neurons (Anderson and Reiner, '87). Thus, the influence of the striatum on target neurons in the substantia nigra is mediated by three different populations of neurons, each using a different set of neurotransmitters and neuromodulators. SS+INPY+ neurons in the hypothalamus. I11 the dorsal part of the lateral hypothalamus, at the level of nucleus rotundus, a population of neurons containing both SS and NPY was present immediately adjacent to a population of SS-only neurons with similar morphology. A ;small population of neurons containing both SS and NPY has been reported in the hypothalamic arcuate nucleus irl rats (Chronwall et al., '84). It is possible that the SS+/NPY+ hypothalamic neurons of birds are homologous to those of

SS AND NPY NEURONS IN THE PIGEON FOREBRAIN the arcuate nucleus in mammals, but the locations of the cell groups are somewhat different. Although some information is available on the functional roles of SS and NPY in the hypothalamo-pituitary axis in birds and mammals (Gray et al.,'86; Levine and Morley, '84; Kuenzel et al., '87; Korf et al., '88), the specific function of hypothalamic neurons containing both SS and NPY is uncertain.

Evolutionaryimplications The existence in cortex-equivalent parts of the pigeon telencephalon of two major types of neurons containing SS or NPY, one containing both SS and NPY and the other containing SS, is consistent with findings in several species of mammals (Chronwall et al., '84; Jones and Hendry, '86) and in red-eared turtles (Reiner and Oliver, '87). Also, as in other amniotes (Smith and Parent, '86; Reiner and Oliver, '871, the pigeon basal telencephalon appears to contain populations of interneurons containing NPY alone (although seemingly in greater abundance in pigeons than in other amniotes studied) and populations of interneurons containing both SS and NPY. The widespread distribution of neurons containing SS and/or NPY throughout the telencephalon in amniotes indicates that such neurons were present in the last common ancestor of amniotes and were inherited by modern-day amniotes from this ancient reptilian ancestor. The survival of such neurons through 300 million years of evolution implies that their disappearance (by mutation) was never associated with an improvement in the adaptive success of individual members of an amniote species. Hence the various populations of neurons containing SS andlor NPY appear to serve important and fundamental roles in various telencephalic neuronal circuits. The conservative nature of these populations indicates that study of their functional role(s)in any member of these groups might have implications for understanding the functional role in members of the other groups. SS+ andlor NPY + neurons also may have been present throughout much of the telencephalon in ancestral amphibians and lobe-finned fish, because SS+ neurons are widely distributed in the telencephalon in frogs (Vandesande and Dierickx, '80; Inagaki et al., '81; Laquerriere et al., '89), and NPY+ neurons have been described in lungfish and in frogs located in telencephalic regions likely homologous to the hippocampal complex (medial pallium) in amniotes, and also in frogs in telencephalic regions likely homologous to parts of the neocortex/DVR/piriform cortex (dorsal and lateral pallium) in amniotes (Reiner and Northcutt, '87; Danger et al., '85). In contrast to the conservative nature of striatal interneurons containing both SS and NPY in amniotes, SS+ striatal projection neurons have now been found in pigeons and guinea pigs (Vincent and von Krosigk, '88), but do not appear to be present in cats (Chesselet and Graybiel, '86) or monkeys (Smith and Parent, '86). Thus, the presence of SS in striatal projection neurons may be a feature that is more phylogenetically variable among birds and mammals, suggesting that such neurons may play a specialized role in the basal ganglia of some but not all mammalian and avian species. Variability in the presence of a neuropeptide in specific types of neurons in the basal ganglia of birds and mammals has been noted before. For example, neurotensin is present in striatopallidal projection neurons in cats and rats, but is not present in these neurons in primates, turtles, or pigeons (Zahm, '87; Sugimoto and Mizuno, '87; Reiner and Carraway, '87; Reiner, unpublished observa-

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tions). Thus, in terms of this population of neurons, pigeons and turtles more closely resemble primates than do some other mammalian species. The present study also provided evidence for the existence of striatal interneurons containing only SS, a population not previously reported in either mammals or reptiles. Such findings indicate that although the organization of the striatal portion of the basal ganglia is fundamentally similar among amniotes in terms of neuropeptides and neurotransmitters (Reiner et al., '841, some variations between species occur that may underlie specialized functions of the basal ganglia particular to certain species.

ACKNOWLEDGMENTS We thank Debora Romeo, Gary Henderson, Ellen Karle, Wes Sweeney, and Donna Purifoy for technical, photographic, and illustrative assistance. This research was supported by NS-19620 (A.R.), the Hereditary Disease Foundation (A.R.), and the Huntington's Disease Society of America (K.D.A.).

Allen, J.M., T.E. Adrian, K. Tatemoto, J.M. Polak, J. Hughes, and S.R. Bloom (1982) Two novel related peptides, neuropeptide Y (NPY) and peptide W (PYY), inhibit the contractions of the electrically field stimulated mouse vas deferens. Neuropeptides 3.5'-77. Allen, J.M., G.P. McGregor, T.E. Adrian, S.R. Bloom, S.Q. Zhong, K.W. Ennis, and W.G. Unger (1983a) Reduction of neuropeptide Y (NPY) in the rabbit iris ciliary body following sympathectomy. Exp. Eye Res. 37;213-215. Allen, Y.S., T.E. Adrian, J.M. Allen, K. Tatemoto, T.J. Crow, S.R. Bloom, and J.M. Pol& (198313) NeuropeptideY distribution in the rat brain. Science 221:877-879. Anderson, K.D., and A. Reiner (1987) Striatonigral projection neurons: A retrograde labeling study of the relative numbers that contain S P or enkephalin. Soc. Neurosci. Abstr. 13:1574. Anderson, K.D., and A. Reiner (1990) The extensive co-occurrence of substance P and dynorphin in striatal projection neurons: An evolutionarily conserved feature of basal ganglia organization. J. Comp. Neurol. 295339-369. Aoki, C., and V.M. Pickel (1988) Neuropeptide Y-containing neurons in the rat striatum: Ultrastructure and cellular relations with tyrosine hydroxylase-containing terminals and with astrocytes. Brain Res. 459:205-225. Arneric, S.P., and D.J. Reis (1986) Somatostatin and cholecystokinin octapeptide differentially modulate the release of [3Hlacetylcholine from caudate nucleus but not cerebral cortex: Role of dopamine receptor activation. Brain Res . 374: 153- 161. Beal, M.F., and J.B. Martin (1983) Effects of lesions on somatostatin-like immunoreactivity in the rat striatum. Brain Res. 266:67-73. Beal, M.F., and J.B. Martin (1984a) The effect of somatostatin on striatal catecholamines. Neurosci. Lett. 44r271-276. Beal, M.F., and J.B. Martin (1984b) Effects of neuroleptic drugs on brain somatostatin-like-immunoreactivity.Neurosci. Lett. 47: 125-130. Beal, M.F., R.C. Frank, D.W. Ellson, and J.B. Martin (1986) The effect of neuropeptide Y on striatal catecholamines. Neurosci. Lett. 71:118-123. Beal, M.F., N.W. Kowall, K.J. Swartz, R.J. Ferrante, and J.B. Martin (1989) Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions. Synapse 3:38-47. Bear, M.F., and F.F. Ebner (1983) Somatostatin-like immunoreactivity in the forebrain of Pseudemys turtles. Neuroscience 9297-307. Bennett-Clarke, C., and S.A. Joseph (1986) Immunocytochemical localization of somatostatin in human brain. Peptides 7:877-884. Bennett-Clarke, C., M.A. Romagnano, and S.A. Joseph (1980) Distribution of somatostatin in the rat brain: Telencephalon and diencephalon. Brain Res. 188:473-486. Berk, M.L. (1987)Projections ofthe lateral hypothalamus and bed nucleus of the stria terminalis to the dorsal vagal complex in the pigeon. J. Comp. Neurol. 2fiOt140-156. Bernath, S., and M.J. Zigmond (1989) Dopamine may influence striatal GABA release via three separate mechanisms. Brain Res. 476:373-376.

280


Gray, T.S., T.L. O’Donohue, and D.J. Magnuson (1986) Neuropeptide Y Blessing, W.W., P.R.C. Howe, T.H. Joh, J.R. Oliver, and J.O. Willoughby (1986) Distribution of tyrosine hydroxylase and neuropeptide Y-like innervation of amygdaloid and hypothalamic neurons that project to the immunoreactive neurons in rabbit medulla oblongata, with attention to dorsal vagd complex in rat. Peptides 7;341-349. co-localization studies, presumptive adrenaline-synthesizing perikarya Graybiel, A. (1984) Neurochemically specified subsystems in the basal and vagal preganglionic cells. J. Comp. Neurol. 248:285-300. ganglia. In: Functions of the Basal Ganglia (Ciba Foundation SympoChan-Palay, V., C. Kohler, U. Haesler, W. Lang, and G. Yasargil (1986) sium 107). London: Pitman, pp. 114-149. Distribution of neurons and axons immunoreactive with antisera against Hendry, S.H.C., E.G. Jones, and P.C. Emson (1984a) Morphology, distribuneuropeptide Y in the normal human hippocampus. J . Comp. Neurol. tion, and synaptic relations of somatostatin- and neuropeptide Y-immu248:360-375. noreactive neurons in rat and monkey neocortex. J. Neurosci. 42497Chesselet, M.-F., and T.D. Reisine (1983) Somatostatin regulates dopamine 2517. release in rat striatal slices and cat caudate nuclei. J. Neurosci. 3.232Hendry, S.H.C., E.G. Jones, J . DeFelipe, D. Schmechel, C. Brandon, and P.C. 236. Emson (1984b) Neuropeptide-containing neurons of the cerebral cortex Chesselet, M.-F., and A.M. Graybiel (1986) Striatal neurons expressing are also GABAergic. Proc. Natl. Acad. Sci. USA 81:6526-6530. somatostatin-like immunoreactivity: Evidence for a peptidergic interneuInagaki, S., S. Shiosaka, K. Takatsuki, M. Sakanaka, H. Takagi, E. Senba, T. ronal system in the cat. Neuroscience 17:547-571. Matsuzaki, and M. Tohyama (1981) Distribution of somatostatin in the Chronwall, B.M., T.N. Chase, and T.L. O’Donohue (1984) Coexistence of frog brain, R a m catesbiana, in relation to location of catecholamineneuropeptide Y and somatostatin in rat and human cortical and rat containing neuron system. J. Comp. Neurol. 20289-101. hypothalamic neurons. Neurosci. Lett. 52:213-217. Ioffe, S.,V. Havlicek, H. Friesen, andV. Chernick (1978) Effect of somatostaChronwall, B.M., D.A. DiMaggio, V.J. Massari, V.M. Pickel, D.A. Ruggiero, tin (SRIF) and L-glutamate on neurons of the sensorimotor cortex in and T.L. O’Donohue (1985) The anatomy of neuropeptide-Y-containing awake habituated rabbits. Brain Res. 153:414418. neurons in rat brain. Neuroscience 15r1159-1181. Johansson, O., T. Hokfelt, and R.P. Elde 11984) Immunohistochemical Colmers, W.F., K. Lukowiak, and Q.J. Pittman (1987) Presynaptic action of distribution of somatostatin-like immunoreactivity in the central nerneuropeptide Y in area CA1 of the rat hippocampal slice. J. Physiol. vous system of the adult rat. Neuroscience 13265-339. (Lond.) 383385-299. Jones, E.G., and S.H.C. Hendry (1986) Peptide-containing neurons of the Corder, R., P.C. Emson, and P.J. Lowry (1984) Purification and characterizaprimate cerebral cortex. In J.B. Martin and J.D. Barchas (eds): Neuropeption of human neuropeptide Y from adrenal-medullary phaeochromocytides in Neurologic and Psychiatric Disease. New York: Raven Press, pp. toma tissue. Biochem. J. 219:699-706. 163-178. Cuello, A.C., G. Galfre, and C. Milstein (1979) Detection of substance P in Karten, H.J., and Hodos, W. (1967) A Stereotaxic Atlas of the Brain of the the central nervous system by a monoclonal antibody. Proc. Natl. Acad. Pigeon (Colurnbaliuia). Baltimore: The Johns Hopkins Press. Sci. USA 76:3532-3536. Kerkerian, L., 0. Bosler, G. Pelletier, and A. Nieoullon (1986) Striatal Danger, J.M., J. Guy, M. Benyamina, S. Jegou, F. Leboulenger, J. Cot6, M.C. neuropeptide Y neurones are under the influence of the nigrostriatal Tonon, G. Pelletier, and H. Vaudry (1985) Localization and identification dopaminergic pathway: Immunohistochemical evidence. Neurosci. Lett. of neuropeptide Y (NPY)-like immunoreactivity in the frog brain. 66: 106-1 12. Peptides 6:1225-1236. Kerkerian, L., P. Salin, and A. Nieoullon (1988) Pharmacological characterD a d a , J.C., S. Guirado, and A. De la Calle (1988) Immunocytocheniical ization of dopaminergic influence on expression of neuropeptide Y localization of somatostatin in the cerebral cortex of lizards. Brain Res. immunoreactivity by rat striatal neurons. Neuroscience 26:809-817. 447:52-59. King, J.A., and R.P. Millar (1979) Phylogenetic and anatomical distribution Davis, B.M., J.E. Krause, N. Bogan, and J.B. Cabot (1988) Intraspinal of somatostatin in vertebrates. Endocrinology 105:1322-1329. substance P-containing pathways to sympathetic preganglionic neuropil Kita, H., and S.T. Kitai (1988) Glutamate decarboxylase immunoreactive in pigeon, Columba liuia: High-performance liquid chromatography neurons in rat neostriatum: Their morphological types and populations. radioimmunoassay and electron microscopic evidence. Neuroscience Brain Res. 447:346-352. 261655-668. Kitt, C.A., and S.E. Brauth (1981) Projections of the paleostriatum upon the Delfs, J.R., and M.A. Dichter (1983) Effects of somatostatin on mammalian midbrain tegmentum in the pigeon. Neuroscience 6:1551-1566. cortical neurons in culture: Physiological actions and unusual dose response characteristics. J. Neurosci. 3:1176-1188. Kohler, C., L. Eriksson, S. Davies, andV. Chan-Palay (1986) NeuropeptideY De Quidt, M.E., and P.C. Emson (1986) Distribution of neuropeptide Y-like innervation of the hippocampal region in the rat and monkey brain. J. immunoreactivity in the rat central nervous system-11. ImmunohisComp. Neurol. 244:384400. tochemical analysis. Neuroscience 18:545-618. Korf, H.-W., G.C. Panzica, C. Viglietti-Panzica, and A. Oksche (1988) DiFiglia, M., and N. Aronin (1982) Ultrastructural features of immunoreacPattern of peptidergic neurons in the avian brain: Clusters-local tive somatostatin neurons in the rat caudate nucleus. J. Neurosci. circuitries-projections. Basic Appl. Histochem. 3255-75. 21267-1274. Kuenzel, W.J., L.W. Douglas, and B.A. Davison (1987) Robust feeding DiFiglia, M., and N. Aronin (1984) Quantitative electron microscopic study following central administration of neuropeptide Y or peptide W in of immunoreactive somatostatin axons in the rat neostriatum. Neurosci. chicks, Gallus domesticus. Peptides 8:823-828. Lett. 50:325-331. Kuljis, R.O., and P. Rakic (1989a) Distribution of neuropeptide Y-containing Erichsen, J.T., A. Reiner, and H.J. Karten (1982) The co-occurrence of perikarya and axons in various neocortical areas in the macaque monkey. substance P-like and leucine enkephalin-like immunoreactivities in J. Comp. Neurol. 280r383-392. neurons and fibers of the avian nervous system. Nature 295:407-410. Kuljis, R.O., and P. Rakic (1989b) Multiple types of neuropeptide Y-containFinley, J.C.W., J.L. Maderdrut, L.J. Roger, and P. Petrusz (1981) The ing neurons in primate neocortex. J. Comp. Neurol. 280:393409. immunocytochemical localization of somatostatin-containing neurons in Laquerriere, A., P. Leroux, B.J. Gonzalez, C. Bodenant, R. Benoit, and H. the rat central nervous system. Neuroscience 62173-2192. Vaudry (1989) Distribution of somatostatin receptors in the brain of the Gaspar, P ., B. Berger, A. Lesur, J.P. Borsotti, and A. Febvret (1987) frog Rana rzdibunda: Correlation with the localization of somatostatinSomatostatin 28 and neuropeptide Y innervation in the septa1 area and containing neurons. J. Comp. Neurol. 280:451467. related cortical and subcortical structures of the human brain. DistribuLevine, A S , and J.E. Morley (1984) Neuropeptide Y A potent inducer of tion, relationships and evidence for differential coexistence. Neuroconsummatory behavior in rats. Peptides 5: 1025-1029. science 2249-73. Lundberg, J.M., L. Terenius, T. Hokfelt, and K. Tatemoto (1984) ComparaGerfen, C.R. (1984) The neostriatal mosaic: Compartmentalization of cortitive immunohistochemical and biochemical analysis of pancreatic polycostriatal input and striatonigral output systems. Nature 311:461464. peptide-like peptides with special reference to presence of neuropeptide Y Gerfen, C.R. (1985)The neostriatal mosaic. I. Compartmental organization in central and peripheral neurons. J , Neurosci. 4.2376-2386. of projections from the striatum to the substantia nigra in the rat. J. Martel, J.C., S. St. Pierre, and R. Quirion (1986)NeuropeptideY receptors in Comp. Neurol. 236:454-476. the rat brain: Autoradiographic localization. Peptides 7~55-60. Gerfen, C.R., M. Herkenham, and J. Thibault (1987) The neostriatal mosaic: Martel, J.C., S. St. Pierre, P.J. Bedard, and R. Quirion (1987) Comparison of 11. Patch- and matrix-directed mesostriatal dopaminergic and non[’251]Bolton-Hunterneuropeptide Y binding sites in the forebrain of dopaminergic systems. J. Neurosci. 7:3915-3934. various mammalian species. Brain Res. 419.403407. Goossens, N., K. Dierickx, and F. Vandesande (1980) Immunocytochemical Massari, V.J., J. Chan, B.M. Chronwall, T.L. O’Donohue, W.H. Oertel, and localization of somatostatin in the brain of the lizard. Ctenosauria V.M. Pickel (1988) Neuropeptide Y in the rat nucleus accumbens: pectinata. Cell Tissue Res. 208:499-505.

SS AND NPY NEURONS IN THE PIGEON FOREBRAIN Ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals. J. Neurosci. Res. 19:171-186. Meyer, D.K., U. Conzelmann, andK. Schultheiss (1989) Effectsof somatostatin-14 on the in vitro release of L3H1GABAfrom slices of rat caudatoputamen. Neuroscience 28:61-68. Nauta, W.J.H., and H.J. Karten (1970) A general profile of the vertebrate brain, with sidelights on the ancestry of cerebral cortex. In G.C. Quarton, T. Melnechuk, and F.O. Schmitt (eds). The Neurosciences: Second Study Program. New York: The Rockefeller University Press, pp. 7-26. Noe, B.D., S.L. Milgram, A. Bdasubramaniam, P.C. Andrews, J. Calka, and J.K. McDonald (1989) Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus).Cell Tissue Res. 257:303-311. Noz&, M., and A. Gorbman (1983) Immunocytochemical localization of somatostatinandvasotocinin the brain ofthe Pacific hagfish, W a t r e t u s stouti. Cell Tissue Res. 229541-550. Ogawa, N., K. Mizukawa, Y. Hirose, S. Kajita, S. Ohara, and Y. Watanabe (1987) MPTP-induced model in mice: pharmacology and behavior. Eur. Neurol. 26:16-23. Olpe, H.-R., V.J. Balcar, H. Bittiger, H. Rink, and P. Sieber (1980) Central actions of somatostatin. Eur. J. Pharmacol. 631127-133. Olschowka, J.A., T.L. O'Donohue, and D.M. Jacobowitz (1981) The distribution of bovine pancreatic polypeptide-like immunoreactive neurons in rat brain. Peptides 2309-331. Penney, J.B., Jr., and A.B. Young (1986) Striatal inhomogeneities and basal ganglia function. Movement Disorders 1:3-15. Phillis, J.W., and J.R. Kirkpatrick (1980) The actions of motilin, luteinizing hormone releasing hormone, cholecystokinin, somatostatin, vasoactive intestinal peptide, and other peptides on rat cerebral cortical neurons. Can. J. Physiol. 58:612-623. Pontet, A., J.M. Danger, P. Dubourg, G. Pelletier, H. Vaudry, A. Galas, and 0 . Kah (1989) Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish. Cell Tissue Res. 255:529-538. Radke, J.M., P . Cumming, and S.R. Vincent (1987) Effects of MPTP poisoning on central somatostatin and substance P levels in the mouse. Eur. J. Pharmacol. 134:105-108. Radke. J.M., A.J. MacLennan, S.R. Vincent, and H.C. Fibiger (1988) Comparison between short- and long-term haloperidol administration on somatostatin and substance P concentrations in the rat brain. Brain Res. 445,5540. Reiner. A, 1986) The co~occurrenceof substance P-like immunoreactivity and dynorphin-like immunoreactivity in striatopallidal and striatonigral projection neurons in birds and reptiles. Brain Res. 371:155-161. Relner, A, ( 1987a) The distribution of proenkepha]in-derived neuropeptides in the central nervous system of turtle. J. Comp. Neurol. 259:65-91. Reiner, A. (1987b) A VIP-like peptide co-occurs with substance P and enkephalin in cholinergic preganglionic terminals of the avian ciliary ganglion. Neurosci. Lett. 78:22-28. Reiner, A. ( 1 9 8 7 ~A) LANT6-like peptide that is distinct from neuromedin N is present in striatal and pallidal neurons in the monkey basal ganglia. Brain Res. 422:18&191. Reiner, A,, and K.D. Anderson (1987) Evidence for the co-occurrence of SP and GABA in striatonigral and striatopallidal projection neurons of the basal ganglia. SOC.Neurosci. Abstr. 13:1574. Reiner, A., and R.E, Carraway (1987) Immunohistochemical and bjochemisin~'~ peptides in the cal studies on L y ~ ~ - A s n ~ - n e u r o t e n (LANTG)-related basal ganglia of pigeons, turtles, and hamsters. J. Comp. Neurol. 257:453476. Reiner, A., and R.G. Northcutt (1987) An immunohistochemical study of the telencephalon of the African lungfish, Protopterus annectens. J. Comp. Neurol. 256,463481. Reiner, A., and J.R. Oliver (1987) Somatostatin and neuropeptide Y are almost exclusively found in the same neurons in the telencephalon of turtles. Brain Res. 426:149-156. Reiner, A,, and R.G. Northcutt (1988)An immunohistochemical study of the Neurosci. Abstr. 14:53. telencephalon of Polypterus. SOC. Reiner, A., H.J. Karten, and A.R. Solina (1983) Substance P: Localization within paleostriatal-tegmental pathways in the pigeon. ~~~~~~~i~~~~ 9:61-85. Reiner, A,, S.E. Brauth, and H.J. Karten (1984) Evolution of the amniote basal ganglia. Trends Neurosci. 7:320-325. Reiner, A,, W,D, Eldred, M . ~ ,~ ~ ~ ~andf J,E, ~ l fiause d , (1985) The co-occurrence of a substance P-like peptide and cholecystokinin-8 in a fiber system of turtle cortex. J. Neurosci. 51527-1544. ~

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Reubi, J.C., and T.M. Jessell (1979) Distribution of substance P in the pigeon brain. J. Neurochem. 31:359-361. Samuel, D., U. Kumar, and A. Nieoullon (1988) Gamma-aminobutyric acid function in the rat striatum is under the double-influence of nigrostriatal dopaminergic and thalamostriatal inputs: Two modes of regulation? J. Neurochem. 51:1704-1710. Scheel-Kruger, J. (1986) Dopamine-GABA interactions: Evidence that GABA transmits, modulates and mediates dopaminergic functions in the basal ganglia and limbic system. Acta Neurol. Scand. 107:73: 1-54. Schmued, L.C., and J.H. Fallon (1986) Fluoro-gold: A new fluorescent retrograde axonal tracer with unique properties, ~~~i~R ~ ~ , 377:147-154, Smith, Y., and A. Parent (1986) Neuropeptide Y-immunoreactive neurons in the striaturn of cat and monkey: Morphological character.stics, intrinsic organization and co-~ocalizationwith somatostatin, Brain Res, 372,.241252. Smith, Y., A. Parent, L. Kerkerian, and G. Pelletier (1985) Distribution of neuropeptide Y immunoreactivity in the basal forebrain and upper brainstem of the squirrel monkey (SamirL sciureus). J. Comp. Neurol. 236:71-89, Sugimoto, T., and N. Mizuno (1987) Neurotensin in projection neurons of the striaturn and nucleus accumbens, with reference to co-existence enkephalin and GAB* An ~mmunohistochemical study in the cat, Comp. Neurol. 257,383-395, J , sornogyi, and A,D, Smith (1983) Fine structural ~ ~ ~H,,gp. somogyi, i , studies on a type of somatostatin-immunoreactive neuron and its synaptic connections in the rat neostriatum: A correlated light and electron microscopic study. J. Comp. Neurol. 214;1-16. Takatsuki, K,, s,Shiosaka,s,Inagaki, M, SakanAa, H, Takagi, E, Senba, T, Matsuzaki, and M. Tohyama (1981)Topographic atlas of somatostatincontainingneuron system in the avian brain in relation to catecholaminecontaining system,1,~ ~ land diencephalon. ~ ~ J. ~ camp, ~ ~ ~ ~202:103-113, ~ ~ ~ l . Tatemoto, K., M. Carlquist, and V. Mutt (1982) Neuropeptide Y-a novel brain peptide with structural similarities for peptide W and pancreatic polypeptide, N~~~~~296,,659-660. Vallarino, M., J.M. Danger, A. Fasolo, G. Pelletier, S. Saint-Pierre, and H. Vaudry (1988) Distribution and characterization of neuropeptide in the brain of an elasmobranch fish,Brain Res, 448,67-76, Vandesande, F., and K. Dierickx (1980) Imrnunocytochemical localization of somatostatin-containing neurons in the brain of Rana temporaria. Cell Tissue Res. 20543-53. Vincent, S.R., and 0. Johansson (1983) Striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APPI-like immunoreactivities and NADPH-diaphorase activity: A light and electron microscopic study. J. Comp. Neurol. 217:264-270. Vincent, S.R., and M. von Krosigk (1988) Two populations of somatostatinimmunoreactive neurons in the guinea pig striatum. Cell Tissue Res. 252:219-222. Vincent, S.R., 0.Johansson, T. Hokfelt, B. Meyerson, C. Sachs, R.P. Elde, L. Terenius, and J. Kimmel (1982a) Neuropeptide coexistence in human cortical neurones' Nature 298'65-67. J,

Vincent, S.R., L. Skirboll, T. Hokfelt, 0. Johansson, J.M. Lundberg, R.P. Elde, L. Terenius, and J. Kimmel (1982b) Coexistence of somatostatinand avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons. Neuroscience 7:439446' Vincent, S.R., C.H.S. McIntosh, A.M.J. Buchan, and J.C. Brown (1985) Central somatostatin systems revealed with monoclonal antibodies. J. Camp. Neurol. 238:16s186. Vuillet, J., L. Kerkerian, P. Salin, and A. Nieoullon (1989a) Ultrastructural features of NPY-containing neurons in the rat striatum. Brain Res. 477:241-251. Vuillet, J., L. Kerkerian, P. Kachidian, 0. Bosler, and A. Nieoullon (1989b) Ultrastructural correlates of functional relationships between nigral dopaminergic O r cortical afferent fibers and neuroPePtide Y-containing neurons in the rat striatum. Neurosci. Lett. 100:99-104. Weindl, A., J. Triepel, and G. Kuchling (1984) Somatostatin in the brain of the turtle Testudo hermanni Gmelin: An immunobistochemical mapping study. Peptides 5.91-100. Weiss, L.T., and M.-F. Chesselet (1989) Regional distribution and regulation of preprosomatostatin messenger RNA in the striatum, as revealed by in situ hybridization histochemistry. Mol. Brain Res. 5121-130. Wessendorf, M.W., and R.P. Elde (1985) Characterization of an immunofluorescence technique for the demonstration of coexisting neurotransmit-

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282

K.D. ANDERSON AND A. REINER.

ters within nerve fibers and terminals. J. Histochem. Cytochem. 33.984Wright, G.M. (1986) Immunocytochemical demonstration of growth hor. 994. mone, prolactin and somatostatin-like immunoreactivities in the brain of larval, young adult and upstream migrant adult sea lamprey, Petrom,yWessendorf, M.W., N.M. Appel, and R. Eide (1987) Simultaneous observazon marinus. Cell Tissue Res. 246:23-31. tion of fluorescent retrogradely labeled neurons and the immunofluoresccntly labeled fibers apposing them using Fluoro-Gold and antisera Young, W.S., 111, T.I. Bonner, and M.R. Brann (1986) Mesencephalic labeled with the blue fluorochrome 7-amino-4-methylcoumarin-3-acetic dopamine neurons regulate the expression of neuropeptide mRNAs in acid (AMCA).Neurosci. Lett. 82.121-126. the rat forebrain. Proc. Natl. Acad. Sci. USA 839827-9831, White, J.D., J.E. Krause, H.J. Karten, and J.F. McKelvy(1985) Presenceand Zahm, D.S. (1987) Neurotensin-immunoreactive neurons in the ventral ontogeny of enkephalin and substance P in the chick ciliary ganglion. J. striatum of the adult rat: Ventromedial caudate-putamen, nucleus Neurochem. 45: 1319-1322. accumbens and olfactory tubercle. Neurosci. Lett. 81:41-47.

Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat.

Immunohistochemical evidence for synaptic connections between neuropeptide Y-containing axons and periventricular somatostatin neurons in the anterior hypothalamus in rats.

Mapping of neuropeptide Y-like immunoreactivity in the human forebrain.

Selective sparing of NADPH-diaphorase-somatostatin-neuropeptide Y neurons in ischemic gerbil striatum.

Ultrastructural relationships between choline acetyltransferase- and neuropeptide y-containing neurons in the rat striatum.

Morphology and laminar distribution of neuropeptide Y immunoreactive neurons in the human striate cortex.

Neurons in the rat medulla oblongata containing neuropeptide Y-, angiotensin II-, or galanin-like immunoreactivity project to the parabrachial nucleus.

Distribution of neuropeptide Y in the prosencephalon of man and cotton-head tamarin (Saguinus oedipus): colocalization with somatostatin in neurons of striatum and amygdala.

Neuropeptide Y stimulates autophagy in hypothalamic neurons.

Old-onset caloric restriction effects on neuropeptide Y- and somatostatin-containing neurons and on cholinergic varicosities in the rat hippocampal formation.

Morphology and distribution of neuropeptide-containing neurons in human cerebral cortex.

Distribution and hypothalamic projection of tyrosine-hydroxylase containing neurons of the nucleus of the solitary tract in the pigeon.

Postnatal development of neuropeptide Y-containing neurons in the visual cortex of normal- and dark-reared rats.

Distributions and colocalization of neuropeptide Y and somatostatin in the goldfish brain.

Extrinsic origins of the somatostatin and neuropeptide Y innervation of the rat basolateral amygdala.

Interaction between neuropeptide Y and noradrenaline on central catecholamine neurons.

Forebrain projections of the pigeon olfactory bulb.

Distribution of neuropeptide Y, substance P, and choline acetyltransferase in the developing visual system of the pigeon and effects of unilateral retina removal.

Distribution of somatostatin immunoreactivity in the forebrain of the squirrel monkey: basal ganglia and amygdala.

GABAergic interneurons containing calbindin D28K or somatostatin are major targets of GABAergic basal forebrain afferents in the rat neocortex.

Identification of a subpopulation of neuropeptide Y-containing locus coeruleus neurons that project to the entorhinal cortex.

Distribution of the neuropeptide galanin in the cat heart and coexistence with vasoactive intestinal peptide, substance P and neuropeptide Y.

Lumbar cerebrospinal fluid concentrations of somatostatin and neuropeptide Y in multiple sclerosis.

Somatostatin and Somatostatin-Containing Neurons in Shaping Neuronal Activity and Plasticity.