Journal of Chemical Neuroanatomy, Vol. 5:441 452 (1992)
The Basal Forebrain Cholinergic System in the Raccoon Gert Briickner, Wiljiqed Schober, Wolfgang Hiirtig, Christei Ostermann-LatiJ ~, Harry H. Webstert, Robert W. Dykest, Douglas D. Rasmusson~ and Dietmar Biesoid Paul Flechsig Institute liar Brain Research, University of Leipzig, Germany *Neurological Clinic, University of G6ttingen, Germany +Departement de Physiologic, Facult6 de M6decine, Univcrsitd de Montrdal. Qudbec, Canada ++Deparmlent of Physiology and Biophysics, Dalhousic [ !niversity. Nova Scotia, Canada
ABSTRACT -l-he distribution o f neurons displaying choline acetyhransl'erase (CHAT) imlnunoreactixity was examined in the raccoon basal forebrain using a rabbit antiserum and at monoclonal antibody. Alternating sections were used for Nissl staining. ChAT-positive neurons were arranged in a conlinuous mass extending from the medial septum to the caudal pole of the pallidum. Based upon spatial relations to fibre tracts, the clustering of neuronal groups, and cytological criteria, the basal l'orebrain magnocellular complex can be subdivided into several distinct regions. Although clear nuclear boundaries ,sere often absent, the ChAT-positive neurons were divided into: the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis): nucleus praeopticus magnocellularis: subslantia innominata: and the nucleus basalis of meynert. ('omparison with Nissl-staincd sections indicated the presence of varying proportions of non-cholinergic neurons clustered or arranged loosely ~ithin these basal forebram subdivisions. These data provide a structural basis for studies concerned with the topographical and physiological aspects o[the raccoon basal l\~rebrain cholincrgic projections and its comparison with the basal forebrains of other species. KEYWORDS: Choline acetyltransferase
hnmunocytochemistry
INTRODUCTION The basal l\~rebrain cholinergic system has been studied intensively for many years because of its likely importance m higher cortical functions such as learning and memory, and in particular because it is affected by disorders during aging and in human neurodegenerative diseases (Bartus el al., 1982: Biesold. 1986: Wenk el a/., 1987: Bigl et al., 1987, 19901. The structural organization of the basal forebrain and the topography of its projections have been analysed in detail in a variety of laboratory animals, including rats (Bigl e; a/., 1982, Armstrong el al., 1983: McKinney el a/., 1983: M e s u l a m et al., 1983b: Woolf ~'t a/., 1983: lrle and Markowitsch, 1984: Saper, 1984: Wainer el a/., 1984: Woolfet al., 1984" Ingham el a/., 1985; Woolf and Butcher, 1985: Woolfe/a/., 1986: Schwaber el al., 1987: Eckenstein el a/.. 1988; Schober el a/., 1988: Gaykema el al., 1990: Koliatsos et al., 1990, Wainer and Mesulam, 1990t, cats (Kimura el a/., 1981 : Vincent and Reincr, Address for correspondenceto: Dr Gert Br/.ickner, Paul Flechsig lnstiluic for Brain Research, Department of Neurochemistry, Uni\ersitv of Leipzig, Jahnallee 59, O-7010 Leipzig, Federal Republic of German>. 0891 0618/92/060441 12511.00 ~ 1992 by John Wiley and Sons Ltd
1987) and monkeys (Mesulam c; al., 1983a: Satoh and Fibiger, 1985: Mesulam. 19881. The mass of the cholinergic magnocellular neurons in the mammalian basal forebrain l\~rms a complex divisible into four nuclei: pars septi medialis, pars \erticalis, pars horizontalis of the nucleus Iractus diagonalis (diagonal band of Broca), and the nucleus basalis of Meynert (Wainer and Mestilam, 1990). In addition to those features of basal t\)rebrain organization common to all mammals, many authors havc noted that there are also important species-dependent peculiarities, perhaps related to the phylogenetic position, and consequently, the state of cortical exolution of the species being considered. Difl'crenl proportions among cortical areas in different species and also differences in their wiring pattern can be expected to be mirrored in the organization of thc spatially and functionally corresponding basal forebrain subunits projecting to the cortex (Woolf el al., 1983. 1984. 1986: Waincr and Mesulam,
1990). We describe here the cholinergic basal forebrain system of the raccoon, mainly with the intention of providing it structural basis l\~r experiments investigating the structural and functional relationships el the basal l\~rebrain to the somatosensorv system. which is highly dexeloped in this species.
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MATERIALS AND METHODS Five young adult female raccoons weighing 3.4 to 4.7 kg were used in this study. The animals were deeply anaesthetized with Rompun (2 mg/kg, i.m.) followed by ketamine (10 mg/kg, i.m.).
Preparation of animals and tissue Transcardial perfusion was initiated with a prerinse of 1 litre of oxygen-saturated saline containing 2500 U/I heparin. Subsequently, the animals were perfused with 3 litres of fixative consisting of 4% paraformaldehyde and 0.2% picric acid in 0.1 M-phosphate buffer, pH7.3. In two of the animals, 0. ! % glutaraldehyde was a component of the fixative. After perfusion, the crown of the skull was removed to expose the brain and the head fixed in a stereotaxic frame (David Kopf Instruments). The brain was transsected at anterior-posterior levels 20, 8 and zero in the coronal plane according to the atlas of Schober and Biesold (1988). The brain was removed from the skull and the forebrain blocks were placed in the fixative, from which glutaraldehyde was omitted. After 2 h of immersion at 4°C, the tissue blocks were penetrated for 6--10 days with 30% sucrose in 0.1 M-phosphate buffer, pH7.3, at 4":C. The blocks were then frozen in n-hexane at -65°C or immediately sectioned on a freezing microtome (Reichert, Austria). Frontal series of sections, 40 ~tm thick, were cut in the coronal plane and collected in phosphate-buffered saline (PBS). After washing several times in PBS, every fourth section was stained with cresyl violet. The adjacent section was used for the immunocytochemical demonstration of choline acetyltransferase (CHAT).
lmmunohistochemical procedure A commercially available polyclonal antiserum (Chemicon, Temecula, CA, USA; AB 143) and a monoclonal antibody (G6ttingen B3.9B3) to ChAT were used. The antiserum was raised in rabbit against the human placental enzyme and the monoclonal antibody was produced against ChAT purified from porcine brain (Ostermann et al., 1990). Biotinylamidocapronyl-goat-anti-rabbitimmunoglobulin G (Bio-GAR) and biotinylamidocapronylgoat-anti-mouse immunoglobulin G (Bio-GAM) were prepared from sera against rabbit and mouse globulin (SIFIN, Berlin, FRG), respectively. The lgG-fraction of sera was isolated by means of a caprylic acid/ammonium sulphate method (McKinney and Parkinson, 1987) and subsequently biotinylated according to Bieber (1985). Streptavidin was purified by affinity chromatography as described by Bayer et al. (1986). Biotinylamidocapronyl-horseradish peroxidase (Bio-HRP) was prepared in the following manner:
8 mg horseradish peroxidase (grade 1, Boehringcr, Mannheim, FRG) in 0.72 ml of 0.1 M-carbonate buffer, pH 8.5, were mixed with 1.4 mg biotinylamidocapronyl-N-hydroxysuccinimide ester in 0.08 ml dimethylformamide. After 2 h, the reaction mixture was dialysed against several changes of PBS. Free-floating sections were placed in 0.6% H~O~ in 0.1 M-Tris-buffered saline (TBS), pH 7.4 for 30rain. Afterwards, sections were transferred to 10% normal goat serum (NGS) in TBS for I h. The sections were subsequently gently moved in rabbitanti-ChAT, diluted 1:2000, or mouse-anti-ChAT~ diluted 1:500 in TBS containing 20% NGS, 2% bovine serum albumin (BSA), 0.1% NaN 3 and 0.3% Triton X-100 at 4 C for 3 days. A subsequent incubation with Bio-GAR or Bio-GAM (10 ~g/ml TBS, containing 2% NGS) was performed for I h. Then the sections were incubated with a preformed complex consisting of streptavidin and Bio-HRP (10Bg+2.51ag/ml TBS, containing 2% BSA) for I h. After transfer of sections into a 0.05% solution of diaminobenzidine (DAB; Serva, Heidelberg, FRG) for 5min, ChAT-positive neurons were visualized by means of DAB/H202 reaction for 2rain. After rinsing in PBS, the sections were mounted onto albumen-glycerol coated slides, airdried and coverslipped. Controls were performed by omitting the first antibody from the immunocytochemical procedure, which resulted in the absence of any cellular staining. To give an overview of the position and extent of the cholinergic nuclei in the basal forebrain, we compiled a cytoarchitectonic atlas (Figs 1-8). On the basis of the micrographs, we have reconstructed the complex of the basal forebrain nuclei (Fig. 17). RESULTS
Topography and extent of the basal forebrain nuclei demonstrated by Nissl staining Nucleus tr. diagonalis (with pars septi medialis, S M DB; pars verticalis, V DB," and pars horizontalis, H DB)
In the raccoon, the complex of the basal forebrain nuclei begins rostrally about 1 mm before the anterior end of the corpus catlosum with the SM DB. No demarcation can be detected between the cells of the SM DB and the more ventral V DB. Each subnucleus is arranged in a thin band under the medioventral surface of the basal hemisphere (Figs 1, 2). Between the dorsoventrally oriented fibres of the diagonal band of Broca, more or less elongated cells are common. The lateral nucleus of the septum is located dorsomedially and laterally to the SM DB. More ventrally lie the nucleus accumbens and the ventral pallidum (Figs 1,2). Ventrolaterally, the V DB merges with the H DB (Figs 2, 3). In more caudal sections, all the subnuclei of the diagonal band are visible but the continuity between V DB and H DB is interrupted (Fig. 3). At the level of the caudal end of V DB and H DB, the
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Raccoon Basal Forebrain Cholinergic System SM DB is still well developed (Figs 3, 4). The SM DB extends caudally as far as the crossing of the cotnmissura anterior, where it is located superior to the commissure (Figs 5, 6). The SM DB ends caudally at the level of the first descending fornix fibres. This caudal part of the SM DB is often called the nucleus triangularis septi (Fig. 6). /Yll(l/~,l~i~li~raeopli('us magnocellularis ( PO M )
This nucleus begins lateral to the caudal end of the H DB. It extends caudally as far as the level of the commissura anterior, where it is connected dorsally with the substantia innominata. Inferiorly, the PO m contacts the ventral surface of the basal forebrain (Figs 3, 6).
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found to be the same if the monoclonal antibody B3.9B3 (G6ttingen) was used: however, this stained the ChAT-positive structures with somewhat lower intensity (Fig. 9a,b). In addition to nerve cell bodies. axons could be detected with both antibodies. The basal forebrain cholinergic cells form a more or less continuous band of ChAT-positive cells. The subdivision of this complex of cholinergic cells into separate nuclear groups is based upon their position and arrangement as well as upon the size and shape of the cell bodies and dendrites. Using these features in coronal sections produces a classification of cholinergic neurons which conforms well with the nuclear subdivisions of the forebrain found in Nissl-stained sections. Nuc/eus tractus diagonalis
Palli~&m renll'ah, ( P I ")
This region is located between the olfactory tubercle and the H DB ventrally, and the nucleus accumbens and the commissura anterior dorsally. Caudally, the PV merges with the substantia innominata (Figs I 3). ,S'uh,~lanlia i n n o n f i n a l a ( Sl )
This nucleus is the caudal extension of the PV, but the PO M spreads caudodorsally into the SI, too (Figs 3 5). The SI extends to the level of the caudal end of the commissura anterior and dorsally it contacts the border of the pallidum (Figs 6, 7). Large, loosely arranged neurons characterize the cellular pattern of the SI. ,\'uch'us hasa/i.s o/Meynerl ( N B M )
Thc large neurons situated between the fibres of the capsula interna and the medial surface of the pallidum, as well as ventrally between the SI and the pallidum, can be attributed to the NBM (Figs 5 7). The5 are arranged within a narrow band or as clusters of neurons, which appear to surround the pallidum almost completely (Fig. 5). As demonstrated in Fig. 17, cell groups of the nucleus basalis of Meynert form a long tail, which continues caudally up to the posterior pole of the pallidum. This tail is also termed the nucleus entopeduncularis (Fig. 8).
Distribution of ChAT-immunoreactive cells in the basal forebrain
The description of the distribution and morphology of the basal forebrain cholinergic neurons performed in this study is based on the immunocytochemical detection of C h A T by a polyclonal rabbit antibody (Chcmicon AB 143). The pattern of labelling was
In this nucleus (Figs 11 13) comprising the SM DB, V DB, and H DB, the cholinergic neurons form a narrow band of cells situated medially and ventrally under the surface of the forebrain (Fig. 12a). A second inner band ofcholinergic cells is visible in the region of the ventral curve of this nucleus parallel to the outer band. The ChAT-containing cells may have a triangular or a multipolar shape, depending on their position. Where they are intermingled with the fibres of the diagonal band, their shape is usually oval, i.e. they are small bipolar cells. The latter cell type is more frequent in the caudal half of the pars septi medialis (Fig. 1 l b,c). The multipolar cells, in the vertical and horizontal subnuclei of the diagonal band, appear to be larger than the bipolar cells. The dendrites are smooth and sparsely branching (Figs 12b~e, 13b,c). The cells of the vertical subnucleus merge with the cholinergic cells of the pars horizontalis (Fig. 12a). Due to the concentration of the ChAT-immunoreactive cells, this part of the diagonal band complex can be clearly distinguished from the ventral pallidum, in which cholinergic cells are scattered with low density (Fig. 10a). Nucleu.~ praeopticus magnocellulari,s'
Posterolaterally, the horizontal cell group of the diagonal band seems to continue into the PO M. However, the cholinergic cells of this nucleus appear different from those of the H DB, particularly due to their size. These multipolar cells are the largest ChAT-positive cells in the ventral forebrain nuclei (Fig. 14a,b). Suhstantia innominata
The loosely arranged cholinergic cells of the PV extend caudally into the SI. The CHATimmunoreactive cells of the SI are multipolar o1 bipolar in shape and are of a medimn size. The
Figs [ 8 Frontal Nissl-staincd sections through the basal forebrain of the raccoon. The figures correspond to the levelsof sections as indicated in Figure 17: 1,20: 2,'23: 3/28; 4,,32: 5/36: 6/40: 7,'44;8/56. Bars- 2 nun.
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Nucleus basalis of Meynert A t the b e g i n n i n g o f the p a l l i d u m , a c o n c e n t r a t i o n o f c h o l i n e r g i c cells occurs on the ventral border o f
this nucleus• arranged in a the p a l l i d u m . o f the N B M pallidum. The b e t w e e n the
M o r e posteriorly, this cell b a n d is m o r e or less circular pattern a r o u n d Posteriorly, the C h A T - p o s i t i v e cells terminate at the caudal end o f the highest density o f cells can be f o u n d p a l l i d u m a n d the capsula interna
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medially and the S1 inreriorly. A main group o1" N B M neurons forms a long, slender column especially along the ventral border o f this region. These ceils are more or less horizontally oriented and arc often bipolar. In contrast to other mammals, the cells o f the basal nucleus are not the largest in the ~entral rorebrain complex (Fig. 16a,b).
n u m b e r and distribution, in tile nucleus lateralis septi, nucleus accumbens, corpus amygdaloideum, hypothalarnus lateralis, and between the fibres of the capsula interna. Also a rcw cells are scattered in the pallidum.
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DISCUSSION considerations
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Fig. 13. (a) Pars horizontalis of the nucleus tractus diagonalis (H DB); compare Fig. 3. Bar = 500 tam. (b,c) ChAT-positive neurons of the H DB. Bar = 50 tam. Fig. 14. (a) Topography of ChAT-positive cells in the nucleus praeopticus magnocellularis (PO M); compare Fig. 4. Bar = 500 tam. (b) ChAT-positive cells of the PO M. Bar = 50 tam. Fig. 15. (a) Pattern of ChAT-positive cells in the substantia innominata (SI); compare Fig. 5. Bar= 500 tam. (b) ChAT-positive neurons in the SI. Bar= 50 lain. Fig. 16. (a) Band of ChAT-positive neurons forming the nucleus basalis of Meynert (NBM); compare Fig. 5. Bar = 500 tam. (b) ChAT-positive cells in the NBM. Bar = 50 tam
R a c c o o n Basal F o r e b r a i n ( ' h o l i n e r g i c S y s t e m
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l:ig. 17. Reconstruction of the coniinuum of nuclei forming the cholinergic basal forcbrain complex in lhc raccoon, fhc Icl'l hemisphere is dcmonslralcd according to lhe levels of sections 16. 20, 23, 28, 32.36, 4(1, 44.48 and 56 of one series. Numbers 20 56 u'orrcspond 1o tigs 1 8.
ABBREVIATIONS A('( AMY A PR B S'I ( (' A (" 1 IX 1t DB I IYI'
nucleus accunlbcllS corpus arnygdaloideum area praeoptica bed nucleus slria lerminalis nucleus caudatus commissura anlerior capsukl intema fornix nucleus iraclus diagonalis, pars horizontalis hypothalamus
LS NBM N TR O PA PAc PAl PC PU PO M PV
USED
IN F I G U R E S
nucleus lateralis septi nucleus basalis of Meyncrl nucleus lractus olfactorius pallidum (globus pallidusl pallidum externum pallidum inlernum cortex piril\mnis putamen nucleus praeopticus magnocellularis pallidum ventrale
o u r l a b o r a t o r y . In t h a t species it p r o d u c e d a s t a i n i n g p a t t e r n o f c h o l i n e r g i c n e u r o n s c o m p a r a b l e to that d e s c r i b e d by n u m e r o u s i n v e s t i g a t o r s . T h e applic a t i o n o f this a n t i s e r u m , in c o m p a r i s o n to the m o n o c l o n a l a n t i b o d y ( G S t t i n g e n B3.9B3), led to a s o m e u h a t h i g h e r i n t e n s i t y o f s t a i n i n g in the rat, just as it did in the r a c c o o n . It c a n be c o n c l u d e d that the d i s t r i b u t i o n o f C h A T - i m m u n o r e a c t i v e cells d e s c r i b e d h e r e r e p r e s e n t s the a c t u a l l o c a t i o n s o f C h A T - c o n t a i n i n g , i.e. c h o l i n e r g i c n e u r o n s , in the r a c c o o n basal f o r e b r a i n .
RET S1 SM DB
nucleu~rcticularis substantia innonlinala nucleus traclus diagonalis, pars sepii mcdialis THAL I]la]alllus 1"O[.F lubcrculum olfactofium TR 0 Ifaclus oplicus TR OLF Iractus olfactorius V DB nucleus lFaclus diagonalis, pars \ erlicalis VL xcnlriculus lalenllis
Delineation of basal forebr,'iin nuclei in Nissl-stained sections T h e i n t e r p r e t a t i o n o f n u c l e a r g r o u p s o f tile basal l'orebrain c o m p l e x p e r f o r n l e d in o u r study agrees closely with d e s c r i p t i o n s o f Nissl studies in o t h e r c a r n i v o r e s (cat: B e r m a n a n d J o n e s . 1982) o r in rats (Bigl et al., 1 9 8 2 : M e s u l a n 3 el al.. 1983b: S a p e r , 1984: l s h i k a w a a n d H i r a t a , 1986: S c h o b e r c t a l . , 1988). O n the basis o f c y t o a r c h i t e c t o n i c criteria, the basal f o r e b r a i n c o m p l e x was s u b d i v i d e d into three
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groups, which may be shown to apply to the raccoon also: (1) the nucleus septi medialis and the nuclei of the diagonal band of Broca, (2) the large neurons of the tuberculum olfactorium, and (3) the basal nucleus proper (Brockhaus, 1942). However, as described in other species, in the raccoon no sharp delineation of nuclei is possible, especially between the second and third group of neurons. This is even more evident in ChAT-stained sections. Distribution of ChAT-immunoreactive neurons
The spatial organization of ChAT-immunoreactive neurons in the raccoon basal forebrain observed in this study basically agrees with that described in other mammals (Kimura et al., 1981; Rye et al., 1984; Wainer et al., 1984; Sofroniew et al., 1987) and especially with the pattern shown in the cat (Vincent and Reiner, 1987). The main nuclei present in the raccoon are the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis), nucleus praeopticus magnocellularis, substantia innominata and the nucleus basatis of Meynert. Mesulam and coworkers referred to these nuclei as the Ch 1-4 regions of the basal forebrain (Mesulam et al., 1983a,b; Mesulam and Geula, 1988). However, as already mentioned by Semba et al. (1988) and discussed by a group of neuroscientists (see Butcher and Semba, 1989), the 'Ch' nomenclature applies best in primates but is not applicable in other species. As in the rat (Schober et al., 1988), the cellular band of the nucleus tractus diagonalis also forms a continuum in the raccoon basal forebrain. Therefore, based on CHAT° immunocytochemical data alone, the description of a separate medial septal nucleus as recognized in other studies (Bigl et al., 1982; Mesulam et al., 1983a,b; Saper, 1984; Ishikawa and Hirata, 1986; Vincent and Reiner, 1987) does not seem to us to be justified. In contrast, the delineation of the nucleus praeopticus magnocellularis, which is often considered as the lateral part of the horizontal limb of the diagonal band in other species (Rye et al., 1984; Zfiborszky et al., 1986), seemed to be a distinct nuclear region in this species because the nucleus praeopticus magnocellularis could be clearly distinguished from the pars horizontalis of the nucleus tractus diagonalis. This was mainly due to the distinctive size and the irregular orientation of the magnocellular preoptic neurons. In comparison with the nucleus praeopticus magnocellularis, the ChAT-immunoreactive neurons contained in the substantia innominata were loosely arranged in a pattern similar to that found in rodents, where it is often referred to as a component or a partial equivalent of the nucleus basalis of Meynert (Bigl et al., 1982; Woolfet al., 1983; Saper, 1984; Sofroniew et al., 1987; Schober et al., 1988). In Nissl-stained sections of the rat basal forebrain, clear nuclear boundaries of the nucleus basalis are
absent (Gorry, 1963; McKinney et al., Rye et al., 1984; Schober et al., 1988). In the raccoon, there is a clearly differentiated band of cells medially and ventrolaterally attached to, and partly surrounding, the pallidum. These groups of neurons correspond to the nucleus basalis of Meynert, which could be recognized in Nissl-stained sections, but which was easier to delineate after immunocytochemical detection of CHAT. By comparing Nissl- and ChAT-stained preparations, it was evident in our study that all of the basal forebrain cell groups in the raccoon contain, to a varying degree, non-cholinergic neurons. In the diagonal band complex and in the magnocellular preoptic nucleus a large portion, or even the majority of neurons, may be GABAergic projection neurons, a situation which has been documented in the rat (K6hler et al., 1984; Brashear et al., 1986; Onteniente et al., 1986; Z~tborszky et al., 1986) and cat (Fisher et al., 1988; Freund and Antal, 1988: Freund and Meskenaite, 1992).
ACKNOWLEDGEMENTS The authors are grateful to Mrs H Gruschka, Mrs M. Schmidt and Mrs M. Volkmann for excellent technical assistance. We thank Professor Dr M. Milder, NeurologicalClinic, University of G6ttingen, for generouslyprovidingthe monoclonalantibody to CHAT.
REFERENCES Armstrong, D. M., Saper, C. B., Levey, A. I., Wainer, B. H. and Terry, R. D. (1983). Distribution ofcholinergic neurons in rat brain: demonstrated by immunocytochemical localization of choline acetyltransferase. J. Comp. Neurol. 216, 53-68. Bartus, R. T., Dean III, R. L., Beer, B. and Lippa, A. S. (1982). The cholinergic hypothesis of geriatric memory dysfunction. Science 217, 408-417. Bayer, E. A., Ben-Hur, H., Gittin, G. and Wilchek, M. (1986). An improved method for the single-step purification of streptavidin. J. Biochem. Biophys. Methods 13, 103-112. Berman, A. L. and Jones, E. G. (1982). The Thalamus and Basal Telencephalon q/ the Cat. University of Wisconsin Press, Madison. Bieber, F. (1985). Biotinylierung monoklonaler Antikfrper. In Monoklonale Antik6rper. Herstellung und Charakterisierung (eds Peters, J. H., Baumgarten, H. and Schulze, M.), pp. 209-212. Springer, Berlin. Biesold, D. (1986). Senile dementia of Alzheimer's type-current concepts and prospects. Z. Psychol. 194, 311-329. Bigl, V., Arendt, T. and Biesold, D. (1990). The nucleus basalis of Meynert during ageing and in dementing neuropsychiatric disorders. In Brain C'tolinergic Systems (eds Steriade, M. and Biesold, D.), pp. 364-386. Oxford University Press. Bigl, V., Arendt, T., Fischer, S., Fischer, S., Werner, M. and Arendt, A. (1987). The cholinergic system in aging. Gerontology 33, 172-180.
R a c c o o n Basal F o r e b r a i n Cholinergic System Bigl, V., Woolf, N. J. and Butcher, L. L. (1982). Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital and cingulate cortices: a combined fluorescent tracer and acetyleholinesterase analysis. Brain Res. Bull. 8, 727 749. Brasbear, H. R., Zaborszky, L. and Heimer, L. (1986). Distribution of GABAergic and cholinergic neurons in the rat diagonal band. Neuroscience 17, 439 451. Brockhaus, H. (1942). Vergleichend-anatornische Untersuchungen fiber den Basalkernkomplex. J. Phrsiol. Neurol. 51, 57 95. Butcher, L. L. and Semba, K. (1989). Reassessing the cholinergic basal forebrain: nomenclature schemata and concepts. Trends in Neurosci. 12, 483 485. Eckenstein, F. P., Baughman, R. W. and Quinn, J. (1988). An anatomical study of cholinergic innervation in rat cerebral cortex. Neuroscience 25, 457 474. Fisher, R. S., Buchwald, N. A., Hull, C. D. and Levine, M. S. (1988). GABAergic basal forebrain neurons project to the neocortex: the localization of glutamie acid decarboxylase and choline acetyltransferase in feline corticopetal neurons. J. Comp. Neurol. 272, 489 502. Freund, T. F. and Antal, M. (1988). GABA-containing neurons in the septum control inhibitory interneurons in the bippocampus. Nature 336, 170 173. Freund. T. F. and Meskenaite, V. (1992). 7-Aminobutyric acid-containing basal forebrain neurons innervate inhibitory interneurons in the neocortex. Pro('. Natl. A(ad. S('i. USA 89, 738 742. Gaykema, R. P. A., Luiten, P. G. M., Nyakas, C. and Traber. J. (1990). Cortical projection pattern of the medial septum diagonal band complex. J. Cmnp. Neurol. 293, 103 124. G o n y . J. D. (1963). Studies on the comparative anatomy of the Ganglion basale of Meynert. Acta Anat. 55, 51 104. lngham. C. A., Bolam. J. P., Wainer, B. H. and Smith, A. D. (1985). A correlated light and electron microseopic study of identified cholinergic basal forebrain neurons that project to the cortex in the rat. J. Comp. N('urol. 239, 176 192. lrle, E. and Markowitsch, H. J. (1984). Basal forebrain efl'crcnts reach the whole cerebral cortex of the cat. Brain Res. Bull. 12,493 512. lshikawa, T. and Hirata, Y. (1986). Organization of choline acetyltransferase-containing structures m the forebrain of the rat. J. Neurosci. 6, 281 292. Kimura, H., McGecr, P. 1., Peng, J. H. and McGeer, E. G. (1981). The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat..l. Comp. Neurol. 200, 151 201. K6hlcr. C., Chan-Palay, V. and Wu, J. Y. (1984). Septal neurons containing glutamic acid decarboxylase immunoreactivity project to the hippocampal region in the rat brain. Anal. Emhrrol. 169, 41 44. Koliatsos, V. E., Martin. L. J. and Price, D. L. (1990). Eft'trent organization of the mammalian basal forebrain. In Brain Cholinergic S.vstems (eds Steriade, M. and Biesold, D.), pp. 120 152. Oxford University Press. McKinney, M. M., Coyle, J. T. and Hedreen, J. C. (1983). Topographic analysis of the innervation of the ra! neocortex and hippocampus by the basal forebrain cholinergic system. J. Comp. Neurol. 217, 103 121.
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McKinney, M. M. and Parkinson, A. (1987). A simplc, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J. hn,mmol. Metho(£" 95, 271 278. Mesulam, M.-M. (1988). Central cholinergic pathways. In Neurotransmitters and Cortical Function (eds Avoii, M., Reader, T. A., Dykes, R. W. and Gloor, P.). pp. 237 259. Plenum Publ. Corp., New York. Mesulam, M.-M. and Geula, C. (1988). Nucleus basalis (Ch4) and cortical cholinergic innervation in the human brain: observations based on the distribution of acetylcholinesterase and choline acetyltransfcrase. J. Comp. Neurol. 275, 216 240. Mesulam, M.-M., Mufson, E. J., Levey. A. 1. and Wainer, B. H. (1983a). Cholinergic innervation of the cortex bv basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata) and hypothalamus in the rhesus monkey. .I. Comp. Ncurol. 214, 170 197. Mesulam, M.-M., Mufson, E. J. and Lcvey, A. 1. (1983b). Central cholinergic pathways in the rat: a n overview based on an alternative nomenclature (('hi Ch6). Neuroscience 10, 1185 1201. Onteniente, B., Tago, H., Kimura. H. and Maeda, T. (1986). Distribution of ?-aminobutyric acidimmunoreactive neurons in the septal region of the ntt brain. JJ Comp. Neurol. 248, 422 430. Ostermann. C., Dickmann, U., Muley, T. and M+ider, M. (1990). Large-scale purification of choline acetyltransferase and production of highly specific antiscra. Eur. ,l. Biochem. 192, 215 218. Rye, D. B., Wainer, B. H., Mesulam, M.-M.. Mufson. E. J. and Saper, C. B. (1984). Cortical projections arising from the basal forebrain. A study o[" cholinergic and non-cholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase. %"('uros(i('n(+(' 13, 627 643. Saper, C. B. (1984). Organization of cerebral cortical afferent systems in the rat. II. Magnocellular basal nucleus. J. Comp. N(,urol. 222, 313 342. Satoh, K. and FiNger, H. C. (1985). Distribution of central cholinergic neurons in the Baboon (Papiopapio). II. A topographic atlas correlated with catecholaminc neurons..1. Comp. Neurol. 236, 215 233. Schober, W., Brauer, K.. Werner, L. and Lfith, H.-J. (1988). Lage und Ausdehnung der magnozelluliiren Kerne im basalen Vorderhirn xon Nagetieren und Hasenartigen. J. ltirt!lbrs('h. 29, 443 459. Schober. W. and Biesold, I). (1988). The brain of the raccoon (Procron lotor) in stereotaxic coordinates. 1. Macroscopic studies. J. llirnlbrs('h. 29, 701 708. Schwaber, J. S., Rogers, W. T., Satoh. K. and Fibigcr, H. C. (1987). Distribution and organization of cholinergic neurons in the rat l\webrain demonstrated by computer-aided data aquisition and threedimensional reconstruction. ,I. ('omp. N('urol. 263, 309 325. Semba, K., Reiner, P. B., McGeer. E. G. and Fibiger, H. C. (1988). Brain stem aflerents to the magnocellular basal lk~rebrain studied by axonal tnmsport, immunohistochemistry, and electrophysiolog5 m the rat. J. ('omp. Neurol. 267,433 453. Sofroniew, M. V., Pearson. R. C. A. and Powell. T. P. S. (1987). The cholinergic nuclei of the basal t\)rcbrain of the rat: normal structure, development and
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experimentally induced degeneration. Brain Res. 411, 310-331. Vincent, S. R. and Reiner, P. B. (1987). The immunohistochemical localization of choline acetyltransferase in the cat brain. Brain Res. Bull. 18, 371 415. Wainer, B. H., Levey, A. I., Mufson, E. J. and Mesulam, M.-M. (1984). Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase. Neurochem. Int. 6, 163 --182. Wainer, B. H. and Mesulam, M.-M. (1990). Ascending cholinergic pathways in the rat. In Brain Cholinergic Systems (eds Steriade, M. and Biesold, D.), pp. 65-119. Oxford University Press. Wenk, G., Hughey, D., Boundy, V., Kim, A., Walker, L. and Olton, D. (1987). Neurotransmitters and memory: role of cholinergic, serotonergic, and noradrenergic systems. Behav. Neurosci. 101,325 332. Woolf, N. J. and Butcher, L. L. (1985). Cholinergic systems in the rat brain: II. Projections to the interpeduncular nucleus. Brain Res. Bull. 14, 63 83.
WoolR N. J., Eckenstein, F. and Butcher, L. i.. (1983). Cholinergic projections from the basal forebrain to the frontal cortex: A combined fluorescent Iraccr and immunohistochemical analysis. Neurosci. Letters 40, 93 98. Woolt, N. J., Eckenstein, F. and Butcher, L. k. (1984). Cholinergic systems in the rat brain: I. Projections to the limbic telencephalon. Brain Res. 13, 751-784. Woolf, N. J., Hernit, M. C. and Butcher, L. L. (1986). Cholinergic and non-cholinergic projections from the rat basal forebrain revealed by combined choline acetyltransferase and Phaseolus vulgaris leucoagglutinin immunohistochemistry. Neurosci. Letters 66, 281-286. Zfiborszky, L., Carlsen, J., Brashear, H. R. and Helmet. L. (1986). Cholinergic and GABAergic afferents to the olfactory bulb in the rat with special emphasis on the projection neurons in the nucleus of the horizontal limb of the diagonal band. J. Comp. Neurol. 243, 488 509.
Professor Dietmar Biesold died on 29 May, 1991, in Leipzig. Our work on the basaljorebrain of the raccoon was initiated by him. Accepted 22 June 1992
The Basal Forebrain Cholinergic System in the Raccoon Gert Briickner, Wiljiqed Schober, Wolfgang Hiirtig, Christei Ostermann-LatiJ ~, Harry H. Webstert, Robert W. Dykest, Douglas D. Rasmusson~ and Dietmar Biesoid Paul Flechsig Institute liar Brain Research, University of Leipzig, Germany *Neurological Clinic, University of G6ttingen, Germany +Departement de Physiologic, Facult6 de M6decine, Univcrsitd de Montrdal. Qudbec, Canada ++Deparmlent of Physiology and Biophysics, Dalhousic [ !niversity. Nova Scotia, Canada
ABSTRACT -l-he distribution o f neurons displaying choline acetyhransl'erase (CHAT) imlnunoreactixity was examined in the raccoon basal forebrain using a rabbit antiserum and at monoclonal antibody. Alternating sections were used for Nissl staining. ChAT-positive neurons were arranged in a conlinuous mass extending from the medial septum to the caudal pole of the pallidum. Based upon spatial relations to fibre tracts, the clustering of neuronal groups, and cytological criteria, the basal l'orebrain magnocellular complex can be subdivided into several distinct regions. Although clear nuclear boundaries ,sere often absent, the ChAT-positive neurons were divided into: the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis): nucleus praeopticus magnocellularis: subslantia innominata: and the nucleus basalis of meynert. ('omparison with Nissl-staincd sections indicated the presence of varying proportions of non-cholinergic neurons clustered or arranged loosely ~ithin these basal forebram subdivisions. These data provide a structural basis for studies concerned with the topographical and physiological aspects o[the raccoon basal l\~rebrain cholincrgic projections and its comparison with the basal forebrains of other species. KEYWORDS: Choline acetyltransferase
hnmunocytochemistry
INTRODUCTION The basal l\~rebrain cholinergic system has been studied intensively for many years because of its likely importance m higher cortical functions such as learning and memory, and in particular because it is affected by disorders during aging and in human neurodegenerative diseases (Bartus el al., 1982: Biesold. 1986: Wenk el a/., 1987: Bigl et al., 1987, 19901. The structural organization of the basal forebrain and the topography of its projections have been analysed in detail in a variety of laboratory animals, including rats (Bigl e; a/., 1982, Armstrong el al., 1983: McKinney el a/., 1983: M e s u l a m et al., 1983b: Woolf ~'t a/., 1983: lrle and Markowitsch, 1984: Saper, 1984: Wainer el a/., 1984: Woolfet al., 1984" Ingham el a/., 1985; Woolf and Butcher, 1985: Woolfe/a/., 1986: Schwaber el al., 1987: Eckenstein el a/.. 1988; Schober el a/., 1988: Gaykema el al., 1990: Koliatsos et al., 1990, Wainer and Mesulam, 1990t, cats (Kimura el a/., 1981 : Vincent and Reincr, Address for correspondenceto: Dr Gert Br/.ickner, Paul Flechsig lnstiluic for Brain Research, Department of Neurochemistry, Uni\ersitv of Leipzig, Jahnallee 59, O-7010 Leipzig, Federal Republic of German>. 0891 0618/92/060441 12511.00 ~ 1992 by John Wiley and Sons Ltd
1987) and monkeys (Mesulam c; al., 1983a: Satoh and Fibiger, 1985: Mesulam. 19881. The mass of the cholinergic magnocellular neurons in the mammalian basal forebrain l\~rms a complex divisible into four nuclei: pars septi medialis, pars \erticalis, pars horizontalis of the nucleus Iractus diagonalis (diagonal band of Broca), and the nucleus basalis of Meynert (Wainer and Mestilam, 1990). In addition to those features of basal t\)rebrain organization common to all mammals, many authors havc noted that there are also important species-dependent peculiarities, perhaps related to the phylogenetic position, and consequently, the state of cortical exolution of the species being considered. Difl'crenl proportions among cortical areas in different species and also differences in their wiring pattern can be expected to be mirrored in the organization of thc spatially and functionally corresponding basal forebrain subunits projecting to the cortex (Woolf el al., 1983. 1984. 1986: Waincr and Mesulam,
1990). We describe here the cholinergic basal forebrain system of the raccoon, mainly with the intention of providing it structural basis l\~r experiments investigating the structural and functional relationships el the basal l\~rebrain to the somatosensorv system. which is highly dexeloped in this species.
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MATERIALS AND METHODS Five young adult female raccoons weighing 3.4 to 4.7 kg were used in this study. The animals were deeply anaesthetized with Rompun (2 mg/kg, i.m.) followed by ketamine (10 mg/kg, i.m.).
Preparation of animals and tissue Transcardial perfusion was initiated with a prerinse of 1 litre of oxygen-saturated saline containing 2500 U/I heparin. Subsequently, the animals were perfused with 3 litres of fixative consisting of 4% paraformaldehyde and 0.2% picric acid in 0.1 M-phosphate buffer, pH7.3. In two of the animals, 0. ! % glutaraldehyde was a component of the fixative. After perfusion, the crown of the skull was removed to expose the brain and the head fixed in a stereotaxic frame (David Kopf Instruments). The brain was transsected at anterior-posterior levels 20, 8 and zero in the coronal plane according to the atlas of Schober and Biesold (1988). The brain was removed from the skull and the forebrain blocks were placed in the fixative, from which glutaraldehyde was omitted. After 2 h of immersion at 4°C, the tissue blocks were penetrated for 6--10 days with 30% sucrose in 0.1 M-phosphate buffer, pH7.3, at 4":C. The blocks were then frozen in n-hexane at -65°C or immediately sectioned on a freezing microtome (Reichert, Austria). Frontal series of sections, 40 ~tm thick, were cut in the coronal plane and collected in phosphate-buffered saline (PBS). After washing several times in PBS, every fourth section was stained with cresyl violet. The adjacent section was used for the immunocytochemical demonstration of choline acetyltransferase (CHAT).
lmmunohistochemical procedure A commercially available polyclonal antiserum (Chemicon, Temecula, CA, USA; AB 143) and a monoclonal antibody (G6ttingen B3.9B3) to ChAT were used. The antiserum was raised in rabbit against the human placental enzyme and the monoclonal antibody was produced against ChAT purified from porcine brain (Ostermann et al., 1990). Biotinylamidocapronyl-goat-anti-rabbitimmunoglobulin G (Bio-GAR) and biotinylamidocapronylgoat-anti-mouse immunoglobulin G (Bio-GAM) were prepared from sera against rabbit and mouse globulin (SIFIN, Berlin, FRG), respectively. The lgG-fraction of sera was isolated by means of a caprylic acid/ammonium sulphate method (McKinney and Parkinson, 1987) and subsequently biotinylated according to Bieber (1985). Streptavidin was purified by affinity chromatography as described by Bayer et al. (1986). Biotinylamidocapronyl-horseradish peroxidase (Bio-HRP) was prepared in the following manner:
8 mg horseradish peroxidase (grade 1, Boehringcr, Mannheim, FRG) in 0.72 ml of 0.1 M-carbonate buffer, pH 8.5, were mixed with 1.4 mg biotinylamidocapronyl-N-hydroxysuccinimide ester in 0.08 ml dimethylformamide. After 2 h, the reaction mixture was dialysed against several changes of PBS. Free-floating sections were placed in 0.6% H~O~ in 0.1 M-Tris-buffered saline (TBS), pH 7.4 for 30rain. Afterwards, sections were transferred to 10% normal goat serum (NGS) in TBS for I h. The sections were subsequently gently moved in rabbitanti-ChAT, diluted 1:2000, or mouse-anti-ChAT~ diluted 1:500 in TBS containing 20% NGS, 2% bovine serum albumin (BSA), 0.1% NaN 3 and 0.3% Triton X-100 at 4 C for 3 days. A subsequent incubation with Bio-GAR or Bio-GAM (10 ~g/ml TBS, containing 2% NGS) was performed for I h. Then the sections were incubated with a preformed complex consisting of streptavidin and Bio-HRP (10Bg+2.51ag/ml TBS, containing 2% BSA) for I h. After transfer of sections into a 0.05% solution of diaminobenzidine (DAB; Serva, Heidelberg, FRG) for 5min, ChAT-positive neurons were visualized by means of DAB/H202 reaction for 2rain. After rinsing in PBS, the sections were mounted onto albumen-glycerol coated slides, airdried and coverslipped. Controls were performed by omitting the first antibody from the immunocytochemical procedure, which resulted in the absence of any cellular staining. To give an overview of the position and extent of the cholinergic nuclei in the basal forebrain, we compiled a cytoarchitectonic atlas (Figs 1-8). On the basis of the micrographs, we have reconstructed the complex of the basal forebrain nuclei (Fig. 17). RESULTS
Topography and extent of the basal forebrain nuclei demonstrated by Nissl staining Nucleus tr. diagonalis (with pars septi medialis, S M DB; pars verticalis, V DB," and pars horizontalis, H DB)
In the raccoon, the complex of the basal forebrain nuclei begins rostrally about 1 mm before the anterior end of the corpus catlosum with the SM DB. No demarcation can be detected between the cells of the SM DB and the more ventral V DB. Each subnucleus is arranged in a thin band under the medioventral surface of the basal hemisphere (Figs 1, 2). Between the dorsoventrally oriented fibres of the diagonal band of Broca, more or less elongated cells are common. The lateral nucleus of the septum is located dorsomedially and laterally to the SM DB. More ventrally lie the nucleus accumbens and the ventral pallidum (Figs 1,2). Ventrolaterally, the V DB merges with the H DB (Figs 2, 3). In more caudal sections, all the subnuclei of the diagonal band are visible but the continuity between V DB and H DB is interrupted (Fig. 3). At the level of the caudal end of V DB and H DB, the
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Raccoon Basal Forebrain Cholinergic System SM DB is still well developed (Figs 3, 4). The SM DB extends caudally as far as the crossing of the cotnmissura anterior, where it is located superior to the commissure (Figs 5, 6). The SM DB ends caudally at the level of the first descending fornix fibres. This caudal part of the SM DB is often called the nucleus triangularis septi (Fig. 6). /Yll(l/~,l~i~li~raeopli('us magnocellularis ( PO M )
This nucleus begins lateral to the caudal end of the H DB. It extends caudally as far as the level of the commissura anterior, where it is connected dorsally with the substantia innominata. Inferiorly, the PO m contacts the ventral surface of the basal forebrain (Figs 3, 6).
445
found to be the same if the monoclonal antibody B3.9B3 (G6ttingen) was used: however, this stained the ChAT-positive structures with somewhat lower intensity (Fig. 9a,b). In addition to nerve cell bodies. axons could be detected with both antibodies. The basal forebrain cholinergic cells form a more or less continuous band of ChAT-positive cells. The subdivision of this complex of cholinergic cells into separate nuclear groups is based upon their position and arrangement as well as upon the size and shape of the cell bodies and dendrites. Using these features in coronal sections produces a classification of cholinergic neurons which conforms well with the nuclear subdivisions of the forebrain found in Nissl-stained sections. Nuc/eus tractus diagonalis
Palli~&m renll'ah, ( P I ")
This region is located between the olfactory tubercle and the H DB ventrally, and the nucleus accumbens and the commissura anterior dorsally. Caudally, the PV merges with the substantia innominata (Figs I 3). ,S'uh,~lanlia i n n o n f i n a l a ( Sl )
This nucleus is the caudal extension of the PV, but the PO M spreads caudodorsally into the SI, too (Figs 3 5). The SI extends to the level of the caudal end of the commissura anterior and dorsally it contacts the border of the pallidum (Figs 6, 7). Large, loosely arranged neurons characterize the cellular pattern of the SI. ,\'uch'us hasa/i.s o/Meynerl ( N B M )
Thc large neurons situated between the fibres of the capsula interna and the medial surface of the pallidum, as well as ventrally between the SI and the pallidum, can be attributed to the NBM (Figs 5 7). The5 are arranged within a narrow band or as clusters of neurons, which appear to surround the pallidum almost completely (Fig. 5). As demonstrated in Fig. 17, cell groups of the nucleus basalis of Meynert form a long tail, which continues caudally up to the posterior pole of the pallidum. This tail is also termed the nucleus entopeduncularis (Fig. 8).
Distribution of ChAT-immunoreactive cells in the basal forebrain
The description of the distribution and morphology of the basal forebrain cholinergic neurons performed in this study is based on the immunocytochemical detection of C h A T by a polyclonal rabbit antibody (Chcmicon AB 143). The pattern of labelling was
In this nucleus (Figs 11 13) comprising the SM DB, V DB, and H DB, the cholinergic neurons form a narrow band of cells situated medially and ventrally under the surface of the forebrain (Fig. 12a). A second inner band ofcholinergic cells is visible in the region of the ventral curve of this nucleus parallel to the outer band. The ChAT-containing cells may have a triangular or a multipolar shape, depending on their position. Where they are intermingled with the fibres of the diagonal band, their shape is usually oval, i.e. they are small bipolar cells. The latter cell type is more frequent in the caudal half of the pars septi medialis (Fig. 1 l b,c). The multipolar cells, in the vertical and horizontal subnuclei of the diagonal band, appear to be larger than the bipolar cells. The dendrites are smooth and sparsely branching (Figs 12b~e, 13b,c). The cells of the vertical subnucleus merge with the cholinergic cells of the pars horizontalis (Fig. 12a). Due to the concentration of the ChAT-immunoreactive cells, this part of the diagonal band complex can be clearly distinguished from the ventral pallidum, in which cholinergic cells are scattered with low density (Fig. 10a). Nucleu.~ praeopticus magnocellulari,s'
Posterolaterally, the horizontal cell group of the diagonal band seems to continue into the PO M. However, the cholinergic cells of this nucleus appear different from those of the H DB, particularly due to their size. These multipolar cells are the largest ChAT-positive cells in the ventral forebrain nuclei (Fig. 14a,b). Suhstantia innominata
The loosely arranged cholinergic cells of the PV extend caudally into the SI. The CHATimmunoreactive cells of the SI are multipolar o1 bipolar in shape and are of a medimn size. The
Figs [ 8 Frontal Nissl-staincd sections through the basal forebrain of the raccoon. The figures correspond to the levelsof sections as indicated in Figure 17: 1,20: 2,'23: 3/28; 4,,32: 5/36: 6/40: 7,'44;8/56. Bars- 2 nun.
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Nucleus basalis of Meynert A t the b e g i n n i n g o f the p a l l i d u m , a c o n c e n t r a t i o n o f c h o l i n e r g i c cells occurs on the ventral border o f
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medially and the S1 inreriorly. A main group o1" N B M neurons forms a long, slender column especially along the ventral border o f this region. These ceils are more or less horizontally oriented and arc often bipolar. In contrast to other mammals, the cells o f the basal nucleus are not the largest in the ~entral rorebrain complex (Fig. 16a,b).
n u m b e r and distribution, in tile nucleus lateralis septi, nucleus accumbens, corpus amygdaloideum, hypothalarnus lateralis, and between the fibres of the capsula interna. Also a rcw cells are scattered in the pallidum.
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Fig. 13. (a) Pars horizontalis of the nucleus tractus diagonalis (H DB); compare Fig. 3. Bar = 500 tam. (b,c) ChAT-positive neurons of the H DB. Bar = 50 tam. Fig. 14. (a) Topography of ChAT-positive cells in the nucleus praeopticus magnocellularis (PO M); compare Fig. 4. Bar = 500 tam. (b) ChAT-positive cells of the PO M. Bar = 50 tam. Fig. 15. (a) Pattern of ChAT-positive cells in the substantia innominata (SI); compare Fig. 5. Bar= 500 tam. (b) ChAT-positive neurons in the SI. Bar= 50 lain. Fig. 16. (a) Band of ChAT-positive neurons forming the nucleus basalis of Meynert (NBM); compare Fig. 5. Bar = 500 tam. (b) ChAT-positive cells in the NBM. Bar = 50 tam
R a c c o o n Basal F o r e b r a i n ( ' h o l i n e r g i c S y s t e m
449
56
/
44
PA
36 32 28
20
CA
16
SM
I
i
TR OLF
PO M
S I
NBM
l:ig. 17. Reconstruction of the coniinuum of nuclei forming the cholinergic basal forcbrain complex in lhc raccoon, fhc Icl'l hemisphere is dcmonslralcd according to lhe levels of sections 16. 20, 23, 28, 32.36, 4(1, 44.48 and 56 of one series. Numbers 20 56 u'orrcspond 1o tigs 1 8.
ABBREVIATIONS A('( AMY A PR B S'I ( (' A (" 1 IX 1t DB I IYI'
nucleus accunlbcllS corpus arnygdaloideum area praeoptica bed nucleus slria lerminalis nucleus caudatus commissura anlerior capsukl intema fornix nucleus iraclus diagonalis, pars horizontalis hypothalamus
LS NBM N TR O PA PAc PAl PC PU PO M PV
USED
IN F I G U R E S
nucleus lateralis septi nucleus basalis of Meyncrl nucleus lractus olfactorius pallidum (globus pallidusl pallidum externum pallidum inlernum cortex piril\mnis putamen nucleus praeopticus magnocellularis pallidum ventrale
o u r l a b o r a t o r y . In t h a t species it p r o d u c e d a s t a i n i n g p a t t e r n o f c h o l i n e r g i c n e u r o n s c o m p a r a b l e to that d e s c r i b e d by n u m e r o u s i n v e s t i g a t o r s . T h e applic a t i o n o f this a n t i s e r u m , in c o m p a r i s o n to the m o n o c l o n a l a n t i b o d y ( G S t t i n g e n B3.9B3), led to a s o m e u h a t h i g h e r i n t e n s i t y o f s t a i n i n g in the rat, just as it did in the r a c c o o n . It c a n be c o n c l u d e d that the d i s t r i b u t i o n o f C h A T - i m m u n o r e a c t i v e cells d e s c r i b e d h e r e r e p r e s e n t s the a c t u a l l o c a t i o n s o f C h A T - c o n t a i n i n g , i.e. c h o l i n e r g i c n e u r o n s , in the r a c c o o n basal f o r e b r a i n .
RET S1 SM DB
nucleu~rcticularis substantia innonlinala nucleus traclus diagonalis, pars sepii mcdialis THAL I]la]alllus 1"O[.F lubcrculum olfactofium TR 0 Ifaclus oplicus TR OLF Iractus olfactorius V DB nucleus lFaclus diagonalis, pars \ erlicalis VL xcnlriculus lalenllis
Delineation of basal forebr,'iin nuclei in Nissl-stained sections T h e i n t e r p r e t a t i o n o f n u c l e a r g r o u p s o f tile basal l'orebrain c o m p l e x p e r f o r n l e d in o u r study agrees closely with d e s c r i p t i o n s o f Nissl studies in o t h e r c a r n i v o r e s (cat: B e r m a n a n d J o n e s . 1982) o r in rats (Bigl et al., 1 9 8 2 : M e s u l a n 3 el al.. 1983b: S a p e r , 1984: l s h i k a w a a n d H i r a t a , 1986: S c h o b e r c t a l . , 1988). O n the basis o f c y t o a r c h i t e c t o n i c criteria, the basal f o r e b r a i n c o m p l e x was s u b d i v i d e d into three
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groups, which may be shown to apply to the raccoon also: (1) the nucleus septi medialis and the nuclei of the diagonal band of Broca, (2) the large neurons of the tuberculum olfactorium, and (3) the basal nucleus proper (Brockhaus, 1942). However, as described in other species, in the raccoon no sharp delineation of nuclei is possible, especially between the second and third group of neurons. This is even more evident in ChAT-stained sections. Distribution of ChAT-immunoreactive neurons
The spatial organization of ChAT-immunoreactive neurons in the raccoon basal forebrain observed in this study basically agrees with that described in other mammals (Kimura et al., 1981; Rye et al., 1984; Wainer et al., 1984; Sofroniew et al., 1987) and especially with the pattern shown in the cat (Vincent and Reiner, 1987). The main nuclei present in the raccoon are the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis), nucleus praeopticus magnocellularis, substantia innominata and the nucleus basatis of Meynert. Mesulam and coworkers referred to these nuclei as the Ch 1-4 regions of the basal forebrain (Mesulam et al., 1983a,b; Mesulam and Geula, 1988). However, as already mentioned by Semba et al. (1988) and discussed by a group of neuroscientists (see Butcher and Semba, 1989), the 'Ch' nomenclature applies best in primates but is not applicable in other species. As in the rat (Schober et al., 1988), the cellular band of the nucleus tractus diagonalis also forms a continuum in the raccoon basal forebrain. Therefore, based on CHAT° immunocytochemical data alone, the description of a separate medial septal nucleus as recognized in other studies (Bigl et al., 1982; Mesulam et al., 1983a,b; Saper, 1984; Ishikawa and Hirata, 1986; Vincent and Reiner, 1987) does not seem to us to be justified. In contrast, the delineation of the nucleus praeopticus magnocellularis, which is often considered as the lateral part of the horizontal limb of the diagonal band in other species (Rye et al., 1984; Zfiborszky et al., 1986), seemed to be a distinct nuclear region in this species because the nucleus praeopticus magnocellularis could be clearly distinguished from the pars horizontalis of the nucleus tractus diagonalis. This was mainly due to the distinctive size and the irregular orientation of the magnocellular preoptic neurons. In comparison with the nucleus praeopticus magnocellularis, the ChAT-immunoreactive neurons contained in the substantia innominata were loosely arranged in a pattern similar to that found in rodents, where it is often referred to as a component or a partial equivalent of the nucleus basalis of Meynert (Bigl et al., 1982; Woolfet al., 1983; Saper, 1984; Sofroniew et al., 1987; Schober et al., 1988). In Nissl-stained sections of the rat basal forebrain, clear nuclear boundaries of the nucleus basalis are
absent (Gorry, 1963; McKinney et al., Rye et al., 1984; Schober et al., 1988). In the raccoon, there is a clearly differentiated band of cells medially and ventrolaterally attached to, and partly surrounding, the pallidum. These groups of neurons correspond to the nucleus basalis of Meynert, which could be recognized in Nissl-stained sections, but which was easier to delineate after immunocytochemical detection of CHAT. By comparing Nissl- and ChAT-stained preparations, it was evident in our study that all of the basal forebrain cell groups in the raccoon contain, to a varying degree, non-cholinergic neurons. In the diagonal band complex and in the magnocellular preoptic nucleus a large portion, or even the majority of neurons, may be GABAergic projection neurons, a situation which has been documented in the rat (K6hler et al., 1984; Brashear et al., 1986; Onteniente et al., 1986; Z~tborszky et al., 1986) and cat (Fisher et al., 1988; Freund and Antal, 1988: Freund and Meskenaite, 1992).
ACKNOWLEDGEMENTS The authors are grateful to Mrs H Gruschka, Mrs M. Schmidt and Mrs M. Volkmann for excellent technical assistance. We thank Professor Dr M. Milder, NeurologicalClinic, University of G6ttingen, for generouslyprovidingthe monoclonalantibody to CHAT.
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Professor Dietmar Biesold died on 29 May, 1991, in Leipzig. Our work on the basaljorebrain of the raccoon was initiated by him. Accepted 22 June 1992
