THE JOURNAL OF COMPARATIVE NEUROLOGY 325~379-387 (1992)
Tyrosine Hydroxylase-Immunoreactive Neurons in the Nucleus Basalis of the Common Marmoset (CaZZithrixjacchus) L. WISNIOWSKI, R.M. RIDLEY, H.F. BAKER, AND A. FINE Neuroscience Institute and Department of Physiology and Biophysics, Dalhousie University Faculty of Medicine, Halifax, Nova Scotia, Canada B3H 4H7 (L.W.,A.F.); Clinical Research Centre, Northwick Park, Harrow, United Kingdom (R.M.R., H.F.B.)
ABSTRACT In the course of characterizing the distribution of putative catecholaminergic neurons in the brain of the common marmoset, we encountered a population of such cells in the basal forebrain. Tyrosine hydroxylase-immunoreactive neurons are abundant within the nucleus basalis magnocellularis throughout its entire rostrocaudal extent, but not in other cholinergic basal forebrain nuclei. Most tyrosine hydroxylase-immunoreactive cells are large and multipolar. Double staining with antibodies to choline acetyltransferaseor nerve growth factor receptor confirmed that these tyrosine hydroxylase-immunoreactive neurons are cholinergic, and compose at least 40% of the nucleus basalis cholinergic cells. The presence of a catecholaminesynthesizing enzyme in the neurons that provide the major cholinergic input to the neocortex may have important consequences for cortical function, and may be relevant to the vulnerability of the nucleus basalis in certain neurodegenerative disorders. Q 1992 Wiley-Liss, Inc. Key words: acetylcholine, catecholamines, choline acetyltransferase, nerve growth factors, primates
The principal source of cholinergic afferents to the mammalian neocortex is the magnocellular neurons of the basal forebrain cholinergic nuclei (reviewed by Semba and Fibiger, '891, in particular the nucleus basalis magnocellularis (BM) and horizontal limb of the diagonal band of Broca (hDB), also referred to as Ch4 and Ch3, respectively (Mesulam et al., '83a,b). The importance of this projection for normal cortical function, particularly learning and memory, has been suggested by experimental studies in rat (Flicker et al., '83; Dunnett et al., '85; Fine et al., '85; Murray and Fibiger, '85) and monkey (Ridley et al., '85, '86). Moreover, degeneration of BM neurons and reductions in cerebral cortical cholinergic markers are seen in Alzheimer's disease and in Parkinson's disease with dementia (e.g., Davies and Maloney, '76; Bartus et al., '82; Mann and Yates, '83; Perry et al., '851, and may be correlated with cognitive deficits (reviewed by Collerton, '86). Acetylcholine may coexist with other neurotransmitters within the basal forebrain cholinergic nuclei. In primates, the neuropeptide galanin has been found within cholinergic BM and hDB neurons in owl monkeys (Melander and Staines, '86), rhesus monkeys (Walker et al., '891, and cebus monkeys (Kordower and Mufson, '901, and there are indications that gamma-aminobutyric acid (GABA)may be present in a small minority of cholinergic basal forebrain neurons in rats (Brashear et al., '86).
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1992 WILEY-LISS, INC.
Catecholamine-containing neurons have been detected by aldehyde-induced fluorescence in the rat (Ungerstedt, '71) and baboon (Satoh and Fibiger, '85) forebrain, but not within the magnocellular cholinergic basal forebrain nuclei. More recently, immunohistochemical detection of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, has provided a more sensitive means of detecting catecholaminergic neurons (Hokfelt et al., '76). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons have been identified in the olfactory and pyriform cortices of Talapoin and African green monkeys (Kohler et al., '831, the subfornical organ of cebus monkeys (Kordower et al., '88b1, the striatum of macaque monkeys (Dubach et al., '87) and rats (Tashiro et al., '891, and human neocortex (Hornung et al., '89), paraolfactory cortex, anterior olfactory nucleus (Gaspar et al., '851, supraoptic nucleus, and paraventricular nucleus (Gaspar et al., '85; Spencer et al., '85; Li et al., '88); none of these studies reported TH-IR neurons within the cholinergic basal forebrain nuclei. Tyrosine hydroxylase immunoreactivity has been found within cholinergic neurons in rat arcuate and periarcuate nuclei Accepted July 19,1992. Address reprint requests to Dr. A. Fine, Neuroscience Institute and Department of Physiology and Biophysics, Dalhousie University Faculty of Medicine, Halifax, Nova Scotia, Canada B3H 4H7.
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(Tinner et al., '89) as well as the dorsal motor nucleus of the vagus (Armstrong et al., '90). Within the cholinergic basal forebrain, TH staining has been observed in a subpopulation of magnocellular neurons in the hamster (Vincent, '881, and medium-sized multipolar TH-IR neurons were seen after colchicine pretreatment in cats (Kitahama et al., '90); colocalization of TH and choline acetyltransferase (ChAT)immunoreactivity has been found in a small proportion of neurons of the ferret BM (Henderson, '87). Here, we provide evidence of widespread TH immunoreactivity colocalized with cholinergic markers in BM neurons of marmoset monkeys.
MATERIALS AND METHODS Our observations were made in 14 marmosets (Cullithrix jucchus) of either sex (13 adults, 300-350 g, and 1 newborn). Seven of these animals were normal, and seven had been rendered chronically parkinsonian 3-9 months previously by intraperitoneal injection of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine(MPTP, cumulative dose, 11.3 mgikg over 3 days) as part of an independent experiment (Fine et al., '88). Following pentobarbital overdose, the animals were perfused transcardially with heparinized phosphate-buffered saline (PBS) followed by phosphatebuffered 4% paraformaldehyde. Their brains were removed and transferred to 30% sucrose in PBS until fully submerged. Coronal frozen sections 40 pm thick were cut on a sliding-knife microtome and collected in wells containing PBS. Sequential sections were stained for the immunohistochemical localization of TH or C U T , or for the colocalization of TH with ChAT or nerve growth factor receptor (NGFr).
were washed thoroughly and incubated overnight at 4°C in biotinylated goat anti-rabbit antiserum (Vector; diluted 1:20).Sections were again washed thoroughly and incubated with avidinibiotinyl-peroxidase complex (ABC; Vector) for 2 hours; bound immunoperoxidase complex was visualized with diaminobenzidine (0.5 mgiml in phosphate buffer). After staining, sections were dehydrated through graded alcohols, cleared with xylene, coverslipped in DPX mountant, and observed by brightfield microscopy. Control sections were processed in the same manner, except that primary antisera were omitted. For visualization of possible colocalization of TH with ChAT or NGFr, we used fluorescence immunohistochemistry on sections of normal marmoset brain. After incubation with primary antibodies against TH and ChAT or NGFr, sections were washed thoroughly in PBS and incubated overnight at 4°C in the dark with rhodamine-conjugated goat anti-rabbit IgG and fluorescein-conjugated goat antimouse (Pierce Chemicals), diluted 1:20. Sections were then rinsed in PBS and coverslipped in glycerol/PBS (1:l by volume). Independent, sequential incubations in each primary and corresponding secondary antibody yielded identical results. Immunofluorescence was observed by standard epifluorescence microscopy and by dual-channel confocal laser scanning microscopy (BioRad MRCSOO), with standard rhodamine and fluorescein filters. Control sections were incubated and observed as described above, except that either one of the primary antisera was omitted. The nonidentical patterns of immunofluorescence observed at the alternate wavelengths provided additional control against the possibility of filter breakthrough (see Results, below).
Immunohistochemistry and immunofluorescence All immunohistochemical procedures were performed on free-floating sections according to, previously described procedures (Fine et al., '88). Sections were incubated for 30 minutes in 0.3% hydrogen peroxide to reduce endogenous peroxidase activity, washed thoroughly, and incubated for 4 hours in PBS containing 0.3% Triton X-100and 1%normal serum of the same species as the secondary antibody. Sections were then incubated with primary and secondary antibodies diluted in a similar PBSiTritoninormal serum solution. Rabbit antiserum directed against bovine adrenal medullary TH was obtained from EugeneTech Intl. (Allendale, NJ), and has been shown to react specifically with TH in diverse species including rat (Armstrong et al., '81)and marmoset (Waters et al., '87). The anti-ChAT antibody we used was the well-characterized monoclonal antibody AB8 (Wainer et al., '851, diluted 1:ZOO. The anti-NGFr antibody we used was the mouse monoclonal antibody ME20.4, raised against human NGFr (Ross et al., '84) and deposited with the American Type Culture Collection as clone 200-3G6-4.This antibody binds the low-affinity subunit of nerve growth factor receptor, which appears to be a component of both high- and low-affinity receptors (Radeke et al., '87). Anti-NGFr antibody was used as the undiluted hybridoma supernatant. For single-label visualization of TH-IR neurons only, imrnunohistochemistry was performed by using the avidinbiotinyl peroxidase method. After incubation in primary antiserum (1:2,000 dilution) for 48 hours at 4"C, sections
Abbreuiations AADC AAm AL ALH BAm BM CAC CC Cd CeAm
CUT CI
cs
DB
GLD GP IAm LAm MAm MEP MIP MPTP NGFr PC Put RT SI so ST TH TL TO VA
VIII ZI
aromatic amino acid decarboxylase area anterior amygdalae ansa lenticuiaris area lateralis hypothalami nucleus basalis amygdalae nucleus basalis magnocellularis (Meynert) commissura anterior cerebri corpus callosum nucleus caudatus nucleus centralis amygdalae choline acetyltransferase capsula interna claustrum nucleus fasciculi diagonalis Brocae, horizontal limb corpus geniculatum laterale dorsale globus pallidus cellulae intercalatae amygdalae nucleus lateralis amygdalae nucleus medialis arnygdalae lamina medullaris externa pallidi lamina medullaris interna pdlidi 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nerve growth factor receptor pedunculus cerebri putamen nucleus reticularis thalami substdntia innominata nucleus supraopticus hypothalami stria terminalis tyrosine hydroxylase nucleus tuberis lateralis tractus opticus nucleus ventralis anterior thalami ventriculus tertius zona incerta
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Analysis The cell bodies of TH-IR cells in randomly selected sections throughout the rostrocaudal extent of the BM were counted at x 100 total magnification; higher magnification was used as necessary to resolve closely apposed or overlapping neurons. Subsequently, coverslips were removed, sections counterstained with cresyl violet for Nissl substance, and the large hyperchromic neurons of the nucleus basalis counted. Counting error was reduced to less than 1%by marking cell bodies with the aid of a camera lucida. Split cell error was corrected according to Abercrombie's formula (Konigsmark, '70). The incidence of TH-IR neurons was expressed as a proportion of BM neurons. Cell size was estimated to the nearest 5 pm with the aid of a calibrated ocular micrometer. Brain structures were identified according to the atlas of Stephan et al. ('80).
RESULTS Cells with TH-like immunoreactivity were seen in normal monkeys within the characteristic catecholaminergic regions previously identified in rats (Hokfelt et al., '76) and marmosets (Waters et al., '87), including substantia nigra pars cornpacta, ventral tegmental area, and paraventricular hypothalamus. TH-IR neurons were greatly reduced in number in the ventral mesencephalon of MPTP-treated marmosets, as previously described (Waters et al., '87). In addition to this expected pattern, numerous intensely TH-IR cells were also seen unexpectedly in the basal forebrain of both normal and MPTP-treated animals (Fig. 1).These cells were generally large (perikaryon diameter, 20-25 km) and multipolar, although spindle-shaped, bipolar morphologies were also encountered (Fig. 2A). The distribution of the TH-IR cells was identical to that of the cholinergic neurons of the BM (Ch4 of Mesulam et al., '83a) (Fig. 2B) in all its subdivisions, as previously mapped in the marmoset (Everitt et al., '88) and cebus monkey (Kordower et al., '89). They extended from a level slightly rostral to the anterior commissure through the full extent of the BM, including the external and internal laminae medularis of the globus pallidus, caudally to the rostral limit of the dorsal lateral geniculate nucleus (Fig. 3). Fibres extended from these cells into the adjacent globus pallidus and striatum, although their full extent and terminations could not be determined. These fibres were most easily distinguished in MPTP-treated animals (as in Fig. 31, where they were not obscured by the massive nigrostriatal projection; however, the possibility cannot be excluded that some of these fibres originated from mesencephalic (or other nonBM) catecholaminergic neurons that survived exposure to MPTP. TH-IR cells were not seen in the other components of the cholinergic basal forebrain system, i.e., within the medial septa1 nucleus (Chl), vertical limb of the diagonal band of Broca (Ch21, or hDB (Ch3). Ch3 is the least distinct of the cholinergic nuclei in the marmoset (Everitt et al., '88), and at its most posterior extent overlaps with the anterolateral and anteromedial subdivisions of Ch4). Thus, in the absence of retrograde-labelling studies, the possibility cannot be excluded that the TH-IR basal forebrain cells include a subgroup of posterior Ch3 neurons. Cell counts of TH-stained, cresyl violet-counterstained 2.5% (s.e.m.1 of BM neurons sections revealed that 43 were TH-IR; this percentage may be only a lower limit, for reasons discussed below. No significant difference was seen between normal and MPTP-treated animals with respect to
*
Fig. 1. TH-immunoreactive neurons in the marmoset BM. Lowmagnification micrograph of the basal forebrain, counterstained with cresyl violet. The darkly stained TH-IR cells are distributed throughout the normal extent of the marmoset BM (Everitt et al., '88). Scale bar = 500 p m .
distribution or proportion of TH-IR neurons; thus cell counts of both groups were pooled for this calculation. To establish whether these TH-IR cells were indeed the cholinergic neurons of the BM, we investigated possible colocalization of TH-immunoreactivity with cholinergic markers (Fig. 4). All TH-IR BM neurons were also ChAT-IR; however, some C U T - I R cells were seen that were not immunoreactive for TH (Fig. 4A,B). Because C U T - I R was not always robust, we also examined the distribution of NGFr immunoreactivity. By using antibodies directed against the low-affinity nerve growth factor receptor (like those used here), NGFr-IR has been shown to colocalize with CUT-IR in the basal forebrain of cebus monkeys and humans (Hefti et al., '86; Kordower et al., '88a; Allen et al., '89; Mufson et al., '89) and thus to be a reliable marker for cholinergic basal forebrain neurons; indeed, we found the distribution of NGFr-IR neurons to be identical to that of the cholinergic BM in the marmoset as well (data not shown). Again, all TH-IR BM neurons were also NGFr-IR (Fig. 4C-F), while a small fraction of NGFr-IR cells were not immunoreactive for TH. Doubly stained TH-IRI NGFr-IR cells appeared to compose substantially more than the 43% of BM neurons suggested by our cresyl
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Fig. 2. A Higher magnification micrograph of TH-IR cells in the normal marmoset BM, without counterstain. Multipolar TH-IR neurons have the typical appearance and distribution of magnocellular neurons ofthe cholinergic BM, as seen in B. B. ChAT-IR neurons in the marmoset BM in a section adjacent to that shown in A. Scale bar = 100 pm.
violet/TH cell counts; this discrepancy may reflect the inclusion of noncholinergic neurons among the "BM" cells counted in the cresyl violet-counterstained sections. The results of these colocalization studies demonstrate that a large proportion of cholinergic neurons of the marmoset BM express TH immunoreactivity.
DISCUSSION Until nucleic acid hybridization studies confirm that TH messenger RNA is synthesized in BM neurons, the possibility must be considered that the TH immunoreactivity described here reflects cross-reactivity with unknown molecular species. The failure to observe TH-IR neurons in the BM of many other species may simply reflect their lack of TH; it is also possible that alternative gene splicing in those cells (Grima et al., '87; Kaneda et al., '87) yields TH isoforms that are not recognized by available antibodies. Indeed, TH staining has been observed in a subpopulation of BM neurons in the hamster (Vincent, '88),cat (Kitahama et al., 'go), and ferret (Henderson, '871, and we have also found TH immunoreactivity colocalized in NGFr-IR and C U T - I R neurons in the raccoon BM (data not shown). We have looked in the marmoset BM for immunohistochemical evidence of other enzymes involved in catecholamine synthe-
sis, and found no staining for dopamine-beta-hydroxylase and only weak staining for aromatic amino acid decarboxylase (AADC). Central nervous system neurons that express TH immunoreactivity either stably or transiently, while lacking catecholamines or catecholamine-synthesizing enzymes, have been identified in a number of species (Jaeger et al., '84; Berger et al., '85; Gaspar et al., '87; Meister et al., '88; Okamura et al., '88b; Kitahama et al., '90; Nagatsu et al., '90; Vincent and Hope, '90). It is also possible that authentic TH may be nonfunctional, due, for example, to aberrant postsynthetic processing or to inadequate levels of essential cofactors such as tetrahydrobiopterin. Nevertheless, the presence of TH immunoreactivity in the cholinergic neurons of the marmoset BM suggests that these neurons may contain functional TH, and are thus able to synthesize L-DOPA from tyrosine. Such appears to be the case in the rat arcuate nucleus, where TH-IR cells that lack AADC immunoreactivity are immunoreactive for L-DOPA (Okamura et al., '88a). The possibility that L-DOPA may function as a neurotransmitter in such cells has been suggested by Vincent and Hope ('90): evoked release of endogenous L-DOPA from striatal slices has been reported (Goshima et al., '881, and iontophoretically applied L-DOPA can affect electrical activity in the spinal cord (Bras et al., '88). The possible direct effects of L-DOPA upon neocortical
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Fig. 4. Double-label immunofluorescence micrographs of BM neurons in a normal marmoset, showing colocalization of TH immunoreactivity (B, D, F) within ChAT-IR (A) and NGFr-IR (C, E) neurons. All the TH-IR neurons in Bare also ChAT-IR in A, other ChAT-IR neurons
in the field (compare arrows) are not TH-IR. Similarly, all TH-IR neurons in D and F are NGFr-IR in C and E, respectively, while other NGFr-IR neurons in those fields are TH-negative. Scale bar = 100 Km (A-D), 150 Km (E, F).
neurons, in the marmoset or other species, remain to be explored. Certain neural crest-derived cells, including sympathetic neurons, ciliary neurons, and chromaffin cells, can express
either cholinergic or catecholaminergic phenotype (Doupe et al., '85; Landis, '88), under the control, at least in part, of the targets they innervate (Schotzinger and Landis, '88). This control may be mediated by factors secreted by target
TYROSINE HYDROXYLASE IN MARMOSET NUCLEUS BASALIS cells (Patterson and Chun, '771, some of which have been at least partially characterized (Fukada, '85; Rao and Landis, 'go), and possibly including NGF (Kessler, '85). Expression of TH in chromaffin cells is also under trans-synaptic control (Kvetnansky et al., '70; Otten et al., '73; Gauthier et al., '79). TH expression can also be induced in cerebral cortical neurons following intracerebral transplantation (Park et al., '86; Herman et al., '88),perhaps also as a result of diffusible target-derived factors (Iacovitti et al., '89). Thus neurotransmitter-phenotypicplasticity may be a property of certain central, as well as peripheral, neurons. It will be of interest to determine whether TH immunoreactivity in BM neurons can be influenced by various pharmacological or surgical manipulations (Henderson, '87); the present observations indicate that at least one such manipulation, MPTP-induced degeneration of dopaminergic input to the adjacent neostriatum, has no gross effect on the appearance of TH-IR BM neurons. Finally, it may be noted that numbers of NGFr-IRI CUT-IR neurons within subfields of the BM, but not of other cholinergic forebrain nuclei, are reduced in Parkinson's disease (Mufson et al., '91); the distribution of affected subfields is reminiscent of the distribution of TH-IR BM neurons in the marmoset as described here. TH-IR BM neurons have not been reported in the human brain, although TH-IR cells assigned to hypothalamic nuclei (Li et al., '88; Panayotacopoulou et al., '91) may have included BM neurons. If the degeneration of dopaminergic substantia nigra neurons in Parkinson's disease is in some way a consequence of their catecholaminergic phenotype (as, for example, through the generation of reactive semiquinone metabolites), the partial expression of this phenotype in BM cells might contribute to their degeneration in that disease. It will thus be of interest to determine, by more sensitive immunohistochemical methods as well as nucleic acid hybridization, whether the pattern of TH expression in the marmoset BM is also present in the human brain.
ACKNOWLEDGMENTS We thank C. Leopold and R. Poirier for excellent technical assistance, and Drs. D.D. Rasmusson, K. Semba, and S.P. Hunt for their valuable comments. This study was supported by the Medical Research Councils of Canada and Great Britain, the Parkinson Foundation of Canada, the Canadian Network of Centres of Excellence in Neural Recovery and Regeneration, and the Whitaker Foundation. L.W. was supported by the Dalhousie Medical Alumni Association.
LITERATURE CITED Allen, S.J., D. Dawbarn, M.G. Spillantini, M. Goedert, G.K. Wilcock, T.H. Moss, and F.M. Semenenko (1989)Distribution of B-nerve growth factor receptors in human basal forebrain. J. Comp. Neurol. 289:626-640. Armstrong, D.M., L. Manley, J.W. Haycock, and L.B. Hersh (1990) Colocalization of choline acetyltransferase and tyrosine hydroxylase within neurons of the dorsal motor nucleus of the vagus. J. Chem. Neuroanat. 3:133-140. Armstrong, D.M., V.M. Pickel, T.H. Joh, D.J. Reis, and R.J. Millerd (1981) Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius of rat brain. J. Comp. Neurol. 196:505-517.
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Bartus, R.T., R.L. Dean, B. Beer, and A.S. Lippa (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 21 7:408-417. Berger, B., C. Verney, P. Gaspar, and A. Febvret (1985)Transient expression of tyrosine hydroxylase immunoreactivity in some neurons of the rat neocortex duringpostnatal development. Dev. Brain Res. 23:141-144. Bras, H., P. Cavallari, and E. Jankowska (1988) An investigation of local actions of iontophoretically applied DOPA in the spinal cord. Exp. Brain Res. 71:447-449. Brashear, H.R., L. Zaborsky, and L.Heimer (1986) Distribution of GABAergic and cholinergic neurons in the rat diagonal band. Neuroscience 17:439-451. Collerton, D. (1986) Cholinergic function and intellectual decline in Alzheimer's disease. Neuroscience 19:l-28. Danes, P., and A.J.F. Maloney (1976) Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet i~:1403. Doupe, A.J., S.C. Landis, and P.H. Patterson (1985) Environmental influences in the development of neural crest derivatives: Glucocorticoids, growth factors and chromaffin cell plasticity. J. Neurosci. 5:2119-2142. Dubach, M., R. Schmidt, D. Kunkel, D.M. Bowden, R. Martin, and D.C. German (1987) Primate neostriatal neurons containing tyrosine hydroxylase: Immunohistochemical evidence. Neurosci. Lett. 75:205-2 10. Dunnett, S.B., G. Toniolo, A. Fine, C.N. Ryan, A. Bjorklund, and S.D. Iversen ( 1985) Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis magnocellularis. 11. Sensorimotor and learning impairments. Neuroscience 16:787-797. Everitt, B.J., T.E.Sirkia, A.C. Roberts, G.H. Jones, andT.W. Robbins (1988) Distribution and some projections of cholinergic neurons in the brain of the common marmoset, Callithrir jacchus. J. Comp. Neurol. 271:533558. Fine, A,, S.B. Dunnett, A. Bjorklund, and S.D. Iversen (1985) Cholinergic ventral forebrain grafts into the neocortex improve passive avoidance memory in a rat model of Alzheimer disease. Proc. Natl. Acad. Sci. USA 825227-5230. Fine, A., S.P. Hunt, W.H. Oertel, M. Nomoto, P.N. Chong, A. Bond, C. Waters, J.A. Temlett, L. Annett, S. Dunnett, P. Jenner, and C.D. Marsden (1988) Transplantation of embryonic marmoset dopaminergic neurons to the corpus striatum of marmosets rendered parkinsonian by l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine. Prog. Brain Res. 78t479489. Flicker, C., R.L. Dean, D.L. Watkins, S.K. Fisher, and R.T. Bartus (1983) Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol. Biochem. Behav. I8:973-981. Fukada, K. (1985) Purification and partial characterization of a cholinergic neuronal differentiation factor. Proc. Natl. Acad. Sci. USA 8287958799. Gaspar, P., B. Berger, C. Alvarez, A. Vigny, and J.P. Henry (1985) Catecholaminergic innervation of the septa1 area in man: Immunocytochemical study using TH and DBH antibodies. J. Comp. Neurol. 241:12-33. Gaspar, P., B. Berger, A. Febvret, A. Vigny, M. Krieger-Poulet, and C. Borri-Voltattorni (1987 Tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex: A novel catecholaminergic group? Neurosci. Lett. 80.957-262. Gauthier, S., J.-P. Gagner, and T.L. Slourkes (1979) Role of descending spinal pathways in the regulation of adrenal tyrosine hydroxylase. Exp. Neurol. 66:42-54. Goshima, Y., T. Kubo, and Y. Misu (1988) Transmitter-like release of endogenous 3,4-dihydroxyphenylalaninefrom rat striatal slices. J. Neurochem. 50: 1725-1 730. Grima, B., A. Lamouroux, C. Boni, J.F. Julien, F. Javoy-Agid, and J. Mallet (1987) A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics. Nature 326:707-711 Hefti, F., J. Hartikka, P. Salvaterra, W.J. Weiner, and D. Mash (1986) Localization of nerve growth factor receptors on cholinergic neurons of the human basal forebrain. Neurosci. Lett. 69:275-281. Henderson, Z. (1987) A small proportion of cholinergic neurones in the nucleus basalis magnocellularis of ferret appear to stain positively for tyrosine hydroxylase. Brain Res. 412:363-369. Herman, J.P., N. Abrous, A. Vigny, J. Dulluc, and M. LeMoal (1988) Distorted development of intraeerebral grafts: Long-term maintenance of tyrosine hydroxylase-containing neurons in grafts of cortical tissue. Dev. Brain Res. 40:81-88. Hokfelt, T., 0. Johansson, K. Fuxe, M. Goldstein, and D. Park (1976)
386 Imrnunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain. I. Tyrosine hydroxylase in the mes- and diencephalon. Med. Bid. 54t427-453. Hornung, J.-P., I. Tork, and N. De Triboler (1989) Morphology of tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex. Exp. Brain Res. 76:lZ-20. Iacovitti, L., M.J. Evinger, T.H. Joh, and D.J. Reis (1989) A muscle-derived factor(s1 induces expression of a catecholamine phenotype in neurons of cultured rat cerebral cortex. J. Neurosci. 9:3529-3537. Jaeger, C.B., D.A. Ruggiero, V.R. Albert, D.H. Park, T.H. Joh, andD.J. Reis (1984)Aromatic L-amino acid decarboxylase in the rat brain: Immunocytochemical localization in neurons of the brain stem. Neuroscience I k691-713. Kaneda, N., K. Kobayashi, H. Ichinose, F. Kishi, A. Kanazawa, Y. Kurosawa, K. Fujita, and T. Nagatsu (1987) Isolation of a novel cDNA clone for human tyrosine hydroxylase: Alternative RNA splicing produces four kinds of mRNA from a single gene. Biochem. Biophys. Res. Commun. 146:971-975. Kessler, J.A. (1985) Parasympathetic, sympathetic and sensory interactions in the iris: Nerve growth factor regulates cholinergic ciliary ganglion innervation in vivo. J. Neurosci. 5.2719-2735. Kitahama, K., M. Geffard, H. Okamura, I. Nagatsu, N. Mans, and M. Jouvet (1990) Dopamine- and DOPA-immunoreactive neurons in the cat forebrain with reference to tyrosine hydroxylase-immunohistochemistry. Brain Res. 518:83-94. Kohler, C., B.J. Everitt, J. Pearson, and M. Goldstein (1983) Immunohistochemical evidence for a new group of catecholamine-containing neurons in the basal forebrain of the monkey. Neurosci. Lett. 37:161-166. Konigsmark, B.W. (1970) Methods for the counting of neurons. In W.J.H. Nauta and S.O.E. Ebbesson (eds): Contemporary Research Methods in Neuroanatomy. New York: Springer-Verlag,pp. 315-337. Kordower, J.H., R.T. Bartus, M. Bothwell, G. Schatteman, and D.M. Gash (1988a) Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus upella): Distribution, morphology, and colocalization with cholinergic enzymes. J. Comp. Neurol. 277:465486. Kordower, J.H., R.T. Bartus, F.F. Marciano, andD.M. Gash (1989)Telencephalic cholinergic system of the New World monkey (Cebus upella): Morphological and cytoarchitectonic assessment and analysis of the projection to the amygdala. J. Comp. Neurol. 279,528-545. Kordower, J.H., and E.J. Mufson (1990) Galanin-like irnnlunoreactivity within the primate basal forebrain: Differential staining patterns between humans and monkeys. J. Comp. Neurol. 294:281-292. Kordower, J.H., J.R. Sladek, Jr., M.S. Fiandaca, G. Bing, and D.M. Gash (1988h) Tyrosine-hydroxylase immunoreactive somata within the primate subfornical organ: Species specificity. Brain Res. 461:221-229. Kvetnansky, R., V.K. Weise, and I.J. Kopin (1970) Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-N-methyl-transferase by repeated immobilization of rats. Endocrinology 87:744-749. Landis, S.C. (1988) Neurotransmitter plasticity in sympathetic neurons and its regulation by environmental factors in vitro and in vivo. In A. Bjorklund, T. Hokfelt, and C. Owrnan (eds): Handbook of Chemical Neuroanatomy, Vol. 6 : The Peripheral Nervous System. Amsterdam: Elsevier, pp. 65-115. Li, Y.W., G.M. Halliday, T.H. Joh, L.B. Geffen, and W.W. Blessing (1988) Tyrosine hydroxylase-containing neurons in the supraoptic and paraventricular nuclei of the adult human. Brain Res. 461:75-86. Mann, D.M.A., and P.O. Yates (1983) Pathological basis for neurotransmitter changes in Parkinson’s disease. Neuropathol. Appl. Neurobiol. 9:3-19. Meister, B., T. Hokfelt, H.M.W. Steinbusch, G. Skagerberg, 0. Lindvall, M. Geffard, T.H. Joh, A.C. Cuello, and M. Goldstein (1988) Do tyrosine hydroxylase-immunoreactive neurons in the ventral arcuate nucleus produce dopamine or only L-DOPA? J. Chem. Neuroanat. 1:59-64. Melander, T., and W.A. Staines (1986) A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complex and in acetylcholinesterase-containing fibers and varicosities within the hippocarnpus in the owl monkey (Antus tciuirgulus). Neurosci. Lett. 68.17-22. Mesulam, M.M., E.J. Mufson, A.I. Levey, and B.H. Wainer (1983a) Cholinergic innervation of cortex by basal forebrain: Qtochemistry and cortical connection for the septa1 area, diagonal band nuclei, nucleus basalis (substantia innominata) and hypothalamus in the rhesus monkey. J. Comp. Neurol. 214:170-197. Mesulam, M.M., E.J. Mufson, B.H. Wainer, and A.I. Levey (1983b) Central
L. WISNIOWSKI ET AL. cholinergic pathways in the r a t An overview based on an alternative nomenclature (Chl-Ch6). Neuroscience 10.1 185-1201. Mufson, E.J., M. Bothwell, L.B. Hersh, and J. Kordower (1989) Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: Colocalization with cholinergic neurons, J. Comp. Neurol. 285:196-217. Mufson, E.J., L.N. Presley, and J.H. Kordower (1991) Nerve growth factor receptor immunoreactivity within the nucleus basalis (Ch4) in Parkinson’s disease: Reduced cell numbers and co-localization with cholinergic neurons. Brain Res. 539:19-30. Murray, C.L., and H.C. Fibiger (1985) Learning and memory deficits after lesions of the nucleus basalis magnocellularis: Reversal by physostigmine. Neuroscience 14: 1025-1032. Nagatsu, I., K. Komori, T. Takeuchi, M. Sakai, K. Yarnada, and N. JSarasawa (1990) Transient tyrosine hydroxylase-immunoreactive neurons in the region of the anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine. Brain Res. 511.55-62. Okamura, H., K. Kitahama, N. Mans, Y. Ibata, M. Jouvet, and M. Geffard (1988a) L-DOPA immunoreactive neurons in the rat hypothalamic tuberal region. Neurosci. Lett. 95;42-46. Okamura, H., K. Kitahama, 1. Nagatsu, andM. Geffard (1988bi Comparative topography of dopamine- and tyrosine hydroxylase-immunoreactive neurons in the rat arcuate nucleus. Neurosci. Lett. 95:347-353. Otten, U., U. Paravicini, F. Oesch, and H. Thoenen (1973) Time requirement for the single steps of transynaptic induction of tyrosine hydroxylase in the peripheral nervous system. Naunyn Schmiedebergs Arch. Pharmacol. 280:117-127. Panayotacopoulou, M.T., R. Guntern, C. Bouras, M.R. Issorides, and J. Constantinidis (1991)Tyrosine hydroxylase-immunoreactive neurons in paraventricular and supraoptic nuclei of the human brain demonstrated by a method adapted to prolonged formalin fixation. J. Neurosci. Methods 39f39-44. Park, J.K., T.H. Joh, and F.F. Ebner (1986) Tyrosine hydroxylase is expressed by neocortical neurons after transplantation. Proc. Natl. Acad. Sci. USA 83:7495-7498. Patterson, P.H., and L.L.Y. Chun (1977) Induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium. Dev. Biol. 56:263-280. Perry, E.K., M. Curtis, D.J. Dick, J.M. Candy, J.R. Atack, C.A. Bloxham, G. Blessed, A. Fairbairn, B.E. Tomlinson, and R.H. Perry (1985) Cholinergic correlates of cognitive impairment in Parkinson’s disease: Comparison with Alzheimer’s disease. J. Neurol. Neurosurg. Psychiatry 48:413421. Radeke, M.J., T.P. Misko, C. Hsu, L. Herzenberg, and E.M. Shooter (1987) Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 325:593-597. Rao, M.S., and S.C. Landis (1990) Characterization of a target-derived neuronal cholinergic differentiation factor. Neuron 5899-910. Ridley, R.M., H.F. Baker, B.S. Drewett, and J.A. Johnson (1985) Effects of ibotenic acid lesions of the basal forebrain on serial reversal learning in the marmoset. Psychopharmacology 86:438449. Ridley, R.M., T.K. Murray, J.A. Johnson, and H.F. Baker (1986) Learning impairment following lesion of the basal nucleus of Meynert in the marmoset: Modification by cholinergic drugs. Brain Res. 376:108-116. Ross, A.H., P. Grob, M. Bothwell, D.E. Elder, C.S. Ernst, N. Marano, B.F.D. Ghrist, C.C. Slemp, M. Herlyn, B. Atkinson, and H. Koprowski (1984) Characterization of nerve growth factors in neural crest tumors using monoclonal antibodies. Proc. Natl. Acad. Sci. USA81:6681-6685. Satoh, K., and H.C. Fibiger (1985) Distribution of central cholinergic neurons in the baboon (Papiopupio). 11. A topographic atlas correlated with catecholamine neurons. J. Comp. Neurol. 236,215-233. Schotzinger, R.J., and S.C. Landis (1988) Cholinergic phenotype developed by noradrenergic sympathetic neurons after innervation of a novel cholinergic target in vivo. Nature 39.5637-639. Semba, K., and H.C. Fihiger (1989) Organization of central cholinergic systems. Prog. Brain Res. 79:37-63. Spencer, S., C.B. Saper, T. Joh, D.J. Reis, M. Goldstein, and J.D. Raese (1985) The distribution of catecholamine-containing neurons in the normal human hypothalamus. Brain Res. 328:73-80. Stephan, H., G. Baron, and W.K. Schwerdtfeger (1980) The Brain of the Common Marmoset (Cullithrtr jucchus): A Stereotaxic Atlas. Berlin: Springer-Verlag. Tashiro, Y., T. Sugimoto, T. Hattori, Y. Uemura, I. Nagatsu, H. Kikuchi, and
TYROSINE HYDROXYLASE IN MARMOSET NUCLEUS BASALIS
387
N. Mizuno (1989) Tyrosine hydroxylase-like immunoreactive neurons in Vincent, S.R., and B.T. Hope (1990) Tyrosine hydroxylase containing the striatum of the rat. Neurosci. Lett. 973-10. neurons lacking aromatic amino acid decarboxylase in the hamster brain. J. Comp. Neurol. 2952990-2998. Tinner, B., K. Fuxe, Ch. Kohler, L. Hersh, K. Andersson, A. Jansson, M. Wainer, B.H., A.I. Levey, D.B. Rye, M.-M. Mesulam,and E.J. Mufson (1985) Goldstein, and L.F. Agnati (1989) Evidence for the existence of a Cholinergic and non-cholinergic septohippocampal pathways. Neurosci. population of arcuate neurons costoring choline acetyltransferase and Lett. 54:45-52. tyrosine hydroxylase immunoreactivities in the male rat. Neurosci. Lett. 99:44-49. Walker, L.C., V.E. Koliatsos, C.A. Kitt, R.T. Richardson, A. Rokaeus, and D.L. Price (1989) Peptidergic neurons in the basal forebrain magnocelluUngerstedt, U. (1971) Stereotaxic mapping of the monoamine pathways in lar complex of the rhesus monkey. J. Comp. Neurol. 280:272-282. the rat brain. Acta Physiol. Scand. Suppl. 367:148. Waters, C.M., S.P. Hunt, P. Jenner, and C.D. Marsden (1987)An immunohisVincent, S.R. (1988) Distributions of tyrosine hydroxylase-, dopamine-Btochemical study of the acute and long-term effects of 1-methyl-4-phenylhydroxylase-, and phenylethanolamine-N-methyltransferase-immunore1,2,3,64etrahydropyridine in the marmoset. Neuroscience 23:1025active neurons in the brain of the hamster (Mesocricetus auratus). J. 1030. Comp. Neurol. 268.584-599.
Tyrosine Hydroxylase-Immunoreactive Neurons in the Nucleus Basalis of the Common Marmoset (CaZZithrixjacchus) L. WISNIOWSKI, R.M. RIDLEY, H.F. BAKER, AND A. FINE Neuroscience Institute and Department of Physiology and Biophysics, Dalhousie University Faculty of Medicine, Halifax, Nova Scotia, Canada B3H 4H7 (L.W.,A.F.); Clinical Research Centre, Northwick Park, Harrow, United Kingdom (R.M.R., H.F.B.)
ABSTRACT In the course of characterizing the distribution of putative catecholaminergic neurons in the brain of the common marmoset, we encountered a population of such cells in the basal forebrain. Tyrosine hydroxylase-immunoreactive neurons are abundant within the nucleus basalis magnocellularis throughout its entire rostrocaudal extent, but not in other cholinergic basal forebrain nuclei. Most tyrosine hydroxylase-immunoreactive cells are large and multipolar. Double staining with antibodies to choline acetyltransferaseor nerve growth factor receptor confirmed that these tyrosine hydroxylase-immunoreactive neurons are cholinergic, and compose at least 40% of the nucleus basalis cholinergic cells. The presence of a catecholaminesynthesizing enzyme in the neurons that provide the major cholinergic input to the neocortex may have important consequences for cortical function, and may be relevant to the vulnerability of the nucleus basalis in certain neurodegenerative disorders. Q 1992 Wiley-Liss, Inc. Key words: acetylcholine, catecholamines, choline acetyltransferase, nerve growth factors, primates
The principal source of cholinergic afferents to the mammalian neocortex is the magnocellular neurons of the basal forebrain cholinergic nuclei (reviewed by Semba and Fibiger, '891, in particular the nucleus basalis magnocellularis (BM) and horizontal limb of the diagonal band of Broca (hDB), also referred to as Ch4 and Ch3, respectively (Mesulam et al., '83a,b). The importance of this projection for normal cortical function, particularly learning and memory, has been suggested by experimental studies in rat (Flicker et al., '83; Dunnett et al., '85; Fine et al., '85; Murray and Fibiger, '85) and monkey (Ridley et al., '85, '86). Moreover, degeneration of BM neurons and reductions in cerebral cortical cholinergic markers are seen in Alzheimer's disease and in Parkinson's disease with dementia (e.g., Davies and Maloney, '76; Bartus et al., '82; Mann and Yates, '83; Perry et al., '851, and may be correlated with cognitive deficits (reviewed by Collerton, '86). Acetylcholine may coexist with other neurotransmitters within the basal forebrain cholinergic nuclei. In primates, the neuropeptide galanin has been found within cholinergic BM and hDB neurons in owl monkeys (Melander and Staines, '86), rhesus monkeys (Walker et al., '891, and cebus monkeys (Kordower and Mufson, '901, and there are indications that gamma-aminobutyric acid (GABA)may be present in a small minority of cholinergic basal forebrain neurons in rats (Brashear et al., '86).
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1992 WILEY-LISS, INC.
Catecholamine-containing neurons have been detected by aldehyde-induced fluorescence in the rat (Ungerstedt, '71) and baboon (Satoh and Fibiger, '85) forebrain, but not within the magnocellular cholinergic basal forebrain nuclei. More recently, immunohistochemical detection of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, has provided a more sensitive means of detecting catecholaminergic neurons (Hokfelt et al., '76). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons have been identified in the olfactory and pyriform cortices of Talapoin and African green monkeys (Kohler et al., '831, the subfornical organ of cebus monkeys (Kordower et al., '88b1, the striatum of macaque monkeys (Dubach et al., '87) and rats (Tashiro et al., '891, and human neocortex (Hornung et al., '89), paraolfactory cortex, anterior olfactory nucleus (Gaspar et al., '851, supraoptic nucleus, and paraventricular nucleus (Gaspar et al., '85; Spencer et al., '85; Li et al., '88); none of these studies reported TH-IR neurons within the cholinergic basal forebrain nuclei. Tyrosine hydroxylase immunoreactivity has been found within cholinergic neurons in rat arcuate and periarcuate nuclei Accepted July 19,1992. Address reprint requests to Dr. A. Fine, Neuroscience Institute and Department of Physiology and Biophysics, Dalhousie University Faculty of Medicine, Halifax, Nova Scotia, Canada B3H 4H7.
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(Tinner et al., '89) as well as the dorsal motor nucleus of the vagus (Armstrong et al., '90). Within the cholinergic basal forebrain, TH staining has been observed in a subpopulation of magnocellular neurons in the hamster (Vincent, '881, and medium-sized multipolar TH-IR neurons were seen after colchicine pretreatment in cats (Kitahama et al., '90); colocalization of TH and choline acetyltransferase (ChAT)immunoreactivity has been found in a small proportion of neurons of the ferret BM (Henderson, '87). Here, we provide evidence of widespread TH immunoreactivity colocalized with cholinergic markers in BM neurons of marmoset monkeys.
MATERIALS AND METHODS Our observations were made in 14 marmosets (Cullithrix jucchus) of either sex (13 adults, 300-350 g, and 1 newborn). Seven of these animals were normal, and seven had been rendered chronically parkinsonian 3-9 months previously by intraperitoneal injection of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine(MPTP, cumulative dose, 11.3 mgikg over 3 days) as part of an independent experiment (Fine et al., '88). Following pentobarbital overdose, the animals were perfused transcardially with heparinized phosphate-buffered saline (PBS) followed by phosphatebuffered 4% paraformaldehyde. Their brains were removed and transferred to 30% sucrose in PBS until fully submerged. Coronal frozen sections 40 pm thick were cut on a sliding-knife microtome and collected in wells containing PBS. Sequential sections were stained for the immunohistochemical localization of TH or C U T , or for the colocalization of TH with ChAT or nerve growth factor receptor (NGFr).
were washed thoroughly and incubated overnight at 4°C in biotinylated goat anti-rabbit antiserum (Vector; diluted 1:20).Sections were again washed thoroughly and incubated with avidinibiotinyl-peroxidase complex (ABC; Vector) for 2 hours; bound immunoperoxidase complex was visualized with diaminobenzidine (0.5 mgiml in phosphate buffer). After staining, sections were dehydrated through graded alcohols, cleared with xylene, coverslipped in DPX mountant, and observed by brightfield microscopy. Control sections were processed in the same manner, except that primary antisera were omitted. For visualization of possible colocalization of TH with ChAT or NGFr, we used fluorescence immunohistochemistry on sections of normal marmoset brain. After incubation with primary antibodies against TH and ChAT or NGFr, sections were washed thoroughly in PBS and incubated overnight at 4°C in the dark with rhodamine-conjugated goat anti-rabbit IgG and fluorescein-conjugated goat antimouse (Pierce Chemicals), diluted 1:20. Sections were then rinsed in PBS and coverslipped in glycerol/PBS (1:l by volume). Independent, sequential incubations in each primary and corresponding secondary antibody yielded identical results. Immunofluorescence was observed by standard epifluorescence microscopy and by dual-channel confocal laser scanning microscopy (BioRad MRCSOO), with standard rhodamine and fluorescein filters. Control sections were incubated and observed as described above, except that either one of the primary antisera was omitted. The nonidentical patterns of immunofluorescence observed at the alternate wavelengths provided additional control against the possibility of filter breakthrough (see Results, below).
Immunohistochemistry and immunofluorescence All immunohistochemical procedures were performed on free-floating sections according to, previously described procedures (Fine et al., '88). Sections were incubated for 30 minutes in 0.3% hydrogen peroxide to reduce endogenous peroxidase activity, washed thoroughly, and incubated for 4 hours in PBS containing 0.3% Triton X-100and 1%normal serum of the same species as the secondary antibody. Sections were then incubated with primary and secondary antibodies diluted in a similar PBSiTritoninormal serum solution. Rabbit antiserum directed against bovine adrenal medullary TH was obtained from EugeneTech Intl. (Allendale, NJ), and has been shown to react specifically with TH in diverse species including rat (Armstrong et al., '81)and marmoset (Waters et al., '87). The anti-ChAT antibody we used was the well-characterized monoclonal antibody AB8 (Wainer et al., '851, diluted 1:ZOO. The anti-NGFr antibody we used was the mouse monoclonal antibody ME20.4, raised against human NGFr (Ross et al., '84) and deposited with the American Type Culture Collection as clone 200-3G6-4.This antibody binds the low-affinity subunit of nerve growth factor receptor, which appears to be a component of both high- and low-affinity receptors (Radeke et al., '87). Anti-NGFr antibody was used as the undiluted hybridoma supernatant. For single-label visualization of TH-IR neurons only, imrnunohistochemistry was performed by using the avidinbiotinyl peroxidase method. After incubation in primary antiserum (1:2,000 dilution) for 48 hours at 4"C, sections
Abbreuiations AADC AAm AL ALH BAm BM CAC CC Cd CeAm
CUT CI
cs
DB
GLD GP IAm LAm MAm MEP MIP MPTP NGFr PC Put RT SI so ST TH TL TO VA
VIII ZI
aromatic amino acid decarboxylase area anterior amygdalae ansa lenticuiaris area lateralis hypothalami nucleus basalis amygdalae nucleus basalis magnocellularis (Meynert) commissura anterior cerebri corpus callosum nucleus caudatus nucleus centralis amygdalae choline acetyltransferase capsula interna claustrum nucleus fasciculi diagonalis Brocae, horizontal limb corpus geniculatum laterale dorsale globus pallidus cellulae intercalatae amygdalae nucleus lateralis amygdalae nucleus medialis arnygdalae lamina medullaris externa pallidi lamina medullaris interna pdlidi 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nerve growth factor receptor pedunculus cerebri putamen nucleus reticularis thalami substdntia innominata nucleus supraopticus hypothalami stria terminalis tyrosine hydroxylase nucleus tuberis lateralis tractus opticus nucleus ventralis anterior thalami ventriculus tertius zona incerta
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Analysis The cell bodies of TH-IR cells in randomly selected sections throughout the rostrocaudal extent of the BM were counted at x 100 total magnification; higher magnification was used as necessary to resolve closely apposed or overlapping neurons. Subsequently, coverslips were removed, sections counterstained with cresyl violet for Nissl substance, and the large hyperchromic neurons of the nucleus basalis counted. Counting error was reduced to less than 1%by marking cell bodies with the aid of a camera lucida. Split cell error was corrected according to Abercrombie's formula (Konigsmark, '70). The incidence of TH-IR neurons was expressed as a proportion of BM neurons. Cell size was estimated to the nearest 5 pm with the aid of a calibrated ocular micrometer. Brain structures were identified according to the atlas of Stephan et al. ('80).
RESULTS Cells with TH-like immunoreactivity were seen in normal monkeys within the characteristic catecholaminergic regions previously identified in rats (Hokfelt et al., '76) and marmosets (Waters et al., '87), including substantia nigra pars cornpacta, ventral tegmental area, and paraventricular hypothalamus. TH-IR neurons were greatly reduced in number in the ventral mesencephalon of MPTP-treated marmosets, as previously described (Waters et al., '87). In addition to this expected pattern, numerous intensely TH-IR cells were also seen unexpectedly in the basal forebrain of both normal and MPTP-treated animals (Fig. 1).These cells were generally large (perikaryon diameter, 20-25 km) and multipolar, although spindle-shaped, bipolar morphologies were also encountered (Fig. 2A). The distribution of the TH-IR cells was identical to that of the cholinergic neurons of the BM (Ch4 of Mesulam et al., '83a) (Fig. 2B) in all its subdivisions, as previously mapped in the marmoset (Everitt et al., '88) and cebus monkey (Kordower et al., '89). They extended from a level slightly rostral to the anterior commissure through the full extent of the BM, including the external and internal laminae medularis of the globus pallidus, caudally to the rostral limit of the dorsal lateral geniculate nucleus (Fig. 3). Fibres extended from these cells into the adjacent globus pallidus and striatum, although their full extent and terminations could not be determined. These fibres were most easily distinguished in MPTP-treated animals (as in Fig. 31, where they were not obscured by the massive nigrostriatal projection; however, the possibility cannot be excluded that some of these fibres originated from mesencephalic (or other nonBM) catecholaminergic neurons that survived exposure to MPTP. TH-IR cells were not seen in the other components of the cholinergic basal forebrain system, i.e., within the medial septa1 nucleus (Chl), vertical limb of the diagonal band of Broca (Ch21, or hDB (Ch3). Ch3 is the least distinct of the cholinergic nuclei in the marmoset (Everitt et al., '88), and at its most posterior extent overlaps with the anterolateral and anteromedial subdivisions of Ch4). Thus, in the absence of retrograde-labelling studies, the possibility cannot be excluded that the TH-IR basal forebrain cells include a subgroup of posterior Ch3 neurons. Cell counts of TH-stained, cresyl violet-counterstained 2.5% (s.e.m.1 of BM neurons sections revealed that 43 were TH-IR; this percentage may be only a lower limit, for reasons discussed below. No significant difference was seen between normal and MPTP-treated animals with respect to
*
Fig. 1. TH-immunoreactive neurons in the marmoset BM. Lowmagnification micrograph of the basal forebrain, counterstained with cresyl violet. The darkly stained TH-IR cells are distributed throughout the normal extent of the marmoset BM (Everitt et al., '88). Scale bar = 500 p m .
distribution or proportion of TH-IR neurons; thus cell counts of both groups were pooled for this calculation. To establish whether these TH-IR cells were indeed the cholinergic neurons of the BM, we investigated possible colocalization of TH-immunoreactivity with cholinergic markers (Fig. 4). All TH-IR BM neurons were also ChAT-IR; however, some C U T - I R cells were seen that were not immunoreactive for TH (Fig. 4A,B). Because C U T - I R was not always robust, we also examined the distribution of NGFr immunoreactivity. By using antibodies directed against the low-affinity nerve growth factor receptor (like those used here), NGFr-IR has been shown to colocalize with CUT-IR in the basal forebrain of cebus monkeys and humans (Hefti et al., '86; Kordower et al., '88a; Allen et al., '89; Mufson et al., '89) and thus to be a reliable marker for cholinergic basal forebrain neurons; indeed, we found the distribution of NGFr-IR neurons to be identical to that of the cholinergic BM in the marmoset as well (data not shown). Again, all TH-IR BM neurons were also NGFr-IR (Fig. 4C-F), while a small fraction of NGFr-IR cells were not immunoreactive for TH. Doubly stained TH-IRI NGFr-IR cells appeared to compose substantially more than the 43% of BM neurons suggested by our cresyl
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Fig. 2. A Higher magnification micrograph of TH-IR cells in the normal marmoset BM, without counterstain. Multipolar TH-IR neurons have the typical appearance and distribution of magnocellular neurons ofthe cholinergic BM, as seen in B. B. ChAT-IR neurons in the marmoset BM in a section adjacent to that shown in A. Scale bar = 100 pm.
violet/TH cell counts; this discrepancy may reflect the inclusion of noncholinergic neurons among the "BM" cells counted in the cresyl violet-counterstained sections. The results of these colocalization studies demonstrate that a large proportion of cholinergic neurons of the marmoset BM express TH immunoreactivity.
DISCUSSION Until nucleic acid hybridization studies confirm that TH messenger RNA is synthesized in BM neurons, the possibility must be considered that the TH immunoreactivity described here reflects cross-reactivity with unknown molecular species. The failure to observe TH-IR neurons in the BM of many other species may simply reflect their lack of TH; it is also possible that alternative gene splicing in those cells (Grima et al., '87; Kaneda et al., '87) yields TH isoforms that are not recognized by available antibodies. Indeed, TH staining has been observed in a subpopulation of BM neurons in the hamster (Vincent, '88),cat (Kitahama et al., 'go), and ferret (Henderson, '871, and we have also found TH immunoreactivity colocalized in NGFr-IR and C U T - I R neurons in the raccoon BM (data not shown). We have looked in the marmoset BM for immunohistochemical evidence of other enzymes involved in catecholamine synthe-
sis, and found no staining for dopamine-beta-hydroxylase and only weak staining for aromatic amino acid decarboxylase (AADC). Central nervous system neurons that express TH immunoreactivity either stably or transiently, while lacking catecholamines or catecholamine-synthesizing enzymes, have been identified in a number of species (Jaeger et al., '84; Berger et al., '85; Gaspar et al., '87; Meister et al., '88; Okamura et al., '88b; Kitahama et al., '90; Nagatsu et al., '90; Vincent and Hope, '90). It is also possible that authentic TH may be nonfunctional, due, for example, to aberrant postsynthetic processing or to inadequate levels of essential cofactors such as tetrahydrobiopterin. Nevertheless, the presence of TH immunoreactivity in the cholinergic neurons of the marmoset BM suggests that these neurons may contain functional TH, and are thus able to synthesize L-DOPA from tyrosine. Such appears to be the case in the rat arcuate nucleus, where TH-IR cells that lack AADC immunoreactivity are immunoreactive for L-DOPA (Okamura et al., '88a). The possibility that L-DOPA may function as a neurotransmitter in such cells has been suggested by Vincent and Hope ('90): evoked release of endogenous L-DOPA from striatal slices has been reported (Goshima et al., '881, and iontophoretically applied L-DOPA can affect electrical activity in the spinal cord (Bras et al., '88). The possible direct effects of L-DOPA upon neocortical
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Fig. 4. Double-label immunofluorescence micrographs of BM neurons in a normal marmoset, showing colocalization of TH immunoreactivity (B, D, F) within ChAT-IR (A) and NGFr-IR (C, E) neurons. All the TH-IR neurons in Bare also ChAT-IR in A, other ChAT-IR neurons
in the field (compare arrows) are not TH-IR. Similarly, all TH-IR neurons in D and F are NGFr-IR in C and E, respectively, while other NGFr-IR neurons in those fields are TH-negative. Scale bar = 100 Km (A-D), 150 Km (E, F).
neurons, in the marmoset or other species, remain to be explored. Certain neural crest-derived cells, including sympathetic neurons, ciliary neurons, and chromaffin cells, can express
either cholinergic or catecholaminergic phenotype (Doupe et al., '85; Landis, '88), under the control, at least in part, of the targets they innervate (Schotzinger and Landis, '88). This control may be mediated by factors secreted by target
TYROSINE HYDROXYLASE IN MARMOSET NUCLEUS BASALIS cells (Patterson and Chun, '771, some of which have been at least partially characterized (Fukada, '85; Rao and Landis, 'go), and possibly including NGF (Kessler, '85). Expression of TH in chromaffin cells is also under trans-synaptic control (Kvetnansky et al., '70; Otten et al., '73; Gauthier et al., '79). TH expression can also be induced in cerebral cortical neurons following intracerebral transplantation (Park et al., '86; Herman et al., '88),perhaps also as a result of diffusible target-derived factors (Iacovitti et al., '89). Thus neurotransmitter-phenotypicplasticity may be a property of certain central, as well as peripheral, neurons. It will be of interest to determine whether TH immunoreactivity in BM neurons can be influenced by various pharmacological or surgical manipulations (Henderson, '87); the present observations indicate that at least one such manipulation, MPTP-induced degeneration of dopaminergic input to the adjacent neostriatum, has no gross effect on the appearance of TH-IR BM neurons. Finally, it may be noted that numbers of NGFr-IRI CUT-IR neurons within subfields of the BM, but not of other cholinergic forebrain nuclei, are reduced in Parkinson's disease (Mufson et al., '91); the distribution of affected subfields is reminiscent of the distribution of TH-IR BM neurons in the marmoset as described here. TH-IR BM neurons have not been reported in the human brain, although TH-IR cells assigned to hypothalamic nuclei (Li et al., '88; Panayotacopoulou et al., '91) may have included BM neurons. If the degeneration of dopaminergic substantia nigra neurons in Parkinson's disease is in some way a consequence of their catecholaminergic phenotype (as, for example, through the generation of reactive semiquinone metabolites), the partial expression of this phenotype in BM cells might contribute to their degeneration in that disease. It will thus be of interest to determine, by more sensitive immunohistochemical methods as well as nucleic acid hybridization, whether the pattern of TH expression in the marmoset BM is also present in the human brain.
ACKNOWLEDGMENTS We thank C. Leopold and R. Poirier for excellent technical assistance, and Drs. D.D. Rasmusson, K. Semba, and S.P. Hunt for their valuable comments. This study was supported by the Medical Research Councils of Canada and Great Britain, the Parkinson Foundation of Canada, the Canadian Network of Centres of Excellence in Neural Recovery and Regeneration, and the Whitaker Foundation. L.W. was supported by the Dalhousie Medical Alumni Association.
LITERATURE CITED Allen, S.J., D. Dawbarn, M.G. Spillantini, M. Goedert, G.K. Wilcock, T.H. Moss, and F.M. Semenenko (1989)Distribution of B-nerve growth factor receptors in human basal forebrain. J. Comp. Neurol. 289:626-640. Armstrong, D.M., L. Manley, J.W. Haycock, and L.B. Hersh (1990) Colocalization of choline acetyltransferase and tyrosine hydroxylase within neurons of the dorsal motor nucleus of the vagus. J. Chem. Neuroanat. 3:133-140. Armstrong, D.M., V.M. Pickel, T.H. Joh, D.J. Reis, and R.J. Millerd (1981) Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius of rat brain. J. Comp. Neurol. 196:505-517.
385
Bartus, R.T., R.L. Dean, B. Beer, and A.S. Lippa (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 21 7:408-417. Berger, B., C. Verney, P. Gaspar, and A. Febvret (1985)Transient expression of tyrosine hydroxylase immunoreactivity in some neurons of the rat neocortex duringpostnatal development. Dev. Brain Res. 23:141-144. Bras, H., P. Cavallari, and E. Jankowska (1988) An investigation of local actions of iontophoretically applied DOPA in the spinal cord. Exp. Brain Res. 71:447-449. Brashear, H.R., L. Zaborsky, and L.Heimer (1986) Distribution of GABAergic and cholinergic neurons in the rat diagonal band. Neuroscience 17:439-451. Collerton, D. (1986) Cholinergic function and intellectual decline in Alzheimer's disease. Neuroscience 19:l-28. Danes, P., and A.J.F. Maloney (1976) Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet i~:1403. Doupe, A.J., S.C. Landis, and P.H. Patterson (1985) Environmental influences in the development of neural crest derivatives: Glucocorticoids, growth factors and chromaffin cell plasticity. J. Neurosci. 5:2119-2142. Dubach, M., R. Schmidt, D. Kunkel, D.M. Bowden, R. Martin, and D.C. German (1987) Primate neostriatal neurons containing tyrosine hydroxylase: Immunohistochemical evidence. Neurosci. Lett. 75:205-2 10. Dunnett, S.B., G. Toniolo, A. Fine, C.N. Ryan, A. Bjorklund, and S.D. Iversen ( 1985) Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis magnocellularis. 11. Sensorimotor and learning impairments. Neuroscience 16:787-797. Everitt, B.J., T.E.Sirkia, A.C. Roberts, G.H. Jones, andT.W. Robbins (1988) Distribution and some projections of cholinergic neurons in the brain of the common marmoset, Callithrir jacchus. J. Comp. Neurol. 271:533558. Fine, A,, S.B. Dunnett, A. Bjorklund, and S.D. Iversen (1985) Cholinergic ventral forebrain grafts into the neocortex improve passive avoidance memory in a rat model of Alzheimer disease. Proc. Natl. Acad. Sci. USA 825227-5230. Fine, A., S.P. Hunt, W.H. Oertel, M. Nomoto, P.N. Chong, A. Bond, C. Waters, J.A. Temlett, L. Annett, S. Dunnett, P. Jenner, and C.D. Marsden (1988) Transplantation of embryonic marmoset dopaminergic neurons to the corpus striatum of marmosets rendered parkinsonian by l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine. Prog. Brain Res. 78t479489. Flicker, C., R.L. Dean, D.L. Watkins, S.K. Fisher, and R.T. Bartus (1983) Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol. Biochem. Behav. I8:973-981. Fukada, K. (1985) Purification and partial characterization of a cholinergic neuronal differentiation factor. Proc. Natl. Acad. Sci. USA 8287958799. Gaspar, P., B. Berger, C. Alvarez, A. Vigny, and J.P. Henry (1985) Catecholaminergic innervation of the septa1 area in man: Immunocytochemical study using TH and DBH antibodies. J. Comp. Neurol. 241:12-33. Gaspar, P., B. Berger, A. Febvret, A. Vigny, M. Krieger-Poulet, and C. Borri-Voltattorni (1987 Tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex: A novel catecholaminergic group? Neurosci. Lett. 80.957-262. Gauthier, S., J.-P. Gagner, and T.L. Slourkes (1979) Role of descending spinal pathways in the regulation of adrenal tyrosine hydroxylase. Exp. Neurol. 66:42-54. Goshima, Y., T. Kubo, and Y. Misu (1988) Transmitter-like release of endogenous 3,4-dihydroxyphenylalaninefrom rat striatal slices. J. Neurochem. 50: 1725-1 730. Grima, B., A. Lamouroux, C. Boni, J.F. Julien, F. Javoy-Agid, and J. Mallet (1987) A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics. Nature 326:707-711 Hefti, F., J. Hartikka, P. Salvaterra, W.J. Weiner, and D. Mash (1986) Localization of nerve growth factor receptors on cholinergic neurons of the human basal forebrain. Neurosci. Lett. 69:275-281. Henderson, Z. (1987) A small proportion of cholinergic neurones in the nucleus basalis magnocellularis of ferret appear to stain positively for tyrosine hydroxylase. Brain Res. 412:363-369. Herman, J.P., N. Abrous, A. Vigny, J. Dulluc, and M. LeMoal (1988) Distorted development of intraeerebral grafts: Long-term maintenance of tyrosine hydroxylase-containing neurons in grafts of cortical tissue. Dev. Brain Res. 40:81-88. Hokfelt, T., 0. Johansson, K. Fuxe, M. Goldstein, and D. Park (1976)
386 Imrnunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain. I. Tyrosine hydroxylase in the mes- and diencephalon. Med. Bid. 54t427-453. Hornung, J.-P., I. Tork, and N. De Triboler (1989) Morphology of tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex. Exp. Brain Res. 76:lZ-20. Iacovitti, L., M.J. Evinger, T.H. Joh, and D.J. Reis (1989) A muscle-derived factor(s1 induces expression of a catecholamine phenotype in neurons of cultured rat cerebral cortex. J. Neurosci. 9:3529-3537. Jaeger, C.B., D.A. Ruggiero, V.R. Albert, D.H. Park, T.H. Joh, andD.J. Reis (1984)Aromatic L-amino acid decarboxylase in the rat brain: Immunocytochemical localization in neurons of the brain stem. Neuroscience I k691-713. Kaneda, N., K. Kobayashi, H. Ichinose, F. Kishi, A. Kanazawa, Y. Kurosawa, K. Fujita, and T. Nagatsu (1987) Isolation of a novel cDNA clone for human tyrosine hydroxylase: Alternative RNA splicing produces four kinds of mRNA from a single gene. Biochem. Biophys. Res. Commun. 146:971-975. Kessler, J.A. (1985) Parasympathetic, sympathetic and sensory interactions in the iris: Nerve growth factor regulates cholinergic ciliary ganglion innervation in vivo. J. Neurosci. 5.2719-2735. Kitahama, K., M. Geffard, H. Okamura, I. Nagatsu, N. Mans, and M. Jouvet (1990) Dopamine- and DOPA-immunoreactive neurons in the cat forebrain with reference to tyrosine hydroxylase-immunohistochemistry. Brain Res. 518:83-94. Kohler, C., B.J. Everitt, J. Pearson, and M. Goldstein (1983) Immunohistochemical evidence for a new group of catecholamine-containing neurons in the basal forebrain of the monkey. Neurosci. Lett. 37:161-166. Konigsmark, B.W. (1970) Methods for the counting of neurons. In W.J.H. Nauta and S.O.E. Ebbesson (eds): Contemporary Research Methods in Neuroanatomy. New York: Springer-Verlag,pp. 315-337. Kordower, J.H., R.T. Bartus, M. Bothwell, G. Schatteman, and D.M. Gash (1988a) Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus upella): Distribution, morphology, and colocalization with cholinergic enzymes. J. Comp. Neurol. 277:465486. Kordower, J.H., R.T. Bartus, F.F. Marciano, andD.M. Gash (1989)Telencephalic cholinergic system of the New World monkey (Cebus upella): Morphological and cytoarchitectonic assessment and analysis of the projection to the amygdala. J. Comp. Neurol. 279,528-545. Kordower, J.H., and E.J. Mufson (1990) Galanin-like irnnlunoreactivity within the primate basal forebrain: Differential staining patterns between humans and monkeys. J. Comp. Neurol. 294:281-292. Kordower, J.H., J.R. Sladek, Jr., M.S. Fiandaca, G. Bing, and D.M. Gash (1988h) Tyrosine-hydroxylase immunoreactive somata within the primate subfornical organ: Species specificity. Brain Res. 461:221-229. Kvetnansky, R., V.K. Weise, and I.J. Kopin (1970) Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-N-methyl-transferase by repeated immobilization of rats. Endocrinology 87:744-749. Landis, S.C. (1988) Neurotransmitter plasticity in sympathetic neurons and its regulation by environmental factors in vitro and in vivo. In A. Bjorklund, T. Hokfelt, and C. Owrnan (eds): Handbook of Chemical Neuroanatomy, Vol. 6 : The Peripheral Nervous System. Amsterdam: Elsevier, pp. 65-115. Li, Y.W., G.M. Halliday, T.H. Joh, L.B. Geffen, and W.W. Blessing (1988) Tyrosine hydroxylase-containing neurons in the supraoptic and paraventricular nuclei of the adult human. Brain Res. 461:75-86. Mann, D.M.A., and P.O. Yates (1983) Pathological basis for neurotransmitter changes in Parkinson’s disease. Neuropathol. Appl. Neurobiol. 9:3-19. Meister, B., T. Hokfelt, H.M.W. Steinbusch, G. Skagerberg, 0. Lindvall, M. Geffard, T.H. Joh, A.C. Cuello, and M. Goldstein (1988) Do tyrosine hydroxylase-immunoreactive neurons in the ventral arcuate nucleus produce dopamine or only L-DOPA? J. Chem. Neuroanat. 1:59-64. Melander, T., and W.A. Staines (1986) A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complex and in acetylcholinesterase-containing fibers and varicosities within the hippocarnpus in the owl monkey (Antus tciuirgulus). Neurosci. Lett. 68.17-22. Mesulam, M.M., E.J. Mufson, A.I. Levey, and B.H. Wainer (1983a) Cholinergic innervation of cortex by basal forebrain: Qtochemistry and cortical connection for the septa1 area, diagonal band nuclei, nucleus basalis (substantia innominata) and hypothalamus in the rhesus monkey. J. Comp. Neurol. 214:170-197. Mesulam, M.M., E.J. Mufson, B.H. Wainer, and A.I. Levey (1983b) Central
L. WISNIOWSKI ET AL. cholinergic pathways in the r a t An overview based on an alternative nomenclature (Chl-Ch6). Neuroscience 10.1 185-1201. Mufson, E.J., M. Bothwell, L.B. Hersh, and J. Kordower (1989) Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: Colocalization with cholinergic neurons, J. Comp. Neurol. 285:196-217. Mufson, E.J., L.N. Presley, and J.H. Kordower (1991) Nerve growth factor receptor immunoreactivity within the nucleus basalis (Ch4) in Parkinson’s disease: Reduced cell numbers and co-localization with cholinergic neurons. Brain Res. 539:19-30. Murray, C.L., and H.C. Fibiger (1985) Learning and memory deficits after lesions of the nucleus basalis magnocellularis: Reversal by physostigmine. Neuroscience 14: 1025-1032. Nagatsu, I., K. Komori, T. Takeuchi, M. Sakai, K. Yarnada, and N. JSarasawa (1990) Transient tyrosine hydroxylase-immunoreactive neurons in the region of the anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine. Brain Res. 511.55-62. Okamura, H., K. Kitahama, N. Mans, Y. Ibata, M. Jouvet, and M. Geffard (1988a) L-DOPA immunoreactive neurons in the rat hypothalamic tuberal region. Neurosci. Lett. 95;42-46. Okamura, H., K. Kitahama, 1. Nagatsu, andM. Geffard (1988bi Comparative topography of dopamine- and tyrosine hydroxylase-immunoreactive neurons in the rat arcuate nucleus. Neurosci. Lett. 95:347-353. Otten, U., U. Paravicini, F. Oesch, and H. Thoenen (1973) Time requirement for the single steps of transynaptic induction of tyrosine hydroxylase in the peripheral nervous system. Naunyn Schmiedebergs Arch. Pharmacol. 280:117-127. Panayotacopoulou, M.T., R. Guntern, C. Bouras, M.R. Issorides, and J. Constantinidis (1991)Tyrosine hydroxylase-immunoreactive neurons in paraventricular and supraoptic nuclei of the human brain demonstrated by a method adapted to prolonged formalin fixation. J. Neurosci. Methods 39f39-44. Park, J.K., T.H. Joh, and F.F. Ebner (1986) Tyrosine hydroxylase is expressed by neocortical neurons after transplantation. Proc. Natl. Acad. Sci. USA 83:7495-7498. Patterson, P.H., and L.L.Y. Chun (1977) Induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium. Dev. Biol. 56:263-280. Perry, E.K., M. Curtis, D.J. Dick, J.M. Candy, J.R. Atack, C.A. Bloxham, G. Blessed, A. Fairbairn, B.E. Tomlinson, and R.H. Perry (1985) Cholinergic correlates of cognitive impairment in Parkinson’s disease: Comparison with Alzheimer’s disease. J. Neurol. Neurosurg. Psychiatry 48:413421. Radeke, M.J., T.P. Misko, C. Hsu, L. Herzenberg, and E.M. Shooter (1987) Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 325:593-597. Rao, M.S., and S.C. Landis (1990) Characterization of a target-derived neuronal cholinergic differentiation factor. Neuron 5899-910. Ridley, R.M., H.F. Baker, B.S. Drewett, and J.A. Johnson (1985) Effects of ibotenic acid lesions of the basal forebrain on serial reversal learning in the marmoset. Psychopharmacology 86:438449. Ridley, R.M., T.K. Murray, J.A. Johnson, and H.F. Baker (1986) Learning impairment following lesion of the basal nucleus of Meynert in the marmoset: Modification by cholinergic drugs. Brain Res. 376:108-116. Ross, A.H., P. Grob, M. Bothwell, D.E. Elder, C.S. Ernst, N. Marano, B.F.D. Ghrist, C.C. Slemp, M. Herlyn, B. Atkinson, and H. Koprowski (1984) Characterization of nerve growth factors in neural crest tumors using monoclonal antibodies. Proc. Natl. Acad. Sci. USA81:6681-6685. Satoh, K., and H.C. Fibiger (1985) Distribution of central cholinergic neurons in the baboon (Papiopupio). 11. A topographic atlas correlated with catecholamine neurons. J. Comp. Neurol. 236,215-233. Schotzinger, R.J., and S.C. Landis (1988) Cholinergic phenotype developed by noradrenergic sympathetic neurons after innervation of a novel cholinergic target in vivo. Nature 39.5637-639. Semba, K., and H.C. Fihiger (1989) Organization of central cholinergic systems. Prog. Brain Res. 79:37-63. Spencer, S., C.B. Saper, T. Joh, D.J. Reis, M. Goldstein, and J.D. Raese (1985) The distribution of catecholamine-containing neurons in the normal human hypothalamus. Brain Res. 328:73-80. Stephan, H., G. Baron, and W.K. Schwerdtfeger (1980) The Brain of the Common Marmoset (Cullithrtr jucchus): A Stereotaxic Atlas. Berlin: Springer-Verlag. Tashiro, Y., T. Sugimoto, T. Hattori, Y. Uemura, I. Nagatsu, H. Kikuchi, and
TYROSINE HYDROXYLASE IN MARMOSET NUCLEUS BASALIS
387
N. Mizuno (1989) Tyrosine hydroxylase-like immunoreactive neurons in Vincent, S.R., and B.T. Hope (1990) Tyrosine hydroxylase containing the striatum of the rat. Neurosci. Lett. 973-10. neurons lacking aromatic amino acid decarboxylase in the hamster brain. J. Comp. Neurol. 2952990-2998. Tinner, B., K. Fuxe, Ch. Kohler, L. Hersh, K. Andersson, A. Jansson, M. Wainer, B.H., A.I. Levey, D.B. Rye, M.-M. Mesulam,and E.J. Mufson (1985) Goldstein, and L.F. Agnati (1989) Evidence for the existence of a Cholinergic and non-cholinergic septohippocampal pathways. Neurosci. population of arcuate neurons costoring choline acetyltransferase and Lett. 54:45-52. tyrosine hydroxylase immunoreactivities in the male rat. Neurosci. Lett. 99:44-49. Walker, L.C., V.E. Koliatsos, C.A. Kitt, R.T. Richardson, A. Rokaeus, and D.L. Price (1989) Peptidergic neurons in the basal forebrain magnocelluUngerstedt, U. (1971) Stereotaxic mapping of the monoamine pathways in lar complex of the rhesus monkey. J. Comp. Neurol. 280:272-282. the rat brain. Acta Physiol. Scand. Suppl. 367:148. Waters, C.M., S.P. Hunt, P. Jenner, and C.D. Marsden (1987)An immunohisVincent, S.R. (1988) Distributions of tyrosine hydroxylase-, dopamine-Btochemical study of the acute and long-term effects of 1-methyl-4-phenylhydroxylase-, and phenylethanolamine-N-methyltransferase-immunore1,2,3,64etrahydropyridine in the marmoset. Neuroscience 23:1025active neurons in the brain of the hamster (Mesocricetus auratus). J. 1030. Comp. Neurol. 268.584-599.
