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Home , Basal forebrain, Basal ganglia, Globus pallidus, Nucleus basalis, Substantia innominata

Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1408-1412, March 1987 Medical Sciences Anatomical relationship between the and the basal of Meynert in and monkey (/acetylcholinesterase//human) SUZANNE HABER Department of Neurobiology and , University of Rochester, Rochester, NY 14642 Communicated by Walle J. H. Nauta, October 20, 1986

ABSTRACT Previous immunohistochemical studies have suggestion that the basal ganglia could serve cognitive as well provided evidence that the external segment of the globus as motor functions (7). Because this notion places the basal pallidus extends ventrally beneath the transverse limb of the ganglia in a functional category comparable, in part at least, into the area of the substantia in- to that of the basal nucleus of Meynert, a more detailed nominata. Enkephalin-positive staining in the form of "woolly description ofthe relationship ofthese two structures to each fibers" has been used as a marker for the and other seemed of interest. its ventral extension. Acetylcholinesterase staining of both The globus pallidus (in particular its most ventral part, the fibers and cell bodies, frequently used as a marker for the basal ventral pallidum) and the basal nucleus of Meynert are nucleus of Meynert, is also found in the area of the substantia adjacent structures (Fig. 2 A-C and 3 A and B). The large innominata. This study describes the differential distribution of acetylcholinesterase (AcChoEase)-positive in the enkephalin-positive woolly fibers and acetylcholinesterase (i.e., the infrapallidal region of the staining on adjacent sections in both the monkey and human ) are regarded as a characteristic marker for basal forebrain area in an attempt to define the relationship the basal nucleus of Meynert and are therefore considered to between the basal ganglia and the basal nucleus of Meynert. define the nucleus in the monkey and human (8, 9). On The results show that while both occupy large regions of the the other hand, enkephalin-staining in the form of "woolly basal forebrain, they overlap very little. In both species fibers" [enkephalin-positive striatal efferents ensheathing investigated, dense concentrations of acetylcholinesterase-pos- each of the long dendrites of pallidal neurons individually itive neurons lie, for the most part, outside the boundaries of (Fig. 1)] has become recognized as an equally characteristic the pallidal fibers. However, some scattered - marker for the globus pallidus in the rat, monkey, and human terase cells do lie within the confines ofthe dorsal pallidum, and forebrain (10-12). Large AcChoEase-positive neurons have a more prominent group is found in the subcommissural been found within the boundaries of the globus pallidus in a ventral pallidum. These cells may constitute a group separate distribution continuous with that of the basal nucleus of from the more densely packed acetylcholinesterase-positive Meynert. Such cells are particularly numerous in a ventral cells in woolly fiber-free regions in that they may receive direct region of the globus pallidus and the ventral pallidum of striatal input. Heimer and Wilson (13) but also occur, more sparsely distributed, in more dorsal pallidal regions. It was initially Parkinson disease and senile of the Alzheimer type assumed that these intrapallidal elements ofthe basal nucleus are two degenerative disorders that occur in mid to late life. ofMeynert are foreign to the globus pallidus and unrelated to Whereas each disease is associated with a specific symptom- the circuitry of the basal ganglia. Recent findings in the rat atology and pathology, a number of cases have recently been forebrain, however, suggest that at least some of these described in which some overlap ofboth disorders is evident intrapallidal neurons receive afferents from the (1-3). For example, a patient may initially exhibit classical (14-16). Such a connection would provide a direct Parkinson-like motor disabilities and later exhibit dementia linkage of the basal ganglia to the basal nucleus of Meynert. characteristic ofAlzheimer disease. Furthermore, cases have This study demonstrates the extent to which the striatal been described in which patients with the symptoms of one projection to the globus pallidus (as defined by enkephalin- disease were found to have the pathology that characterizes positive woolly fibers), and the basal nucleus of Meynert (as the other disease, either in addition to, or in the absence of, defined by its AcChoEase-positive neurons), overlap in the the pathology of the diagnosed disease. human and nonhuman primate brain. The rationale for the has been known to reflect a disor- study was the assumption that striatal efferents establish Parkinson disease long synaptic contact with neurons in the basal nucleus of der of the basal ganglia, whereas Alzheimer disease has Meynert only within such areas of overlap. Moreover, it recently become widely attributed, at least in part, to a seemed possible that pathology affecting the function of such pathology of the basal nucleus of Meynert (4). The basal areas might be reflected in symptoms that represent both ganglia (composed primarily of the striatum and globus Parkinson and Alzheimer disease. pallidus) and the basal nucleus of Meynert are traditionally viewed as separate functional entities-the former involved in somatic movement, the latter associated with cognitive EXPERIMENTAL PROCEDURES functions. Recent anatorhical findings, however, have raised Five from neurologically normal patients were ob- questions regarding a purely motoric function of the basal tained between 8 and 10 hr after death. Coronal slabs of ganglia; in particular, the existence ofconnections linking the approximately 15-mm thickness were cut immediately, fixed striatopallidal complex not only to the but also by immersion in buffered fornialin for 2 weeks, then trans- to the (5-7) and has led to the ferred from lower to higher concentrations of sucrose (5%, 10%, and 20%), and finally stored in 30% sucrose in 0.1 M The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: AcChoEase, acetylcholinesterase; ChoAcTase, in accordance with 18 U.S.C. §1734 solely to indicate this fact. choline acetyltransferase.

Downloaded by guest on September 29, 2021 1408 Medical Sciences: Haber Proc. Natl. Acad. Sci. USA 84 (1987) 1409

a drawing tube. The drawing was then photographically reduced and taped to the wall for projection of the adjacent

ill enkephalin-stained section onto it. To further verify regions of overlap the two sections were viewed on separate micro- I. scopes equipped with a Leitz comparator bridge to allow superimposition of the images from the two slides. Finally, key sections were double-stained by the fluorescent-antibody method and the Geneser-Jensen and Blackstad technique for AcChoEase to verify the observations in the adjacent sec- tions. Due to the weak and fading fluorescence it was difficult

A' to analyze double-stained sections in detail.

0. RESULTS I Numerous large AcChoEase-positive neurons were found in 1. t ~ the basal forebrain of all brains examined. Their distribution a;*I r pattern was consistent with that described previously (8, 9). In areas 4 many the individual cell bodies were somewhat ., 0 obscured by the vast number of cholinergic fibers passing through the region; within such areas the actual number of AcChoEase-positive neurons may have been underestimat- ed. Adjacent Nissl-stained sections confirmed the location and distribution of these neurons originally described by Meynert in 1872 (20). Enkephalin-like immunoreactivity appeared in its characteristic pattern of woolly fibers in the region of the globus pallidus. Fig. 2 A-C illustrates the relationship between the enkeph- alin-positive fibers and the AcChoEase-positive neurons in FIG. 1. Single enkephalin-positive striatal efferent fiber joining the monkey forebrain, and Fig. 3 A-E illustrates this in the other fibers wrapping a pallidal dendrite to form a woolly fiber. Open human forebrain. arrow indicates single efferent fiber; filled arrow indicates woolly In the , large AcChoEase-positive neurons fiber. appear within the fiber laminae forming the boundaries ofthe globus pallidus. Such cells are found within the internal phosphate buffer, pH 7.2 for 1-3 days. Four adult African capsule and within both the external medullary lamina that green monkeys (Cercopithecus aethiops) were anesthetized separates the from the external segment of the and perfused with 4% paraformaldehyde, and their brains globus pallidus and the internal medullary lamina that sepa- were then transferred through the concentrations of sucrose rates the external and internal pallidal segments. Whereas listed above. these cells are distributed throughout the thickness of the Antisera to enkephalin were provided by R. Elde (Univer- laminae, most are located directly adjacent to the external sity of Minnesota). Characterization of these antibodies has segment of the globus pallidus (Figs. 2A and 3B). A few been described in detail elsewhere (17). These antisera do not scattered AcChoEase-positive magnocellular neurons are cross-react with either ofthe other two classes ofendogenous also seen within the external and internal segment of the opiates, endorphin or (see ref. 12 for this descrip- globus pallidus. These appear to be more numerous in the tion and a comparison between the differential distribution of external segment than in the internal segment and also appear enkephalin and dynorphin in human globus pallidus). to be more numerous in monkey than in human. The number Sections were cut at 50-pum thicknesses on a freezing of cells seen within the boundaries of the dorsal globus microtome and incubated in primary antisera diluted 1:1000 pallidus represents approximately 6% of the large Ac- in 0.3% Triton X-100/0.1 M phosphate buffer (PB/T) at pH ChoEase-positive neurons appearing in these cross-sections 7.2 for 3-5 days at 40C. After being rinsed, the tissue was (Figs. 2 and 3). This, however, is likely to be an overestimate incubated in goat anti-rabbit IgG (diluted 1:500 in PB/T) because, in the dorsal pallidum, such neurons are easily overnight at 40C, and then rinsed well and incubated in rabbit identified, whereas those lying ventral to the anterior com- peroxidase-anti-peroxidase antisera diluted 1:50 in phos- missure are often masked by a great crowding ofAcChoEase- phate buffer for 1 hr at room temperature. After rinsing with positive fiber bundles. In both monkey and human, the main 0.05 M Tris buffer, pH 7.6, the tissue was incubated in concentration of the large AcChoEase-positive neurons of 3,3'-diaminobenzidine tetrahydrochloride (0.05 mg/ml) in the basal forebrain is located ventral to the conventionally Tris buffer/0.01% H202. Sections were rinsed, dehydrated, defined globus pallidus and stretches rostralward to the level and coverslipped with Permount (Fisher). Adjacent sections of the anterior commissure (Figs. 1-3). were stained for AcChoEase by the Geneser-Jensen and Enkephalin-positive woolly fibers are densely distributed Blackstad (18) technique. Although in brain stem regions throughout the external segment of the globus pallidus and cholinesterase-positive staining does not correspond well to extend ventralward to include the ventral pallidum located the staining observed using antisera against the acetylcho- beneath the anterior commissure. This ventral region of line-synthesizing enzyme choline acetyltransferase (ChoAc- dense staining extends both ventrolaterally into the amyg- Tase), this does not appear to be the case in the basal daloid complex (Fig. 2 B and C and Fig. 3 C and E) and forebrain. The decision to use the AcChoEase method of medially into the bed nucleus of the (Fig. 3 A marking the cholinergic forebrain system was based on the and B). Most of the internal pallidal segment is much less observation that neurons in the basal forebrain of the rat that densely innervated with enkephalin-positive woolly fibers, are strongly positive for AcChoEase are also ChoAcTase but its medial portion does contain a prominent plexus of positive (19). such fibers (Fig. 2C and Fig. 3 B and C). Analysis entailed several approaches. For the line draw- Large AcChoEase-positive neurons located within the ings, individual AcChoEase-positive cells were plotted with fiber laminae bordering the globus pallidus lie entirely outside Downloaded by guest on September 29, 2021 1410 Medical Sciences: Haber Proc. Natl. Acad. Sci. USA 84 (1987) A the plexus of woolly fibers. Even where such cells appear to lie directly on the border of the globus pallidus (see Figs. 2A and 3 B-D), closer inspection reveals that they are adjacent to, but not within, the plexus of woolly fibers. Within the main body of the globus pallidus, the scattered AcChoEase- positive cells are all embedded within the enkephalin-positive fiber plexus, but this relationship is most obvious in the external segment (Figs. 2B and 3 B, D, and E). At several levels shown here the largest concentrations of cells belonging to the basal nucleus of Meynert lie outside (although adjacent to) regions identified by enkephalin- positive woolly fibers as part of the ventral pallidum. Exam- ples of such nonoverlapping distribution are indicated in Figs. 2 and 3 by solid arrowheads. In several restricted locations a circumscript void in the woolly-fiber plexus coincides pre- cisely with a cluster of basal-nucleus neurons (Fig. 2C). In contrast, a substantial overlap of the basal nucleus with the woolly-fiber plexus-and hence, with the distribution of enkephalin-positive striatofugal fibers-is evident in a region extending longitudinally underneath the globus pallidus. The region is indicated in Figs. 3 B-E by open arrows. Although the number of cells embedded in this zone of enkephalin- positive fibers in any given cross-section is relatively small (18%, computed from the coronal section at the levels shown) in comparison with those lying outside the fiber plexus, the overlap is consistent throughout the rostrocaudal extent of the ventral pallidum. It involves a greater number of basal- nucleus cells at its more caudal levels. In sections stained for both AcChoEase and enkephalin, those AcChoEase-positive cells that are embedded within the woolly-fiber plexus appear to be closely surrounded by enkephalin-like immunoreactivity. Whereas it is not possible to determine at the light microscopic level whether or not the immunoreactive tissue components include that actu- ally are making direct contact with the AcChoEase-positive neurons, the histological picture suggests the possibility of such a relationship (Fig. 4).

DISCUSSION This evidence indicates that the basal nucleus of Meynert largely lies outside the massive efferent fiber system of the striatum in both monkey and human. In several places the AcChoEase-positive neurons of the basal nucleus lie imme- diately adjacent to, but not directly within, the plexus of enkephalin-positive striatofugal fibers. Areas that lack the staining of one substance are often positive for the other. This observation conveys the impression that the two systems do not interact. However, two exceptions to this rule can be mentioned. (i) A few scattered cholinergic cells are located within the enkephalin-positive plexus permeating the dorsal globus pallidus. Whereas these cells form only a very small percent- age of the total population of the , their presence raises questions as to the origin of their afferents. It seems unlikely that cells so sparsely and widely disseminated are innervated by an afferent fiber system separate from the circuitry of the basal ganglia. As for the nucleus basalis neurons lying in the laminae bordering the FIG. 2. Line drawing of coronal section through rhesus monkey globus pallidus-although their cell bodies are outside the basal ganglia. Large black dots indicate AcChoEase-positive neurons of intrapallidal plexus of striatofugal fibers-it must be consid- the basal nucleus of Meynert, and stippling indicates enkephalin- ered that these cells may send one or more of their long positive woolly fibers marking the globus pallidus. Closed arrows dendrites into the globus pallidus. Thus, there may indeed be indicate areas where AcChtEase-positive cell bodies lie outside the a dorsal group of AcChoEase-positive nucleus basalis neu- woolly-fiber plexus. Open arrows indicate regions of overlap between rons a small that are in some way involved in the AcChoEase-positive neurons and woolly fiber plexus. A, amygdala; (albeit one) AC, anterior commissure; C, ; GPe, globus pallidus- circuitry of the basal ganglia. external segment; GPj, globus pallidus-internal segment; IC, internal (it) Within the region of the ventral pallidum, a larger capsule; NB, basal nucleus of Meynert; P, putamen; and VP, ventral number ofAcChoEase-positive neurons are found embedded pallidum. among enkephalin-positive striatofugal fibers. Although Downloaded by guest on September 29, 2021 Medical Sciences: Haber Proc. Nati. Acad. Sci. USA 84 (1987) 1411

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FIG. 3. Line drawing of coronal section through human basal ganglia. Legend as for Fig. 2. these cells, too, compose a relatively modest percentage of have been further subdivided with respect to their afferent the basal-nucleus population, they form a rather consistent and efferent projections. Those neurons that are embedded in group clustered in a specific region ofthe enkephalin-positive enkephalin-positive woolly fibers correspond best, but not plexus of woolly fibers. They may represent a specific group completely, to the anterior lateral group (Ch4A1) and the of cholinergic neurons that either receive input from, or send dorsal group of the nucleus of the ansa peduncularis (Ch4id) projections to, a particular brain region. The AcChoEase- described by Mesulam and co-workers (8, 21). positive neurons that constitute the basal nucleus of Meynert This group of cells is of particular interest. AcChoEase- Downloaded by guest on September 29, 2021 1412 Medical Sciences: Haber Proc. Natl. Acad. Sci. USA 84 (1987) themselves both in dyskinesia and in cognitive disorder ofthe Alzheimer type. I thank Jeanine Schu for her technical assistance and Dr. N. Kowell, Massachusetts General Hospital, and the Brain Tissue Resource Center, McLean Hospital, Belmont, MA, for making human material available for study. This study was supported by National Institutes of Health Grants NS 22511 and Research Career Development Award K04 NS 01071. 1. Chou, H. C., Teng, E. L., Henderson, V. W. & May, A. C. (1985) Neurology 35, 1544-1550. 2. Mayeux, R., Stern, Y. & Spanton, S. (1985) Neurology 35, 453-461. 3. Price, D. L., Whitehouse, P. J. & Struble, R. G. (1986) Trends tm,, Neurosci. 9, 29-33. 4. Whitehouse, P. J., Price, D. L., Struble, R. G., Clark, A. W., embede Coyle, J. T. & DeLong, M. R. (1982) Science 215, 1237-1239. 5. Goldschmidt, R. B. & Heimer, L. (1980) Soc. Neurosci. Abstr. 6, 271. 6. Young, W. S., Alheid, G. F. & Heimer, L. (1984) J. Neurosci. 4, 1626-1638. 7. Haber, S. N., Groenewegen, H., Grove, E. A. & Nauta,

" W. J. H. (1985) J. Comp. Neurol. 235, 322-335. 8. Mesulam, M.-M., Mufson, E. J., Levey, E. J. & Wainer, B. H. (1983) J. Comp. Neurol. 214, 170-197. 9. Saper, C. B. & Chelimsky, T. C. (1984) Neuroscience 13, 1023-1038. 10. Haber, S. N. & Nauta, W. J. H. (1983) Neuroscience 9, 245-260. 11. Beach, T. G. & McGeer, E. G. (1984) Neuroscience 13, 29-52. 12. Haber, S. N. & Watson, S. J. (1985) Neuroscience 14, nern Vnaiigti ein foelpwudeal h 1011-1024. stratmhAbeuaafetoanuceuhechoinrgcthhoae-psthaelamuros,nnevaioteamygdala,o th aygalamnd 13. Heimer, L. & Wilson, R. D. (1975) Golgi Centennial Sympo- ananedialoffotlcriaregionit s t hetara.Teefero nta corteoinoxs includinguhcnattuicthe edill sium, ed. Santini, M. (Raven, New York), pp. 177-193. 14. Zaborszky, L., Eckenstein, F., Leranth, C. S., Oertel, W., prfonitale courtex andth anadjoining mediaio stipnothemoeranoto Schmeckel, D., Alones, V. & Heimer, L. (1984) Soc. Neurosci. Abstr. 10, 8. Studraies byacmbeddinathreion ofhitohemicntal panlduretogade 15. Haber, S. N. (1984) Soc. Neurosci. Abstr. 10, 7. laeeings methodsi havfersowetatthprojectionsfromthenulsac 16. Grove, E. A. & Nauta, W. J. H. (1984) Soc. Neurosci. Abstr. regionsi questio(2)n thecorextandoramyogdaaarhicseud the 10, 7. mosapt(7 fromiatheaindwelingijchoiergict cells rgoftherbsa 17. Haber, S. N. & Elde, R. (1982) Neuroscience 7, 1049-1095. noucleu (23-26),del whreansptostedntolto thethlmsuventrali 18. Geneser-Jensen, F. A. & Blackstad, T. W. (1971) Z. evidence (A5and 27), butcurrenstlye npeuros tombeddeceiving Zellforsch. Mikrosk. Anat. 114, 460-481. morepdefinpoitivetuegmntlaraandsubsathalamgrcultrastructufrentsuppors.nucljeusoaresoArrow appeaatrignateyindicateywooll 19. Levey, A. I., Wainer, B. H., Mufson, E. J. & Mesulam, neuprons popuatntheetregpionl(6s7e5)gEientlyuasynaticth M.-M. (1983) Neuroscience 9, 9-22. 20. Meynert, T. (1872) ed. Stricker, S. A Manual of Histology psitivecontractlonemurnsintealouscation;thgensrital in.2)with,I olthebaioosalthussemorbanceivnucleusan able thatliertiemeddysfnctheonsioof the venitralcould thates (English transl., J. J. Putnam) (Wood, New York), pp. naeurons ihabitingr thles rgonofe overlapus enygable, 650-766. tegon the hoinergi innrvatinclofn theamygdiala 21. Mesulam, M.-M. & Mufson, E. J. (1984) Brain Res. 107, andoffrontal corticaldaresnhadonotio ofiasuchp contactmthus 253-274. 22. Wilson, R. D. (1972) Dissertation, Massachusetts Institute of frhStureste exaclusivelytuonofhsugghesielgtmicaanreroscopic Technology, Cambridge, MA. evideince (15,od16a2),bu currentltappartoebejetin freceivin 23. Nagai, T., Kimura, H., Maeda, T., McGeer, P. L., Peng, F. & McGeer, E. G. (1982) J. Neurosci. 2, 513-520. segona communiction;trhef 28).rte woud thusal seiem concteiv 24. Woolf, N. J. & Butcher, L. L. (1982) Brain Res. Bull. 8, 751-763. 25. Grove, E. A., Haber, S. N., Domesick, V. B. & Nauta, W. J. H. (1983) Soc. Neurosci. Abstr. 10, 7. ablethacts certain ysfunctionofferethestriatum coubasld mnfleust 26. Carlsen, J., Zaborszky, L. & Heimer, L. (1985) J. Comp. Neurol. 234, 155-167. 27. Grove, E. A., Domesick, V. B. & Nauta, W. J. H. (1986) Brain Res. 367, 379-384. 28. Grove, E. A. & Ingham, C. A. (1986) Soc. Neurosci. Abstr. 12, 1328. Downloaded by guest on September 29, 2021