S.-M. Aquilonius and P.-G. Gillberg (Eds.) Progress in Brain Research, Vol. 84 0 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
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CHAPTER 26
Human brain cholinergic pathways M.-Marsel Mesulam Bullard and Denny-Brown Laboratories, Division of Neuroscience and Behavioral Neurologv, Hamard Neurology Department and the Dana Research Institute of the Beth Israel Hospital, Boston, MA 02215, U.S.A.
Introduction The past decade has witnessed considerable advances in unravelling the organization of central cholinergic pathways. The availability of new anatomical methodology and the observation that cortical cholinergic innervation is markedly depleted in Alzheimer’s disease constitute two important factors that have catalysed much of the recent progress in this field. Acetylcholine is one of the most ubiquitous neurotransmitters in the mammalian central nervous system. Neuroanatomical experiments indicate that the cholinergic innervation of a given brain structure can be intrinsic or extrinsic. The innervation of the striatum, for example, is almost exclusively intrinsic and arises from cholinergic interneurons. In contrast, the cholinergic innervation of limbic structures, neocortex, thalamus and superior colliculus is predominantly extrinsic. In the rodent, as much as a third of presynaptic cholinergic markers in the cerebral cortex originates from choline acetyltransferase (CUT)positive interneurons. Such putatively cholinergic interneurons may also exist in the fetal primate brain but apparently not during adulthood (Hendry et al, 1987). In the adult primate, it appears that the cortical cholinergic innervation is exclusively extrinsic. The major cholinergic innervation for limbic structures and neocortex arises from four groups of cholinergic neurons in the basal forebrain; for
the thalamus from two cholinergic cell groups in the pontomesencephalic brainstem; for the interpeduncular nucleus (at least in part) from the medial habenula; and for the superior colliculus from the parabigeminal nucleus. In addition to these major pathways, there are also lesser cholinergic projections from the basal forebrain to the striatum, thalamus (especially the reticular and medodorsal nuclei), and the interpeduncular nucleus; from the pontomesencephalic cell groups to the cerebral cortex and the superior colliculus; and from the parabigeminal nucleus to the thalamus. The cholinergic cells that provide the major ascending projections to the cerebral cortex and .thalamic nuclei are not entirely confined within traditional nuclear groups and they are also frequently intermingled with noncholinergic neurons. We therefore proposed an alternative designation of Chl-Ch8 in order to specificially designate the major groups of cholinergic projection neurons (Fig. 1). According to this nomenclature, Chl-Ch4 designate the cholinergic cell groups centered around the general area of the medial septal nucleus (Chl), nucleus of the diagonal band of Broca (Ch2), nucleus of the horizontal band of Broca (Ch3) and the nucleus basalis of Meynert (Ch4); Ch5 and Ch6 designate the cholinergic cells centered around the pedunculopontine and laterodorsal nuclei, respectively; Ch7 designates the cholinergic cells in the medial habenula and Ch8 designates the cholinergic neurons in the
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RETICULAR FORMATION DE~CENDING PROJECTIONS TOBRAINSTEM AND? SPINAL CORD
Fig. 1. Diagrammatic representation of some chohergic pathways. The solid arrows indicate major pathways and the broken arrows minor pathways. The open circle and arrow indicate that the thalamocortical pathway is non-cholinergic.
parabigeminal nucleus. Chl and Ch2 are major sources of cholinergic projections to the hippocampus, Ch3 to the olfactory bulb, Ch4 to the amygdala and the cerebral cortex, Ch5-Ch6 to the thalamic nuclei, Ch7 to the interpeduncular nucleus and Ch8 to the superior colliculus. The Ch4, Ch5, Ch6 cell groups are particularly extensive in the primate brain. These three cell groups contain a compact center and also interstitial elements that extend into adjacent fiber bundles and nuclei. The density of cholinergic fibers in n m r t e x and in thalamic nuclei shows major regional variations. For example, limbic and paralimbic areas of the cerebral cortex contain a far denser concentration of presynaptic cholinergic markers than immediately adjacent sensory association areas. These limbic and paralimbic areas also seem to be the only parts of the cerebral cortex that have substantial projections back into the basal forebrain cholinergic cell groups. Of all the cholinergic cell groups in the primate brain, the Ch4 cell group, located predominantly within the nucleus basalis of Meynert, is the
largest. This is in keeping with the marked cortical development in the primate line of evolution. On morphological grounds, the Ch4 complex in the monkey brain has been subdivided into anteromedial (Ch4am), anterolateral (Ch4al), intermediate (Ch4i) and posterior (Ch4p) sectors. The corticopetal projections from Ch4 display considerable overlap. However, each part of the cerebral cortex appears to receive its major cholinergic innervation from specific subsectors of Ch4. In that sense, the ascending projections from Ch4 to the cortical surface are topographically organized. This information on the organization of cholinergic pathways has been obtained in laboratory animals and has been reviewed elsewhere (Mesulam, 1988). Recent observations are indicating that much of this organization is also shared in the human brain (Mesulam et al., 1983; Pearson et al., 1983; Nagai et al., 1983; Hedreen et al., 1984; Saper and Chelimsky, 1984; German et al., 1985; Mizukawa et al., 1986; Mesulam and Geula, 1988; Mesulam et al., 1989). The purpose of this report is to concentrate on the information that has been derived in the human brain, especially on the Ch4, Ch5 and Ch6 cell groups. Human chemical neuroanatomy Initial experiments with monoclonal antibodies to ChAT did not yield satisfactory immunostaining in the human basal forebrain. Subsequent immunohistochemical preparations based on a polyclonal antibody have demonstrated the existence of numerous ChAT-rich neurons in this region (Mesulam and Geula, 1988). We recently studied the constellation of cholinergic neurons centered around the nucleus basalis of Meynert (Fig. 2). In keeping with the nomenclature that we proposed for the monkey brain, these cholinergic neurons were designated as Ch4. Concurrent histochemical and immunological staining demonstrated that all ChAT-positive Ch4 neurons also contained AChE. However, there is a small number of AChE-rich magnocellular cell bodies in the region of the nucleus basalis of
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Fig. 2. CUT-immunoreactive (i.e. cholinergic) neurons of the human nucleus basalis (Ch4). From a 76-year-old man. Polyclona antibody, gift of L. Hersh. Magnification X 150 (From Mesulam and Geula, 1988).
Meynert which appeared ChAT-negative. Our observations show that approximately 90% of the magnocellular hyperchromic neurons in the human nucleus basalis are CUT-positive and therefore belong to Ch4. The Ch4 complex of the human brain has a compact part that overlaps with the nucleus basalis of Meynert and numerous additional interstitial elements embedded within the adjacent fiber bundles such as the anterior commissure, inferior thalamic peduncle, ansa peduncularis, diagonal band of Broca, and the internal capsule. On morphological grounds, the compact part of the human Ch4 complex could be divided into anteromedial (Ch4am), anterolateral (Ch4al), anterointermediate (Ch4ai), intermediate (Ch4i) and
posterior (Ch4p) sectors. In addition to its larger size, it also appeared that the human Ch4 displays a greater level of differentiation than the Ch4 complex in the monkey brain. Previous observations had already noted that the nucleus basalis of Meynert shows a gradual increase in size and differentiationin the course of phylogenetic evolution (Gorry, 1963). Each hemisphere of the human brain contains approximately 200,000 magnocellular basal forebrain neurons, most of which belong to Ch4 (Arendt et al., 1985). The human basal forebrain is cytochemically heterogeneous. For example, ChAT-containing Ch4 neurons of the nucleus basalis are intermingled with noncholinergic but NADPHd-positive neurons (Mesulam et al., 1989). Our observations
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show that some nucleus basalis neurons (e.g. those that are CUT-negative and NADPHd-positive) do not belong to Ch4 and that some Ch4 neurons (e.g. interstitial CUT-positive neurons embedded within the internal capsule) are located outside the traditional boundaries of the nucleus basalis. This cytochemical heterogeneity and the lack of confinement within cytoarchitectonic boundaries provide two of the most important justifications for the alternative Ch terminology. Immunohistochemicalobservations in the reticular formation of the human pontomesencephalic region have revealed the presence of many
CUT-rich neurons. These neurons form two major complexes with a morphological organization very similar to the one described for the Ch5 and Ch6 cell groups in the brain of rodents and nonhuman primates (Mesulam et al., 1989). One of these cholinergic cell groups, corresponding to the Ch5 complex of other animals, reaches its peak density within the compact pedunculopontine nucleus of the human brain but also extends into the regions through which the superior cerebellar peduncle and central tegmental tract course (Fig. 3). The second constellation, designated Ch6, is centered on the laterodorsal tegmental nucleus
Fig. 3. ChAT immunoreactive neurons in the human pedunculopontine nucleus (Ch5). Same brain and same antibody as in Fig. 2. Magnification X 175. (From Mesulam et al, 1989).
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and spreads into the central gray and medial longitudinal fasciculus. The two groups are related to each other in the form of partially overlapping constellations rather than discrete nuclei with firm boundaries. The Ch5 and Ch6 groups display a compact central core within the traditional nuclear boundaries of the pedunculopontine nucleus (pars compacta) and the laterodorsal tegmental nucleus, respectively. Both cholinergic cell groups also have interstitial elements that extend into surrounding fiber bundles. As in the case of Ch4, cytochemical heterogeneity is evident. For example, there is considerable interminghng of cholinergic Ch6 neurons with adjacent catecholaminergic neurons of the nucleus locus coeruleus complex. As in the basal forebrain, the lack of confinement within conventional nuclear boundaries and the cytochemical heterogeneity provide the justification for the Ch designation of these cholinergic cell groups. The majority of the basal forebrain and brainstem cholinergic cells are magnocellular and hyperchromic. However, the Ch4 neurons are considerably larger than those in Ch5 and Ch6. The Ch4, Ch5 and Ch6 cell groups fit the description of “open” nuclei since their constituent neurons display considerable cytological heteromorphism, overlapping and isodendritic branching patterns and a propensity for extending into adjacent fiber bundles in the form of interstitial elements. These properties, shared by other cell groups of the reticular formation, support the suggestion of Ramon-Moliner and Nauta that the magnocellular cells of the basal forebrain (corresponding to the nucleus basalis and its cholinergic elements) represent a telencephalic extension of the brainstem isodendritic reticular core (RamonMoliner and Nauta, 1966). The brainstem and forebrain cholinergic cell groups in the human brain share the cytochemical feature of being AChE-rich. However, there are also differences in the cytochemical signature of the individual cholinergic cell groups. For example, Ch5 and Ch6 but not Ch4 neurons are NADPHd-rich. Furthermore, all Ch4 neurons contain receptors for nerve growth factor (NGFr)
whereas Ch5 and Ch6 neurons do not contain immunohistochemically detectable NGFr. The NGFr is synthesized by the Ch4 perikarya and is transported anterogradely into the cholinergic axons that innervate the cerebral cortex. It is thought that Ch4 neurons require the trophic effect of NGF for survival (Hefti et al., 1986). This growth factor is synthesized by cortical neurons and binds to the NGF receptor molecules in cholinergic axons. The NGF-NGFr complex is then transported retrogradely to Ch4 cell bodies in the basal forebrain. The Ch5-Ch6 neurons display cytological features that are identical to those of Ch4 but do not require the trophic effect of NGF. The cholinergic neurons of Ch4, Ch5 and Ch6 also contain a number of neuropeptides. It appears that atriopeptin is present in almost all Ch5 and Ch6 neurons (at least in the rat) while calbindin and galanin are present in Ch4 neurons, at least in the monkey (Celio and Norman, 1985; Standaert et al., 1986; Walker et al., 1987). These overall differences in cytochemical signature probably influence the selective vulnerability of each cholinergic cell group to various physiological and degenerative processes. Two kinds of experiments have been used to chart the anatomical organization of ascending cholinergic projections in experimental animals. One is based on the concurrent demonstration of choline acetyltransferase and retrogradely transported horseradish peroxidase. In another group of experiments, lesions in various cholinergic cell groups have been combined with subsequent histochemical and biochemical determinations of cholinergic markers at the target sites of ascending projections. These approaches are not applicable to the human brain. However, some relatively indirect observations provide pertinent information. For example, patients with Alzheimer’s disease show a profound depletion of presynaptic cortical cholinergic markers and also a loss of cell bodies in Ch4. There is a significant positive correlation between the extent of loss in cortical presynaptic markers and the extent of cell loss in Ch4. This
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indirectly supports the notion that Ch4 cells are the source of the corticopetal cholinergic innvervation (Etienne et al., 1986). In many cases of Alzheimer’s disease, the depletion of cortical cholinergic markers is very widespread and uniformly severe. Such cases are not very useful for studying the topography of corticopetal projections from Ch4. However, in some patients, the loss of presynaptic cholinergic markers is relatively selective. In such cases, it becomes possible to match the topography of cholinergic depletion in the cerebral cortex with the topography of cell loss in Ch4. For example, we described two cases in whom the cell loss in Ch4 was unevenly distributed (Mesulam and Geula, 1988). In these patients, the cell loss in Ch4 was greatest (80-88%) in the posterior sector of Ch4 (Ch4p) and least (20-54%) in its anterior sector (Ch4a). Experiments based on retrograde transport combined with ChAT immunohistochemistry in the monkey had shown that Ch4p provides the major cholinergic innervation for the superior temporal gyrus and temporopolar cortex, whereas Ch4a provides the major cholinergic input for frontoparietal opercular cortex, the amygdala, and medial frontoparietal cortex (Mesulam et al., 1983; 1986b). Of these regions, sections from temporopolar and opercular cortex were available in these two cases of Alzheimer’s disease and were stained with a sensitive acetylcholinesterase histochemical method that allowed us to count cortical AChE-rich (putatively cholinergic) axons. When compared to brains from age-matched, non-demented individuals, cholinergic axons in both cases of Alzheimer’s disease were dramatically depleted in temporopolar cortex but virtually unaltered in the opercular cortex. These observations provide indirect support for the notion that the major cholinergic innervation of temporopolar cortex in the human brain is likely to emanate from Ch4p rather than from Ch4a, a conclusion consistent with the anatomical relationships that had been shown with experimental methods in the rhesus monkey. Many additional cases with relatively selective loss of corti-
cal cholinergic innervation will need to be studied in order to determine how closely the organization of ascending cholinergic projections from Ch4 in the human parallels the overall organization determined in the monkey brain. Brains of patients who have suffered structural damage to portions of Ch4 (as a consequence of stroke, tumor and so on) can also provide very useful information. Ascending cholinergic projections from Ch5Ch6 to thalamic nuclei have been demonstrated conclusively in several subprimate species with the help of retrograde tracing methods combined with immunohistochemistry (e.g. Hallanger et al., 1987). We assume that analogous projections exist in the primate brain but a definitive demonstration is yet to be published in either monkey or man. There are major regional variations in the distribution of cholinergic projections to cortical areas and thalamic nuclei. Recent observations, based mostly on AChE histochemistry, have demonstrated that the regional variations described in
Fig. 4. AChE-rich (putatively cholinergic) axons in paralimbic temporopolar cortex in a 91-year-old man. Magnification X 162. (From Mesulam and Geula, 1988).
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Fig. 5. This patient had a stroke which also damaged the uncus and amygdala. The brain was then processed with the Nauta method for the selective impregnation of degenerated fibers. The photomicrograph shows degenerated fibers (arrow) within the nucleus basalis, demonstrating the presence of a neural projection from the damaged uncal-amygdaloid area to the nucleus basalis.
the monkey brain also exist in the human (Mesulam and Geula, 1988). Our histochemical preparations in the neurologically normal human brain show that paralimbic areas such as the caudal orbitofrontal cortex, insula, temporopolar cortex, entorhinal cortex, and parts of the cingulate gyms contain a more intense concentration of presynaptic cholinergic markers than immediately adjacent sensory association areas (Fig. 4). Core limbic areas such as the hippocampus and amygdala also display a very high intensity of cholinergic markers. Experiments based on the anterograde transport of tritiated amino acids in monkeys, indicate that the major cortical input to the Ch4 region originates from a very limited set of regions, almost all of which belong to the limbic-paralimbic group of areas (Mesulam and Mufson, 1984; Russchen et al., 1985). It is unknown whether a simi-
lar organization exists in the human brain. We have examined the brains of two patients, one with a surgical lesion in the cingulate area and the other with a vascular lesion in the uncus and amygdala. In these brains, we detected anterograde degeneration (revealed with the Nauta method) in the region of Ch4. These observations, subject to all the caveats that are associated with axonal degeneration methods, indicate that the human Ch4 may also receive limbic and paralimbic input (Fig. 5). Many additional cases are needed, however, to determine if the type of selectivity demonstrated in the monkey is also present in the human brain. Behavioral implications Experimental observations in several species of animals have implicated central cholinergic path-
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ways in the regulation of many behaviors including extrapyramidal motor control, arousal, sleep, mood, and especially memory. The relationship to memory and learning h e attracted a great deal of interest. In humans, cholinergic antagonists cause a disruption of memory capacity (Drachman and Leavitt, 1974). In laboratory animals, basal forebrain lesions which deplete cortical cholinergic innervation cause memory impairments that can be reversed by the administration of cholinomimetic drugs (Flicker et al., 1983; Ridley et al., 1986). The importance of cholinergic innervation to learning and memory m a y reflect several mechanisms. One possibility is that acetylcholineplays a special role in the cellular events that underly learning. For example, cholinergic transmission appears to play a direct role in the establishment of hippocampal long term potentiation (Tanaka et al., 1989). Alternatively, the influence upon memory could reflect the higher density of cholinergic pathways within limbic and paralimbic areas. Because of this selective concentration, cholinergic agonists may have a relatively greater impact on limbic and paralimbic parts of the brain, areas which are known to play a major role in the organization of memory and learning. Based on an analysis of regional variations in cholinergic innervation, we made the suggestion that cortical cholinergic pathways could gate sensory information into and out of the limbic system (Mesulam et al., 1986). Many lines of investigation have shown that the transfer of information from sensory association cortex to limbic structures plays a pivotal role in memory and learning. Lesions that directly disrupt corticopetal cholinergic pathways, systemically administered cholinergic antagonists or disease conditions that involve basal forebrain cholinergic neurons may each interfere with memory and learning by disrupting this putative sensory-limbic gating mechanism. The existence of a major cortical cholinergic depletion in Alzheimer’s disease is well known (Bowen et al., 1976; Davies and Maloney, 1976).
Conceivably, cortical cholinergic fibers may have a special vulnerability to the pathology of Alzheimer‘s disease but it is unlikely that the cholinergic lesion is a prime mover in the pathophysiology of this complex degenerative condition (Mesulam, 1986). In addition to the cholinergic depletion, Alzheimer’s disease is also associated with major neuronal and axonal degeneration in many limbic and association areas. These additional lesions can also underlie many of the behavioral changes seen in these patients. While the loss of cholinergic innervation undoubtedly contributes to the amnesias and other mental state deficits seen in Alzheimer’s disease, it is not likely to constitute their primary anatomical substrate. The ascending cholinergicinnervation from Ch4 to cortex appears to be affected early and very severely in Alzheimer‘s disease. However, there is no evidence to favor a transmitter-specific vulnerability. For example, other cholinergic projections (including the intrinsic innervation in the striatum and the axending projection from Ch5-Ch6 to thalamus) appear to be relatively spared. Furtherm e , cholinergic innervation is only one of several corticopetal pharmacosystems involved in Alzheimer’s disease. Just about every other widespread transmitter system that innervates cortex (e.g. serotonergic, norepinephrinergic) is also depleted, albeit to a lesser extent and probably not as early in the course of the disease (Mann et al., 1982). These are some of the reasons for suggesting that the prime mover in the pathology of this complex disease may originate in cortex and then cause retrograde alterations in cell groups that have widespread corticopetal projections. If there is a special relationship between Alzheimer’s disease and the basal forebrain cholinergic neurons, this relationship may reflect the very widespread projections of these neurons to cerebral cortex, and perhaps their special dependency on cortically synthesized NGF. The relationship of age to the cholinergic innervation of cortex is complex. Some investigators have reported a decline of cholinergic markers in the cortex of aging animals. Others have failed to
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confirm these observations. Some reports, including a few in the human brain, describe an age-related decrease in the number of Ch4 neurons while others suggest that the major alteration takes the form of a decrease in perikaryal volume rather than a loss in the number of Ch4 neurons (see Mesulam et al., 1987 for review). We performed a semiquantitative study of AChE-rich (putatively cholinergic) axons in three cytoarchitectonic subregions of the human brain (Geula and Mesulam, 1989). There was an overall age-related decline in the density of these cholinergic axons but this was both modest and regionally selective. When we compared the density of these cholinergic axons from the second to the ninth decade of life, we found that the greatest loss did not exceed 20-25%. This contrasts sharply with the 80-905% loss that occurs in Alzheimer’s disease. There was also some regional specificity to the age-related changes. For example, the changes in cingulate cortex were of much lesser intensity than those in entorhinal and inferotemporal cortex. Aging is frequently associated with modest but clearly measurable deficits in memory function. Drachman and Leavitt (1974) showed that the administration of anticholinergic agents to young volunteers elicited memory impairments similar to those that arise during normal aging. These observations suggest that age-related memory changes may be caused by a depletion of cholinergic innervation. The very severe cholinergic depletion in Alzheimer’s disease, the basal forebrain neuronal loss and the additional non-cholinergic degenerative changes (e.g. plaques, tangles), make it unlikely that cholinergic therapies alone will have a major impact in treating the mental changes. However, the situation may be quite different in normal aging where the cortical cholinergic depletion is modest, the basal forebrain cell loss probably absent and the additional degenerative changes relatively inconspicuous. These considerations suggest that cholinomimetic therapies may well reverse (or even pervent) agerelated alterations of memory and learning.
Anatomical experiments show that the cortical input into Ch4 originates from limbic and paralimbic areas but not from other motor, sensory and association areas. Thus, most cortical areas have no direct feedback control over the cholinergic input that they receive whereas a handful of limbic-paralimbic areas are in a position to exert substantial monosynaptic control over their own cholinergic input and also over the cholinergic input directed to all other parts of the cerebral cortex. Because of this arrangement, the Ch4 group can act as a pivotal cholinergic relay for rapidly switching the physiological state of the entire neocortex in a way that primarily reflects the internal emotional and motivational state of the organism as encoded by limbic and paralimbic areas. This limited corticofugal control of widespread corticopetal projections is also a feature of other subcortical nuclei such as the nucleus locus coeruleus and the brainstem raphe nuclei. This skewed organization appears to be a key feature of brain structures that regulate behavioral states such as mood, arousal and attention (Mesulam, 1987). While much has been learned about the organization of cholinergic pathways, much remains to be discovered. The human brain has a unique anatomy that underlies a behavioral repertoire not found in other species. So far our observations show that the overall plan of organization for ascending cholinergic pathways in the human brain displays many similarities to that of other primates. However, it is important to realize that the cholinergic pathways in the human brain may also possess distinctive and unique features. Much of the research in trying to reveal these finer details of human cholinergic neuroanatomy will depend on the fortuitous availability of appropriate cases, will not be as clear cut as animal experiments and will take a much longer time to conduct. Nonetheless, it is essential to obtain this information in order to understand further how this pharmacosystem is organized in the human brain, how it differs from the organization of cholinergic pathways in other animals and in what way these
240 afferents to the thalamus in the rat. J. Comp. Neurol. 262: 105-124. Hedreen, J.C., Struble, R.G., Whitehouse, P.J. and Price, D.L. (1984) Topography of the magnocellular basal forebrain system in human brain. J. Neuroputhol. Exp. Neurol. 43: Admowledgements 1-21. Hefti, F., Hartikka, J., Salvaterra, A., Weiner, W.J. and Mash, We thank Leah Christie, Kristin Loud and D.C. (1986) Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain. NeuAnnemarie Dineen for expert secretarial and techrosci. Lett. 69: 37-41. nical assistance. Supported in part by the McHendry, S.H., Jones, E.G., Wackey, H.P. and Chalupa, L.M. Knight Foundation, Javits Neuroscience Investi(1987) Choline acetyltransferase-hmunoreactive neurons in fetal monkey cerebral cortex. Bruin Res., 465: 313-317. gator Award (NS20285)’ an Alzheimer’s Disease Mann, D.A., Yates, P.O. and Hawkes, J. (1982) The Research Center Grant (AGO5134) and the Alznoradrenergic system in Alzheimer‘s disease. J. Neurol. heimer’s Disease and Related Disorders AssociaN e u r w g . Psychiut., 45: 115-119. tion (ADRDA). Mesulam, M-M. (1986) Alzheimer plaques and cortical cholinergic innervation. Neuroscience, 17: 275-276. Mesulam, M-M. (1987) Asymmetry of neural feedback in the References organization of behavioral states. Science, 237: 537-538. Mesulam, M-M. (1988) Central cholinergic pathways: NeuroArendt, T., Bigl, V., Tennstedt, A. and Arendt, A. (1985) anatomy and some behavioral implications. In M. Avoli, Neuronal loss in different parts of the nucleus basalis is T.A. Reader, RW. Dykes and P. Gloor, (Eds.), Neurotrunsrelated to neuritic plaque formation in cortical target areas mitters und Cortical Function, Chapter 15, Plenum, New in Alzheimer’s disease.Neuroscience, 14: 1-14. York, pp. 237-260. Bowen, D.M., Smith, C.B., White, P. and Davison, A.N. (1976) Mesulam, M-M. and Geula, C. 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Saper, C.B. and Chelimsky, T.C. (1984) A cytoarchitectonic and histochemical study of nucleus basalis and associated cell groups in the normal human brain. Neuroscience, 13: 1023-1037. Standaert, D.G., Saper, C.B., Rye, D.C. and Wainer, B.H. (1986) Colocalization of atnopeptin-like immunoreactivity with choline acetyltransferase- and substance P-like immunoreactivity in the pedunculopontine and laterodorsal tepental nuclei in the rat. Bruin Res., 382: 163-168. Tanaka, Y., Sakurai, M. and Hayashi, S. (1989) Effect of scopolamine and HP 029, a cholinesterase inhibitor, on long-term potentiation in hippocampal slices of the guinea pig. Neurosci. Lett., 98: 179-183. Walker, L.C., Koliatsos, V.E., Kitt, C.A., Richardson, R.T. and Price,D.L. (1987) Galanin-containingsomata in the nucleus basalis/diagonal band complex. SOC.N w o s c i . Abstr. 13: 995.
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CHAPTER 26
Human brain cholinergic pathways M.-Marsel Mesulam Bullard and Denny-Brown Laboratories, Division of Neuroscience and Behavioral Neurologv, Hamard Neurology Department and the Dana Research Institute of the Beth Israel Hospital, Boston, MA 02215, U.S.A.
Introduction The past decade has witnessed considerable advances in unravelling the organization of central cholinergic pathways. The availability of new anatomical methodology and the observation that cortical cholinergic innervation is markedly depleted in Alzheimer’s disease constitute two important factors that have catalysed much of the recent progress in this field. Acetylcholine is one of the most ubiquitous neurotransmitters in the mammalian central nervous system. Neuroanatomical experiments indicate that the cholinergic innervation of a given brain structure can be intrinsic or extrinsic. The innervation of the striatum, for example, is almost exclusively intrinsic and arises from cholinergic interneurons. In contrast, the cholinergic innervation of limbic structures, neocortex, thalamus and superior colliculus is predominantly extrinsic. In the rodent, as much as a third of presynaptic cholinergic markers in the cerebral cortex originates from choline acetyltransferase (CUT)positive interneurons. Such putatively cholinergic interneurons may also exist in the fetal primate brain but apparently not during adulthood (Hendry et al, 1987). In the adult primate, it appears that the cortical cholinergic innervation is exclusively extrinsic. The major cholinergic innervation for limbic structures and neocortex arises from four groups of cholinergic neurons in the basal forebrain; for
the thalamus from two cholinergic cell groups in the pontomesencephalic brainstem; for the interpeduncular nucleus (at least in part) from the medial habenula; and for the superior colliculus from the parabigeminal nucleus. In addition to these major pathways, there are also lesser cholinergic projections from the basal forebrain to the striatum, thalamus (especially the reticular and medodorsal nuclei), and the interpeduncular nucleus; from the pontomesencephalic cell groups to the cerebral cortex and the superior colliculus; and from the parabigeminal nucleus to the thalamus. The cholinergic cells that provide the major ascending projections to the cerebral cortex and .thalamic nuclei are not entirely confined within traditional nuclear groups and they are also frequently intermingled with noncholinergic neurons. We therefore proposed an alternative designation of Chl-Ch8 in order to specificially designate the major groups of cholinergic projection neurons (Fig. 1). According to this nomenclature, Chl-Ch4 designate the cholinergic cell groups centered around the general area of the medial septal nucleus (Chl), nucleus of the diagonal band of Broca (Ch2), nucleus of the horizontal band of Broca (Ch3) and the nucleus basalis of Meynert (Ch4); Ch5 and Ch6 designate the cholinergic cells centered around the pedunculopontine and laterodorsal nuclei, respectively; Ch7 designates the cholinergic cells in the medial habenula and Ch8 designates the cholinergic neurons in the
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RETICULAR FORMATION DE~CENDING PROJECTIONS TOBRAINSTEM AND? SPINAL CORD
Fig. 1. Diagrammatic representation of some chohergic pathways. The solid arrows indicate major pathways and the broken arrows minor pathways. The open circle and arrow indicate that the thalamocortical pathway is non-cholinergic.
parabigeminal nucleus. Chl and Ch2 are major sources of cholinergic projections to the hippocampus, Ch3 to the olfactory bulb, Ch4 to the amygdala and the cerebral cortex, Ch5-Ch6 to the thalamic nuclei, Ch7 to the interpeduncular nucleus and Ch8 to the superior colliculus. The Ch4, Ch5, Ch6 cell groups are particularly extensive in the primate brain. These three cell groups contain a compact center and also interstitial elements that extend into adjacent fiber bundles and nuclei. The density of cholinergic fibers in n m r t e x and in thalamic nuclei shows major regional variations. For example, limbic and paralimbic areas of the cerebral cortex contain a far denser concentration of presynaptic cholinergic markers than immediately adjacent sensory association areas. These limbic and paralimbic areas also seem to be the only parts of the cerebral cortex that have substantial projections back into the basal forebrain cholinergic cell groups. Of all the cholinergic cell groups in the primate brain, the Ch4 cell group, located predominantly within the nucleus basalis of Meynert, is the
largest. This is in keeping with the marked cortical development in the primate line of evolution. On morphological grounds, the Ch4 complex in the monkey brain has been subdivided into anteromedial (Ch4am), anterolateral (Ch4al), intermediate (Ch4i) and posterior (Ch4p) sectors. The corticopetal projections from Ch4 display considerable overlap. However, each part of the cerebral cortex appears to receive its major cholinergic innervation from specific subsectors of Ch4. In that sense, the ascending projections from Ch4 to the cortical surface are topographically organized. This information on the organization of cholinergic pathways has been obtained in laboratory animals and has been reviewed elsewhere (Mesulam, 1988). Recent observations are indicating that much of this organization is also shared in the human brain (Mesulam et al., 1983; Pearson et al., 1983; Nagai et al., 1983; Hedreen et al., 1984; Saper and Chelimsky, 1984; German et al., 1985; Mizukawa et al., 1986; Mesulam and Geula, 1988; Mesulam et al., 1989). The purpose of this report is to concentrate on the information that has been derived in the human brain, especially on the Ch4, Ch5 and Ch6 cell groups. Human chemical neuroanatomy Initial experiments with monoclonal antibodies to ChAT did not yield satisfactory immunostaining in the human basal forebrain. Subsequent immunohistochemical preparations based on a polyclonal antibody have demonstrated the existence of numerous ChAT-rich neurons in this region (Mesulam and Geula, 1988). We recently studied the constellation of cholinergic neurons centered around the nucleus basalis of Meynert (Fig. 2). In keeping with the nomenclature that we proposed for the monkey brain, these cholinergic neurons were designated as Ch4. Concurrent histochemical and immunological staining demonstrated that all ChAT-positive Ch4 neurons also contained AChE. However, there is a small number of AChE-rich magnocellular cell bodies in the region of the nucleus basalis of
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Fig. 2. CUT-immunoreactive (i.e. cholinergic) neurons of the human nucleus basalis (Ch4). From a 76-year-old man. Polyclona antibody, gift of L. Hersh. Magnification X 150 (From Mesulam and Geula, 1988).
Meynert which appeared ChAT-negative. Our observations show that approximately 90% of the magnocellular hyperchromic neurons in the human nucleus basalis are CUT-positive and therefore belong to Ch4. The Ch4 complex of the human brain has a compact part that overlaps with the nucleus basalis of Meynert and numerous additional interstitial elements embedded within the adjacent fiber bundles such as the anterior commissure, inferior thalamic peduncle, ansa peduncularis, diagonal band of Broca, and the internal capsule. On morphological grounds, the compact part of the human Ch4 complex could be divided into anteromedial (Ch4am), anterolateral (Ch4al), anterointermediate (Ch4ai), intermediate (Ch4i) and
posterior (Ch4p) sectors. In addition to its larger size, it also appeared that the human Ch4 displays a greater level of differentiation than the Ch4 complex in the monkey brain. Previous observations had already noted that the nucleus basalis of Meynert shows a gradual increase in size and differentiationin the course of phylogenetic evolution (Gorry, 1963). Each hemisphere of the human brain contains approximately 200,000 magnocellular basal forebrain neurons, most of which belong to Ch4 (Arendt et al., 1985). The human basal forebrain is cytochemically heterogeneous. For example, ChAT-containing Ch4 neurons of the nucleus basalis are intermingled with noncholinergic but NADPHd-positive neurons (Mesulam et al., 1989). Our observations
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show that some nucleus basalis neurons (e.g. those that are CUT-negative and NADPHd-positive) do not belong to Ch4 and that some Ch4 neurons (e.g. interstitial CUT-positive neurons embedded within the internal capsule) are located outside the traditional boundaries of the nucleus basalis. This cytochemical heterogeneity and the lack of confinement within cytoarchitectonic boundaries provide two of the most important justifications for the alternative Ch terminology. Immunohistochemicalobservations in the reticular formation of the human pontomesencephalic region have revealed the presence of many
CUT-rich neurons. These neurons form two major complexes with a morphological organization very similar to the one described for the Ch5 and Ch6 cell groups in the brain of rodents and nonhuman primates (Mesulam et al., 1989). One of these cholinergic cell groups, corresponding to the Ch5 complex of other animals, reaches its peak density within the compact pedunculopontine nucleus of the human brain but also extends into the regions through which the superior cerebellar peduncle and central tegmental tract course (Fig. 3). The second constellation, designated Ch6, is centered on the laterodorsal tegmental nucleus
Fig. 3. ChAT immunoreactive neurons in the human pedunculopontine nucleus (Ch5). Same brain and same antibody as in Fig. 2. Magnification X 175. (From Mesulam et al, 1989).
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and spreads into the central gray and medial longitudinal fasciculus. The two groups are related to each other in the form of partially overlapping constellations rather than discrete nuclei with firm boundaries. The Ch5 and Ch6 groups display a compact central core within the traditional nuclear boundaries of the pedunculopontine nucleus (pars compacta) and the laterodorsal tegmental nucleus, respectively. Both cholinergic cell groups also have interstitial elements that extend into surrounding fiber bundles. As in the case of Ch4, cytochemical heterogeneity is evident. For example, there is considerable interminghng of cholinergic Ch6 neurons with adjacent catecholaminergic neurons of the nucleus locus coeruleus complex. As in the basal forebrain, the lack of confinement within conventional nuclear boundaries and the cytochemical heterogeneity provide the justification for the Ch designation of these cholinergic cell groups. The majority of the basal forebrain and brainstem cholinergic cells are magnocellular and hyperchromic. However, the Ch4 neurons are considerably larger than those in Ch5 and Ch6. The Ch4, Ch5 and Ch6 cell groups fit the description of “open” nuclei since their constituent neurons display considerable cytological heteromorphism, overlapping and isodendritic branching patterns and a propensity for extending into adjacent fiber bundles in the form of interstitial elements. These properties, shared by other cell groups of the reticular formation, support the suggestion of Ramon-Moliner and Nauta that the magnocellular cells of the basal forebrain (corresponding to the nucleus basalis and its cholinergic elements) represent a telencephalic extension of the brainstem isodendritic reticular core (RamonMoliner and Nauta, 1966). The brainstem and forebrain cholinergic cell groups in the human brain share the cytochemical feature of being AChE-rich. However, there are also differences in the cytochemical signature of the individual cholinergic cell groups. For example, Ch5 and Ch6 but not Ch4 neurons are NADPHd-rich. Furthermore, all Ch4 neurons contain receptors for nerve growth factor (NGFr)
whereas Ch5 and Ch6 neurons do not contain immunohistochemically detectable NGFr. The NGFr is synthesized by the Ch4 perikarya and is transported anterogradely into the cholinergic axons that innervate the cerebral cortex. It is thought that Ch4 neurons require the trophic effect of NGF for survival (Hefti et al., 1986). This growth factor is synthesized by cortical neurons and binds to the NGF receptor molecules in cholinergic axons. The NGF-NGFr complex is then transported retrogradely to Ch4 cell bodies in the basal forebrain. The Ch5-Ch6 neurons display cytological features that are identical to those of Ch4 but do not require the trophic effect of NGF. The cholinergic neurons of Ch4, Ch5 and Ch6 also contain a number of neuropeptides. It appears that atriopeptin is present in almost all Ch5 and Ch6 neurons (at least in the rat) while calbindin and galanin are present in Ch4 neurons, at least in the monkey (Celio and Norman, 1985; Standaert et al., 1986; Walker et al., 1987). These overall differences in cytochemical signature probably influence the selective vulnerability of each cholinergic cell group to various physiological and degenerative processes. Two kinds of experiments have been used to chart the anatomical organization of ascending cholinergic projections in experimental animals. One is based on the concurrent demonstration of choline acetyltransferase and retrogradely transported horseradish peroxidase. In another group of experiments, lesions in various cholinergic cell groups have been combined with subsequent histochemical and biochemical determinations of cholinergic markers at the target sites of ascending projections. These approaches are not applicable to the human brain. However, some relatively indirect observations provide pertinent information. For example, patients with Alzheimer’s disease show a profound depletion of presynaptic cortical cholinergic markers and also a loss of cell bodies in Ch4. There is a significant positive correlation between the extent of loss in cortical presynaptic markers and the extent of cell loss in Ch4. This
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indirectly supports the notion that Ch4 cells are the source of the corticopetal cholinergic innvervation (Etienne et al., 1986). In many cases of Alzheimer’s disease, the depletion of cortical cholinergic markers is very widespread and uniformly severe. Such cases are not very useful for studying the topography of corticopetal projections from Ch4. However, in some patients, the loss of presynaptic cholinergic markers is relatively selective. In such cases, it becomes possible to match the topography of cholinergic depletion in the cerebral cortex with the topography of cell loss in Ch4. For example, we described two cases in whom the cell loss in Ch4 was unevenly distributed (Mesulam and Geula, 1988). In these patients, the cell loss in Ch4 was greatest (80-88%) in the posterior sector of Ch4 (Ch4p) and least (20-54%) in its anterior sector (Ch4a). Experiments based on retrograde transport combined with ChAT immunohistochemistry in the monkey had shown that Ch4p provides the major cholinergic innervation for the superior temporal gyrus and temporopolar cortex, whereas Ch4a provides the major cholinergic input for frontoparietal opercular cortex, the amygdala, and medial frontoparietal cortex (Mesulam et al., 1983; 1986b). Of these regions, sections from temporopolar and opercular cortex were available in these two cases of Alzheimer’s disease and were stained with a sensitive acetylcholinesterase histochemical method that allowed us to count cortical AChE-rich (putatively cholinergic) axons. When compared to brains from age-matched, non-demented individuals, cholinergic axons in both cases of Alzheimer’s disease were dramatically depleted in temporopolar cortex but virtually unaltered in the opercular cortex. These observations provide indirect support for the notion that the major cholinergic innervation of temporopolar cortex in the human brain is likely to emanate from Ch4p rather than from Ch4a, a conclusion consistent with the anatomical relationships that had been shown with experimental methods in the rhesus monkey. Many additional cases with relatively selective loss of corti-
cal cholinergic innervation will need to be studied in order to determine how closely the organization of ascending cholinergic projections from Ch4 in the human parallels the overall organization determined in the monkey brain. Brains of patients who have suffered structural damage to portions of Ch4 (as a consequence of stroke, tumor and so on) can also provide very useful information. Ascending cholinergic projections from Ch5Ch6 to thalamic nuclei have been demonstrated conclusively in several subprimate species with the help of retrograde tracing methods combined with immunohistochemistry (e.g. Hallanger et al., 1987). We assume that analogous projections exist in the primate brain but a definitive demonstration is yet to be published in either monkey or man. There are major regional variations in the distribution of cholinergic projections to cortical areas and thalamic nuclei. Recent observations, based mostly on AChE histochemistry, have demonstrated that the regional variations described in
Fig. 4. AChE-rich (putatively cholinergic) axons in paralimbic temporopolar cortex in a 91-year-old man. Magnification X 162. (From Mesulam and Geula, 1988).
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Fig. 5. This patient had a stroke which also damaged the uncus and amygdala. The brain was then processed with the Nauta method for the selective impregnation of degenerated fibers. The photomicrograph shows degenerated fibers (arrow) within the nucleus basalis, demonstrating the presence of a neural projection from the damaged uncal-amygdaloid area to the nucleus basalis.
the monkey brain also exist in the human (Mesulam and Geula, 1988). Our histochemical preparations in the neurologically normal human brain show that paralimbic areas such as the caudal orbitofrontal cortex, insula, temporopolar cortex, entorhinal cortex, and parts of the cingulate gyms contain a more intense concentration of presynaptic cholinergic markers than immediately adjacent sensory association areas (Fig. 4). Core limbic areas such as the hippocampus and amygdala also display a very high intensity of cholinergic markers. Experiments based on the anterograde transport of tritiated amino acids in monkeys, indicate that the major cortical input to the Ch4 region originates from a very limited set of regions, almost all of which belong to the limbic-paralimbic group of areas (Mesulam and Mufson, 1984; Russchen et al., 1985). It is unknown whether a simi-
lar organization exists in the human brain. We have examined the brains of two patients, one with a surgical lesion in the cingulate area and the other with a vascular lesion in the uncus and amygdala. In these brains, we detected anterograde degeneration (revealed with the Nauta method) in the region of Ch4. These observations, subject to all the caveats that are associated with axonal degeneration methods, indicate that the human Ch4 may also receive limbic and paralimbic input (Fig. 5). Many additional cases are needed, however, to determine if the type of selectivity demonstrated in the monkey is also present in the human brain. Behavioral implications Experimental observations in several species of animals have implicated central cholinergic path-
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ways in the regulation of many behaviors including extrapyramidal motor control, arousal, sleep, mood, and especially memory. The relationship to memory and learning h e attracted a great deal of interest. In humans, cholinergic antagonists cause a disruption of memory capacity (Drachman and Leavitt, 1974). In laboratory animals, basal forebrain lesions which deplete cortical cholinergic innervation cause memory impairments that can be reversed by the administration of cholinomimetic drugs (Flicker et al., 1983; Ridley et al., 1986). The importance of cholinergic innervation to learning and memory m a y reflect several mechanisms. One possibility is that acetylcholineplays a special role in the cellular events that underly learning. For example, cholinergic transmission appears to play a direct role in the establishment of hippocampal long term potentiation (Tanaka et al., 1989). Alternatively, the influence upon memory could reflect the higher density of cholinergic pathways within limbic and paralimbic areas. Because of this selective concentration, cholinergic agonists may have a relatively greater impact on limbic and paralimbic parts of the brain, areas which are known to play a major role in the organization of memory and learning. Based on an analysis of regional variations in cholinergic innervation, we made the suggestion that cortical cholinergic pathways could gate sensory information into and out of the limbic system (Mesulam et al., 1986). Many lines of investigation have shown that the transfer of information from sensory association cortex to limbic structures plays a pivotal role in memory and learning. Lesions that directly disrupt corticopetal cholinergic pathways, systemically administered cholinergic antagonists or disease conditions that involve basal forebrain cholinergic neurons may each interfere with memory and learning by disrupting this putative sensory-limbic gating mechanism. The existence of a major cortical cholinergic depletion in Alzheimer’s disease is well known (Bowen et al., 1976; Davies and Maloney, 1976).
Conceivably, cortical cholinergic fibers may have a special vulnerability to the pathology of Alzheimer‘s disease but it is unlikely that the cholinergic lesion is a prime mover in the pathophysiology of this complex degenerative condition (Mesulam, 1986). In addition to the cholinergic depletion, Alzheimer’s disease is also associated with major neuronal and axonal degeneration in many limbic and association areas. These additional lesions can also underlie many of the behavioral changes seen in these patients. While the loss of cholinergic innervation undoubtedly contributes to the amnesias and other mental state deficits seen in Alzheimer’s disease, it is not likely to constitute their primary anatomical substrate. The ascending cholinergicinnervation from Ch4 to cortex appears to be affected early and very severely in Alzheimer‘s disease. However, there is no evidence to favor a transmitter-specific vulnerability. For example, other cholinergic projections (including the intrinsic innervation in the striatum and the axending projection from Ch5-Ch6 to thalamus) appear to be relatively spared. Furtherm e , cholinergic innervation is only one of several corticopetal pharmacosystems involved in Alzheimer’s disease. Just about every other widespread transmitter system that innervates cortex (e.g. serotonergic, norepinephrinergic) is also depleted, albeit to a lesser extent and probably not as early in the course of the disease (Mann et al., 1982). These are some of the reasons for suggesting that the prime mover in the pathology of this complex disease may originate in cortex and then cause retrograde alterations in cell groups that have widespread corticopetal projections. If there is a special relationship between Alzheimer’s disease and the basal forebrain cholinergic neurons, this relationship may reflect the very widespread projections of these neurons to cerebral cortex, and perhaps their special dependency on cortically synthesized NGF. The relationship of age to the cholinergic innervation of cortex is complex. Some investigators have reported a decline of cholinergic markers in the cortex of aging animals. Others have failed to
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confirm these observations. Some reports, including a few in the human brain, describe an age-related decrease in the number of Ch4 neurons while others suggest that the major alteration takes the form of a decrease in perikaryal volume rather than a loss in the number of Ch4 neurons (see Mesulam et al., 1987 for review). We performed a semiquantitative study of AChE-rich (putatively cholinergic) axons in three cytoarchitectonic subregions of the human brain (Geula and Mesulam, 1989). There was an overall age-related decline in the density of these cholinergic axons but this was both modest and regionally selective. When we compared the density of these cholinergic axons from the second to the ninth decade of life, we found that the greatest loss did not exceed 20-25%. This contrasts sharply with the 80-905% loss that occurs in Alzheimer’s disease. There was also some regional specificity to the age-related changes. For example, the changes in cingulate cortex were of much lesser intensity than those in entorhinal and inferotemporal cortex. Aging is frequently associated with modest but clearly measurable deficits in memory function. Drachman and Leavitt (1974) showed that the administration of anticholinergic agents to young volunteers elicited memory impairments similar to those that arise during normal aging. These observations suggest that age-related memory changes may be caused by a depletion of cholinergic innervation. The very severe cholinergic depletion in Alzheimer’s disease, the basal forebrain neuronal loss and the additional non-cholinergic degenerative changes (e.g. plaques, tangles), make it unlikely that cholinergic therapies alone will have a major impact in treating the mental changes. However, the situation may be quite different in normal aging where the cortical cholinergic depletion is modest, the basal forebrain cell loss probably absent and the additional degenerative changes relatively inconspicuous. These considerations suggest that cholinomimetic therapies may well reverse (or even pervent) agerelated alterations of memory and learning.
Anatomical experiments show that the cortical input into Ch4 originates from limbic and paralimbic areas but not from other motor, sensory and association areas. Thus, most cortical areas have no direct feedback control over the cholinergic input that they receive whereas a handful of limbic-paralimbic areas are in a position to exert substantial monosynaptic control over their own cholinergic input and also over the cholinergic input directed to all other parts of the cerebral cortex. Because of this arrangement, the Ch4 group can act as a pivotal cholinergic relay for rapidly switching the physiological state of the entire neocortex in a way that primarily reflects the internal emotional and motivational state of the organism as encoded by limbic and paralimbic areas. This limited corticofugal control of widespread corticopetal projections is also a feature of other subcortical nuclei such as the nucleus locus coeruleus and the brainstem raphe nuclei. This skewed organization appears to be a key feature of brain structures that regulate behavioral states such as mood, arousal and attention (Mesulam, 1987). While much has been learned about the organization of cholinergic pathways, much remains to be discovered. The human brain has a unique anatomy that underlies a behavioral repertoire not found in other species. So far our observations show that the overall plan of organization for ascending cholinergic pathways in the human brain displays many similarities to that of other primates. However, it is important to realize that the cholinergic pathways in the human brain may also possess distinctive and unique features. Much of the research in trying to reveal these finer details of human cholinergic neuroanatomy will depend on the fortuitous availability of appropriate cases, will not be as clear cut as animal experiments and will take a much longer time to conduct. Nonetheless, it is essential to obtain this information in order to understand further how this pharmacosystem is organized in the human brain, how it differs from the organization of cholinergic pathways in other animals and in what way these
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