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Neuropsychopharmacology: The Fifth Generation of Progress

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Structure and Function of Cholinergic Pathways in the Cerebral Cortex, Limbic System, Basal Ganglia, and Thalamus of the Human Brain

Marek-Marsel Mesulam


The cerebral cortex, thalamus, and basal ganglia of the human brain provide neural templates for the transformation of simple movements and sensations into exceedingly complex psychological acts and experiences. These transformations occur through the orderly transfer of information along parallel and serial pathways that lead to the formation of large-scale distributed networks. The thousands of neural pathways that contribute to the formation of these networks can be divided into two major groups. One group contains point-to-point (discrete) projections such as those that interconnect individual thalamic nuclei with their cortical targets. The second group contains equally important regulatory (diffuse) neural projections which (a) innervate the entire cerebral cortex, (b) arise from relatively small nuclei, and (c) employ small amines such as dopamine, histamine, norepinephrine, serotonin, and acetylcholine as the transmitter substances. These regulatory pathways are less involved in determining the specific contents of experience than in modulating its general flavor and impact on the individual. Each of these regulatory pathways has been implicated in the modulation of global behavioral states such as emotion, motivation, and arousal (39; see also Introduction to Preclinical Neuropsychopharmacology).

Every complex psychological phenomenon represents an intermingling of contributions from discrete and regulatory pathways. The content of a memory, for example, is probably dependent on the specific information transported along discrete point-to-point pathways that interconnect association cortex with limbic structures. The emotional tone of the recollection and perhaps the speed and efficiency of recall, on the other hand, may be determined by the activity of regulatory pathways that innervate the same parts of the brain. Among the various components of complex behavior, those that represent the contributions of the regulatory pathways are the most amenable to psychopharmacological treatment. In fact, the vast majority of modern neuropharmacology is based on drugs that alter the activity along one or more of these transmitter-specific regulatory pathways. This is one reason why regulatory pathways have attracted such a great deal of interest. This chapter will deal with one of these regulatory pathways, namely, the one that employs acetylcholine as its transmitter substance.

Cholinergic pathways are phylogenetically ancient and anatomically ubiquitous. Their presence is identified by markers such as acetylcholinesterase (AChE), muscarinic and nicotinic receptors (see Neuronal Nicotinic Acetylcholine Receptors: Novel Targets for CNS Therapeutics, Molecular Biology, Pharmacology, and Brain Distribution of Subtypes of the Muscarinic Receptor, and Cholinergic Transduction), and choline acetyltransferase (ChAT). Of these markers, ChAT is confined to presynaptic cholinergic elements whereas the other two are found in both presynaptic cholinergic neurons and also in the postsynaptic cholinoceptive neurons. All peripheral motor nerves (cranial and spinal) and a substantial portion of the autonomic nervous system are cholinergic. In this chapter, however, the emphasis will be on cholinergic pathways that are exclusively distributed within the central nervous system.

There are eight major cholinergic cell groups that project to other central nervous system structures. Most of these cholinergic cell groups do not respect traditional nuclear boundaries, and their constituent cholinergic cells are intermixed with other noncholinergic neurons. We have therefore introduced the Ch1–Ch8 nomenclature in order to classify the cholinergic neurons within these eight cell groups (38).

According to this nomenclature, Ch1 designates the cholinergic cells associated with the medial septal nucleus, Ch2 those associated with the vertical nucleus of the diagonal band, Ch3 those associated with the horizontal limb of the diagonal band nucleus, Ch4 those associated with the nucleus basalis of Meynert, Ch5 those associated with the pedunculopontine nucleus of the rostral brainstem, Ch6 those associated with the laterodorsal tegmental nucleus also in the rostral brainstem, Ch7 those in the medial habenula, and Ch8 those in the parabigeminal nucleus.

Tracer experiments in a number of animal species have shown that Ch1 and Ch2 provide the major cholinergic innervation for the hippocampal complex, Ch3 for the olfactory bulb, Ch4 for the cerebral cortex and amygdala, Ch5 and Ch6 for the thalamus, Ch7 for interpeduncular nucleus, and Ch8 for the superior colliculus. There are also lesser connections from Ch1–Ch4 and Ch8 to the thalamus and from Ch5–Ch6 to the cerebral cortex (38, 72).

All basal ganglia display widespread cholinergic innervation. The cholinergic innervation of the striatum is mostly intrinsic, arising from cholinergic interneurons. The striatum also receives a lesser cholinergic innervation from Ch4 and from Ch5–Ch6. The cholinergic innervation of other basal ganglia such as the globus pallidus, the subthalamic nucleus, and the pars compacta of the substantia nigra is exclusively extrinsic, probably originating mostly from Ch5–Ch6.

In the rodent brain, there are intrinsic cholinergic interneurons which may provide up to 30% of the cholinergic innervation in the cerebral cortex. No such cholinergic interneurons have been reported in the adult primate cerebral cortex or in the thalamus of any species studied this far. Therefore, the cholinergic innervation of the adult primate cerebral cortex and thalamus is exclusively extrinsic.


The basal forebrain of the primate brain contains four overlapping constellations of cholinergic projection neurons. Studies in the monkey brain show that approximately 10% of perikarya within the boundaries of the medial septal nucleus are cholinergic and belong to the Ch1 cell group, approximately 70% of neurons in the vertical limbic nucleus of the diagonal band are cholinergic and belong to the Ch2 cell group, approximately 1% of neurons in the horizontal nucleus of the diagonal band are cholinergic and belong to the Ch3 cell group, and approximately 90% of the large neurons in the nucleus basalis of the substantia innominata are cholinergic and belong to the Ch4 cell group. Of these four cholinergic cell groups, the Ch4 group is by far the largest and the one that has been most extensively studied in the human brain (41, 47).

Because nearly 90% of the nucleus basalis (NB) neurons in the human brain express choline acetyltransferase (and therefore belong to Ch4), this cell group can also be designated as the NB–Ch4 complex. The more general "NB" term can be used to designate all of the components in this nucleus (large and small cells, cholinergic and noncholinergic), whereas the more restrictive Ch4 designation is reserved for the contingent of cholinergic NB neurons as revealed by ChAT immunohistochemistry.

The human NB–Ch4 extends from the level of the olfactory tubercle to that of the anterior hippocampus, spanning a distance of 13–14 mm in the sagittal plane. It attains its greatest mediolateral width of 18 mm within the substantia innominata (subcommissural gray) (FIG. 1. AChE histochemistry of a coronal section through the anterior sector of NB–Ch4 showing its anteromedial (am) and anterolateral (al) subsector. Medial is to the left, dorsal is to the top. At this level, the NB–Ch4 complex is surrounded by the anterior commissure (ac), the amygdala (ag), and the ventral globus pallidus (gpv). This section also passes through the putamen (pt) and the more dorsal part of the globus pallidus (gp). From the brain of a 91-year-old subject. Magnification x5. (From ref. 41.) ). Arendt et al. (1) have estimated that the human NB–Ch4 complex contains 200,000 neurons in each hemisphere. Thus, the NB–Ch4 contains at least ten times as many neurons as the nucleus locus coeruleus, which has approximately 15,000 neurons in the adult human brain (69). On topographical grounds, the constituent neurons of the human NB–Ch4 complex can be subdivided into six sectors that occupy its anteromedial (Ch4am), anterolateral (NB–Ch4al) anterointermediate (NB–Ch4ai), intermediodorsal (NB–Ch4id) intermedioventral (NB–Ch4iv), and posterior (NB–Ch4p) regions.

Gorry (16) has pointed that the NB displays a progressive evolutionary trend, becoming more and more extensive and differentiated in more highly evolved species, especially in primates and cetacea. Our observations are consistent with this general view and show that the human NB–Ch4 is a highly differentiated and relatively large structure. Although many morphological features of the human NB–Ch4 are similar to those described for the rhesus monkey, there is also a sense of increased complexity and differentiation. For example, a prominent Ch4ai sector is easily identified in the human brain but not in the rhesus monkey. In addition to these "compact" neuronal sectors, the Ch4 complex also contains "interstitial" elements which are embedded within the internal capsule, the diagonal band of Broca, the anterior commissure, the ansa peduncularis, the inferior thalamic peduncle, and the ansa lenticularis. The physiological implications of this intimate association with fiber bundles are unknown. Conceivably, the NB–Ch4 complex, and especially its interstitial components, could monitor and perhaps influence the physiological activity along these fiber tracts. The presence of these interstitial components outside the traditional boundaries of the nucleus basalis is another reason why Ch4 and NB are not synonymous terms.

There is no strict delineation between the boundaries of NB–Ch4 and adjacent cell groups such as those of the olfactory tubercle, preoptic area, hypothalamic nuclei, striatal structures, nuclei of the diagonal band, amygdaloid nuclei, and globus pallidus. In addition to this "open" nuclear structure, the neurons of NB–Ch4 are heteromorphic in shape and have an isodendritic morphology with overlapping dendritic fields, many of which extend into fiber tracts traversing the basal forebrain. These characteristics are also present in the nuclei of the brainstem reticular formation and have led to the suggestion that the NB–Ch4 complex could be conceptualized as a telencephalic extension of the brainstem reticular core (58).

All neurons of the Ch1–Ch4 cell groups contain AChE and ChAT in the perikarya, dendrites, and axons. Approximately 90% of Ch1–Ch4 neurons express the p75 low-affinity nerve growth factor receptor (NGFr) (44, 53). Nearly all Ch1–Ch4 cholinergic neurons of the human brain also express calbindin D28K (14). There are considerable interspecies differences in the cytochemical signature of basal forebrain cholinergic neurons (14). For example, 20–30% of cholinergic neurons in the basal forebrain of the rat contain reduced nicotinamideadenine-dinucleotide-phosphate-diaphorase (NADPHd) activity [which is now known to overlap with nitric oxide synthase activity (70)], whereas none of the basal forebrain cholinergic neurons in the monkey or human brain do so. Furthermore, the basal forebrain cholinergic neurons of the rat do not express calbindin D28K, whereas almost all Ch1–Ch4 neurons of the monkey and human do. There are differences even within primates. For example, Ch1–Ch4 neurons of the monkey express galanin, whereas this does not occur in the human brain (30). Such cytochemical differences need to be taken into account when developing animal models for human diseases that effect the basal forebrain cholinergic cell groups.

The Ch1–Ch4 groups are the only neurons which regularly express large amounts of NGFr in the adult human central nervous system. The NGFr is expressed in the perikaryon and is transported intraaxonally to the cerebral cortex, where it binds cortically produced NGF (23). The NGF–NGFr complex is then transported retrogradely to the Ch1–Ch4 cell body, which is dependent on this retrogradely transported NGF for its survival. Because all Ch1–Ch8 cholinergic neurons express ChAT, the presence of this enzyme in an axon designates it as cholinergic but does not help to identify its origin. Because only Ch1–Ch4 neurons express NGFr, axonal NGFr helps to identify the axon not only as cholinergic but also as originating in the basal forebrain. However, because not all Ch1–Ch4 neurons are NGFr-positive, the absence of NGFr in a cholinergic axon does not rule out the possibility that the axon originates in the basal forebrain.


Axonal transport experiments combined with AChE histochemistry or ChAT immunocytochemistry in the monkey have shown that Ch1 and Ch2 provide the major source of cholinergic innervation for the hippocampal complex, that Ch3 provides the major source of cholinergic innervation for the olfactory bulb, and that Ch4 is the major source of cholinergic projections for the entire cerebral cortex and the amygdala. This type of experimental evidence is not available for the human brain. However, there is indirect support for the existence of a similar organization. For example, in patients with Alzheimer's disease, cell loss in the NB–Ch4 complex is almost always significantly correlated with the magnitude of ChAT depletion in the cerebral cortex (11).

Experimental neuroanatomical methods in the monkey brain have shown that different cortical areas receive their major cholinergic input from individual sectors of the NB–Ch4 complex. Thus, Ch4am provides the major source of cholinergic input to medial cortical areas including the cingulate gyrus; Ch4al to frontoparietal and opercular regions and the amygdaloid nuclei; Ch4id–Ch4iv to laterodorsal frontoparietal, peristriate and midtemporal regions; and Ch4p to the superior temporal and temporopolar areas (47). The experimental methods that are needed to reveal this topographic arrangement cannot be used in the human brain. However, indirect evidence for the existence of a similar topographical arrangement can be gathered from patients with Alzheimer's disease. We described two patients in whom extensive loss of cholinergic fibers in temporopolar but not frontal opercular cortex was associated with marked cell loss in the posterior (Ch4p) but not the anterior (Ch4am + Ch4al) sectors of Ch4 (41). This relationship is consistent with the topography of the projections in the monkey brain.

The distribution of cholinergic innervation in the human cerebral cortex has been studied in detail with the help of AChE histochemistry, ChAT immunocytochemistry, and NGFr immunocytochemistry (13, 43, 45). All cytoarchitectonic regions and layers of the cerebral cortex display a dense cholinergic innervation (FIG. 2. AChE histochemistry in layer III of inferotemporal visual association cortex of 22-year-old subject. Dorsal is to the top. The AChE-rich axons (which are also ChAT-positive) form a dense plexus. The axons (curved arrows) display numerous swellings which may constitute sites of axonal varicosities and synaptic contact. The axons come in close association with cortical neurons that are AChE-rich (double arrow) as well as with those that are not (single arrow). x415. (From ref. 41.) ). These fibers have numerous varicosities and, on occasion, complex preterminal profiles arranged in the form of dense clusters. The density of cholinergic axons is higher in the more superficial layers (layers I and II and the upper parts of layer III) of the cerebral cortex. Distinct patterns of lamination exist in individual cytoarchitectonic regions.

There are also major and statistically significant differences in the overall density of cholinergic axons among the various cytoarchitectonic areas (FIG. 3. Dark-field photomicrograph showing the differential density and lamination of cholinergic axons in primary visual cortex (V1) (A) and in enterorhinal cortex (B). Note the very substantial difference in overall density. Magnification x62. (From ref. 45.) ). The cholinergic innervation of primary sensory, unimodal and heteromodal association areas is lighter than that of paralimbic and limbic areas. Within unimodal association areas, the density of cholinergic axons and varicosities is lower in the upstream (parasensory) sectors than in the downstream sectors. Within paralimbic regions, the nonisocortical sectors have a higher density of cholinergic innervation than the isocortical sectors. The highest density of cholinergic axons occurs in core limbic structures such as the hippocampus and the amygdala.

Within the hippocampal complex, the highest density of AChE-rich cholinergic fibers is seen in a thin band along the inner edge of the molecular layer of the dentate gyrus and within parts of the CA2, CA3, and CA4 sectors. The subiculum has a cholinergic innervation that is lighter than that of the other hippocampal sectors (17). In the amygdala, each nucleus has a slightly different profile of cholinergic innervation (10). The density is highest in the central and basal lateral nuclei and lightest in the lateral nucleus. The medial nucleus is the only region of the amygdala that has virtually no cholinergic innervation.

In all cortical and hippocampal fields, NGFr axonal staining is of approximately equivalent density as that of axonal ChAT, providing further evidence that the majority of cholinergic innervation to these regions arises from the Ch1–Ch4 cell groups (46). The one exception occurs in the amygdala, especially in the basolateral nucleus, which contains very light NGFr staining, raising the possibility that the cholinergic innervation to this nucleus and perhaps to other parts of the amygdala arises from NGFr negative Ch1–Ch4 neurons or from cholinergic neurons in the brainstem.


Electronmicroscopic studies in rodents indicate that most cortical cholinergic axons are unmyelinated and that they make symmetrical and asymmetrical synaptic contacts with large numbers of cortical neurons (12, 71). It is also thought that some acetylcholine may be re-leased outside of traditional synaptic contacts and that it may exert its effect by diffusion into receptor-containing sites (68).

The acetylcholine released from presynaptic cholinergic axons of the cerebral cortex exerts its neurotransmitter effects through the mediation of nicotinic and muscarinic receptors. Muscarinic receptors predominate in the mammalian cerebral cortex. Five subtypes of muscarinic cholinergic receptors (m1–m5) have been recognized, each the product of a different gene (3, 26). Three muscarinic receptor subtypes have been characterized pharmacologically (M1–M3), and of these the M1 and M2 subtypes have received the greatest attention. Autoradiographic experiments in the rhesus monkey showed that the pirenzepine-sensitive M1 receptors were far more numerous than M2 receptors. The M1 receptor density reaches the highest levels in components of limbic and association cortex. In contrast, the M2 receptors reach their highest densities in primary sensory and motor areas of the cortex (34).

Immunocytochemical studies in the human brain have identified cortical neurons which express nicotinic and muscarinic receptors. Such neurons are localized predominantly in the pyramidal neurons of layers III and V. Approximately 30% of immunopositive pyramidal neurons were found to display immunoreactivity for both muscarinic and nicotinic receptors (62).

It is thought that all cholinoceptive neurons express AChE in order to hydrolyze acetylcholine. However, only a subset of cholinoceptive neurons give an AChE-rich histochemical reaction (21, 41, 42). Some of these AChE-rich neurons are polymorphic in shape and are distributed preferentially in the deeper cortical layers and the subjacent white matter. Others are pyramidal in shape and are located in layers III and V, especially in association cortex. It is interesting to note that such AChE-rich neurons have a low density in limbic and paralimbic areas, although these parts of the cerebral cortex contain the highest presynaptic cholinergic innervation.

In addition to postsynaptic receptors, cholinergic axons are also thought to contain presynaptic autoreceptors. These presynaptic autoreceptors may be involved in the autoregulation of acetylcholine release.


Nearly all of the hyperchromic neurons in the pedunculopontine nucleus (pars compacta) of the human brain are intensely ChAT-positive and constitute the compact sector of the Ch5 cell group (Ch5c). The Ch5c component is surrounded by a diffuse-interstitial component (Ch5d) containing slightly smaller ChAT-positive neurons embedded within passing fiber systems such as the superior cerebellar peduncle and especially the central tegmental tract. Isolated Ch5 neurons are also seen in the traditional boundaries of the cuneiform nucleus, the parabrachial nuclei, the subcoeruleus zone, and the lateral lemniscus. The interstitial Ch5 neurons are at least as numerous as those within the compact pedunculopontine region (44).

Approximately 10% of neurons within the compact sector of the pedunculopontine nucleus fail to give a detectable ChAT reaction, even though they have an identical appearance in Nissl preparations. The frequency of noncholinergic neurons can be as high as 75% in regions containing the interstitial components of Ch5. Some of the noncholinergic neurons intermingled with Ch5 are catecholaminergic (tyrosine hydroxylase-positive), but the transmitter identity of most of these neurons remains unknown.

The Ch6 complex has its center of gravity within the laterodorsal tegmental nucleus, but its neurons spread into the adjacent parts of the central gray, into the medial longitudinal fasciculus, and even into the regions of the nucleus locus coeruleus. The Ch6 complex reaches peak density at a level somewhat more caudal than the region of peak density for Ch5. The Ch6 neurons are slightly smaller than those of Ch5c. The laterodorsal tegmental nucleus contains a relatively pure population of cholinergic neurons. However, the surrounding zones of the central gray which contain interstitial Ch6 neurons also contain many tyrosine hydroxylase-positive catecholaminergic neurons.

There is no precise delineation between Ch5 and Ch6. The two groups are related to each other in the form of partially overlapping constellations rather than discrete nuclei with firm boundaries. Here as in the basal forebrain, the lack of correspondence with traditional cytoarchitectonic boundaries and the intermingling of cholinergic with noncholinergic neurons justifies the use of the Ch designation.

Experiments in a number of animal species indicate that Ch5–Ch6 neurons provide the vast majority of cholinergic innervation for the thalamus and that this thalamic projection constitutes the major output for Ch5–Ch6 (18, 47). The Ch5–Ch6 cell groups may also provide a relatively sparse projection to parts of the cerebral cortex, the basal forebrain, and a number of extrapyramidal structures such as the striatum, globus pallidus, subthalamic nucleus, and substantia nigra (see refs. 44 and 46 for review).

Animal experiments indicate that the connectivity of Ch5 is not identical to that of Ch6. The Ch5 neurons appear to be more closely interconnected with extrapyramidal structures than the Ch6 neurons (75). Furthermore, the Ch6 group sends relatively few projections to sensory relay nuclei of the thalamus, but it figures prominently in projections to its limbic nuclei (18, 75). In keeping with the limbic affiliations of Ch6, projections have been described from the laterodorsal tegmental nucleus of the baboon to such extrathalamic limbic structures as the cingulate and subicular cortices but not to parietal neocortex (49). Furthermore, the cholinergic neurons of the laterodorsal tegmental nucleus (Ch6) have more extensive projections than those of Ch5 to the lateral septum and medial prefrontal cortex (60). Therefore, it appears that Ch6 is more closely associated with the limbic system, whereas Ch5 participates more extensively in the neural systems subserving sensory processing and extrapyramidal motor control. This distinction further justifies the delineation of Ch5 from Ch6 despite the absence of strict cytoarchitectonic demarcations.


Choline acetyltransferase-positive axonal staining shows that the human thalamus receives substantial and widespread cholinergic innervation (22). The highest levels of ChAT axonal staining are found in intralaminar nuclei (except for the parafascicular nucleus), the reuniens nucleus, the anterodorsal nucleus, medially situated patches in the mediodorsal nucleus, the lateral geniculate nucleus, and the reticular nucleus. The lowest levels are found in the pulvinar and the medial geniculate nucleus. The remaining nuclei display an intermediate density of ChAT-positive cholinergic staining. The cholinergic axons in these nuclei display multiple varicosities and other complex preterminal profiles.

Based on animal experiments, we assume that the majority of these thalamic projections originate in the Ch5–Ch6 cell groups. The existence of NGFr-immunoreactive axonal profiles suggested that some thalamic nuclei of the human brain also receive a second cholinergic innervation from the basal forebrain. Nuclei with the most extensive NGFr immunoreactivity included the mediodorsal nucleus (especially the medially situated patches) as well as the intralaminar and reticular nuclei. The other thalamic nuclei contained only rare NGFr-positive axonal profiles. In each of these areas, the density of ChATimmunoreactive axonal profiles was always higher, and in most instances much higher, than the density of NGFr-immunoreactive profiles, suggesting that even in the nuclei with a prominent dual cholinergic innervation, the preponderant cholinergic input arises from the brainstem. These observations show that some thalamic nuclei— especially the mediodorsal, intralaminar, and reticular— are under complex dual cholinergic influence, one arising from the basal forebrain and the other arising from the brainstem. In contrast to the cerebral cortex, the thalamus contains more M2 than M1 receptors (6) and also a high level of nicotinic binding (76).


The distribution of ChAT-positive varicose axons indicated that the human striatum, globus pallidus, subthalamic nucleus, red nucleus, and substantia nigra receive substantial cholinergic innervation (46). The density of cholinergic innervation is very high in the striatum, high in the subthalamic nucleus and red nucleus, moderate in the globus pallidus and ventral tegmental area, and low in the pars compacta of the substantia nigra. This cholinergic innervation displays a very orderly but also complex organization within each of these subcortical structures.

The overall cholinergic innervation of the four striatal components (caudate, putamen, olfactory tubercle, and nucleus accumbens) is of comparable intensity, but each component shows a complex mosaic of ChAT-positive varicosities organized in the form of light and dark patches. Numerous ChAT-positive perikarya are distributed throughout the striatal components (FIG. 4. Choline acetyltransferase (ChAT) immunocytochemistry in the putamen. A: Multipolar cholinergic neurons (curved arrows) are interspersed throughout the putamen. The ChAT-positive varicosities display intricate variations of density. The lightest areas correspond to the striatal patches (or striasomes), and the more intensely stained areas correspond to the matrix. There is no obvious relationship between the density of ChAT-positive neurons and the density of neuropil staining. Dorsal towards the top, lateral towards the left. Magnification 64. B: Detail of putaminal ChAT immunopositivity. Two ChAT-positive multipolar neurons are embedded in a dense bed of ChAT-positive preterminal profiles (or varicosities). From the brain of a 55-year-old subject. Magnification 343. (From ref. 46.) ).

Animal experiments indicate that the vast majority of striatal cholinergic innervation arises from these cholinergic interneurons. However, we also found that the striatum, especially the putamen, contains NGFrimmunoreactive axons, suggesting that there is a second but lesser cholinergic input from the Ch1–Ch4 neuronal groups. Observations in monkeys show that the striatum may also receive cholinergic projections from the Ch5–Ch6 cell groups of the brainstem (63).

The density of ChAT-positive axons and varicosities in the globus pallidus was modest in comparison to that of the striatum. The rarity of ChAT-positive axonal staining within the striatopallidal bundles eliminates the striatum as the major source of this input. The presence of some NGFr-positive varicosities shows that part of this cholinergic input (especially in the anterior and external pallidal segments) is likely to arise from the Ch1–Ch4 cell groups of the basal forebrain. The majority of the cholinergic innervation for the globus pallidus, especially for its internal sector (which corresponds to the entopeduncular nucleus of nonprimates), however, appears to arise from cholinergic cells outside of the forebrain or from NGFr-negative neurons of the Ch1–Ch4 cell groups. Most of this projection is likely to come from the Ch5–Ch6 cell groups.

The perikarya of the subthalamic nucleus and red nucleus were embedded within a dense matrix of ChAT-positive varicosities. The paucity of NGFr-like immunoreactivity in these two structures suggested that almost all of this cholinergic innervation arises from sources outside of the basal forebrain. A substantial component of this input probably originates from the Ch5 and Ch6 cell groups. There has been considerable controversy, however, concerning the magnitude of the Ch5–Ch6 projection to the subthalamic nucleus (see ref. 46 for review). Conceivably, the subthalamic nucleus and the red nucleus could receive cholinergic input from brainstem sources other than Ch5–Ch6. It has been suggested (but not yet confirmed), for example, that the red nucleus may receive the bulk of its cholinergic input from ChAT-positive neurons of the cerebellum (27).

The melanin-containing (presumably dopaminergic) neurons of the human substantia nigra also receive ChAT-positive axonal innervation. Axonal NGFr is nearly absent in the substantia nigra. Some experiments in rats have suggested that the Ch5–Ch6 cell groups provide the major source of nigral cholinergic input while other reports provide conflicting evidence. Within the substantia nigra, we found that the medially situated pigmented neurons of the ventral tegmental area received a more intense cholinergic innervation than the more laterally situated neurons of the pars compacts. Such neurochemical differences may underlie the different behavioral affiliations displayed by these two major groups of dopaminergic neurons. The pars compacta of the substantia nigra plays a major role in extrapyramidal motor control, whereas the ventral tegmental area seems to be more closely affiliated with the modulation of motivation and related functions of the limbic system.

Electron microscopic investigations in the striatum of the rat show that the dominant mode of cholinergic neurotransmission occurs through symmetrical synapses upon the medium-sized and noncholinergic (presumably GABAergic) projection neurons (55, 57). Assuming that symmetrical synapses are mostly inhibitory, such an input on inhibitory GABAergic projection neurons would have a net excitatory effect upon the targets of striatofugal GABAergic pathways such as the substantia nigra and the globus pallidus. Only 2–3% of cholinergic terminals in the striatum were found to make synaptic contact with cholinergic interneurons (57). It is not known if the extrinsic cholinergic terminals that arise from the basal forebrain have a synaptic organization that sets them apart from the far more numerous intrinsic terminals. In the monkey, muscarinic receptor autoradiography shows that the striatum contains considerably more M1 than M2 receptors (34). Physiological experiments in tissue slices show that muscarinic agonists have complex excitatory and inhibitory effects on striatal neurons (9).

In contrast to the predominance of symmetrical contact within the striatum, the predominant type of cholinergic contact in the subthalamic nucleus and substantia nigra is of the asymmetrical and presumably excitatory type (33, 66). In the pars compacta of the substantia nigra and in the ventral tegmental area, cholinergic neurotransmission is mediated predominantly by M1-like receptors and tends to increase the rate of spontaneous action potential of the dopaminergic neurons (31).

Clinical evidence shows that cholinergic agents tend to produce tremor, whereas anticholinergic agents are quite effective for treating the rigidity and bradykinesia of Parkinsonism (54). The influence of cholinoactive substances upon motor activity has traditionally been attributed to the well-established cholinergic innervation of the striatum. Our observations show that the globus pallidus, red nucleus, substantia, and subthalamic nucleus may also participate in mediating the effects of cholinergic agents upon extrapyramidal function.


The physiological effect of acetylcholine on cholinoceptive cortical neurons is exceedingly complex. The major effect of acetylcholine is to cause a relatively prolonged reduction of potassium conductance so as to make cortical cholinoceptive neurons more susceptible to other excitatory inputs (36, 64); see also Electrophysiology. However, the effect of acetylcholine on cortical neurons can also be inhibitory, either directly or through the mediation of GABAergic interneurons.

Because all regions of the cerebral cortex receive intense cholinergic innervation, it is not surprising that all aspects of cortical function are influenced by cholinergic neurotransmission. In primary visual cortex, for example, cholinergic stimulation does not alter the orientation specificity of a given neuron but increases the likelihood that the neuron will fire in response to its preferred stimulus (61). An analogous effect has been described in somatosensory cortex (48).

The Ch1–Ch4 cell groups of the basal forebrain can be considered as a telencephalic extension of the brainstem reticular formation and also as a direct extension of basomedial limbic cortex. This dual identity helps to understand why arousal and memory are the two major behavioral affiliations of the Ch1–Ch4 cell groups. Thus, experiments in rats have shown that the cortical cholinergic projections from the basal forebrain play a major role in sustaining at least one component of the hippocampal theta rhythm and also the arousal-related low-voltage fast activity of the cortical electroencephalogram (EEG) (5, 65). In a number of animal species, lesions of the Ch4 cell group can cause severe impairments of memory that can be reversed by the systemic administration of agonists (28, 59). We are beginning to understand the cellular bases for these two behavioral affiliations.

Single-unit studies in monkeys have shown that the neurons of the nucleus basalis (Ch4) are particularly sensitive to stimulus novelty and to the motivational relevance of sensory cues (73, 74). The novelty and behavioral significance of a sensory event can therefore influence the cortical release of acetylcholine, which, in turn, modulates the cortical response to the sensory event. Cortical cholinergic pathways are thus in a position to alter the neural impact of sensory experiences according to their behavioral significance. It is easy to see how such a circuitry would have a major influence on cortical arousal. In keeping with this interpretation, the muscarinic blocking agent scopolamine attenuates the cortical P-300 arousal response that is normally elicited by novel or surprising stimuli (19).

The relationship of the Ch1–Ch4 cell groups and of cortical cholinergic innervation to memory function is quite complex. Limbic and paralimbic regions of the cerebral cortex are known to play a critical role in memory and learning. The preferential concentration of cholinergic innervation in these parts of cortex may explain why cholinergic antagonists and cholinoactive drugs seem to have a preferential effect on memory, learning, and other limbic functions such as mood, reward, and aggressive behaviors (15, 77, 78). The role of acetylcholine in hippocampal long-term potentiation (67) may provide another mechanism that underlies the relationship of cholinergic pathways to memory. Recent brain-slice experiments in piriform cortex of the rat have shown that acetylcholine can selectively suppress excitatory intrinsic synaptic transmission through a presynaptic mechanism, while leaving excitatory afferent input unaffected. In a computation model, this selective suppression can prevent interference from previously stored patterns during the learning of new patterns. Hasselmo et al. (20) have argued that this could provide a novel mechanism through which cortical cholinergic innervation could participate in new learning. Buzsaki (4) has proposed a different model according to which the cholinergic innervation, especially of the hippocampal complex, plays a major role in switching from on-line attentive processing, characterized by the hippocampal theta rhythm, to an off-line period of consolidation, characterized by sharp wave activity (see ref. 7 for review). Cholinergic innervation may even participate in cortical plasticity and axonal sprouting (2, 32). We have speculated, for example, that the exceedingly complex presynaptic "dense cluster" profiles displayed by cholinergic axons of the cerebral cortex may reflect events of local plasticity and reorganization in response to individual experience (45).

Another mechanism that links cholinergic axons to memory and learning may be related to the differential regional density of cortical cholinergic innervation. Experimental evidence leads to the conclusion that sensory–limbic pathways play pivotal roles in a wide range of behaviors related to emotion, motivation, and especially memory (37, 50). The primary sensory areas of the cerebral cortex which provide a portal for the entry of sensory information into cortical circuitry. These primary areas project predominantly to upstream (parasensory) unimodal sensory association areas, which, in turn, project to downstream unimodal areas and heteromodal cortex. The heteromodal and downstream unimodal areas collectively provide the major source of sensory information into paralimbic and limbic areas of the brain. Our observations show that the density of cholinergic innervation is lower within unimodal and heteromodal association areas than in paralimbic areas of the brain. Moreover, in the unimodal areas the downstream sectors had a higher density of cholinergic innervation than the upstream sectors. Within all major paralimbic areas, we found that the nonisocortical subsectors, known to have the more extensive interconnections with limbic structures, also had a higher density of cholinergic innervation. Core limbic areas such as the amygdala and hippocampus had the highest densities of cholinergic innervation.

This pattern of differential distribution led us to suggest that sensory information is likely to come under progressively greater cholinergic influence as it is conveyed along the multisynaptic pathways leading to the limbic system. As a consequence of this arrangement, cortical cholinergic innervation may help to channel (or gate) sensory information into and out of the limbic system in a way that is sensitive to the behavioral relevance of the associated experience. The memory disturbances that arise after damage to the Ch1–Ch4 cell groups or after the systemic administration of cholinergic antagonists may therefore reflect a disruption of sensory–limbic interactions which are crucial for effective memory and learning.

In addition to Ch1–Ch4, the cholinergic neurons of the upper brainstem are also intimately involved in the modulation of arousal. Moruzzi and Magoun (52) had introduced the concept of an ascending reticular activating system (ARAS) that acted to desynchronize the cortical electroencephalogram via a relay in the thalamus. Subsequent work revealed that a most important component in this system consists of a cholinergic reticulothalamic pathway that facilitates the activation of corticopetal relay neurons in the thalamus (8, 24, 25, 35, 56). The physiological relevance of this pathway to the reticular activating system was demonstrated by Kayama et al. (29). They identified the Ch5–Ch6 neurons with NDPHd histochemistry and showed that electrical stimulation of these neurons causes a scopolamine-sensitive activation of lateral geniculate neurons and even an occasional enhancement of their response to photic stimulation. Thus, the Ch5–Ch6 neurons can facilitate the transthalamic (and ultimately corticopetal) processing of sensory information in ways that could modulate arousal and attention.

Electrical stimulation of Ch5 causes a hyperpolarization of GABAergic neurons in the reticular nucleus of the thalamus. Because the neurons of the reticular nucleus have an inhibitory effect on thalamic relay neurons, the net effect of Ch5 stimulation is to facilitate transthalamic processing by the excitation of relay nuclei and also through a process of disinhibition (64). The reticular nucleus, which plays a pivotal role in the regulation of many processes related to arousal, attention, and sleep, receives a dual cholinergic innervation, one from Ch5–Ch6 and a second from Ch4.

Acetylcholine appears to be the neurotransmitter responsible for switching the burst firing mode of thalamic neurons during EEG-synchronized sleep toward a tonic firing mode associated with waking and rapid eye movement (REM) sleep 64). The Ch5–Ch6 cell groups are also directly involved in brainstem mechanisms which trigger REM sleep (51).


These observations show that the original concept of the ARAS needs to be expanded to include at least two sources of ascending cholinergic projections, a traditional one in the upper brainstem (Ch5–Ch6) and a second one in the basal forebrain (Ch1–Ch4). Noncholinergic regulatory pathways which arise from the hypothalamus (histaminergic; see Histamine), ventral tegmental area (dopaminergic; see Electrophysiological Properties of Midbrain Dopamine Neurons, Dopaminergic Neuronal Systems in the Hypothalamus, and Mesocorticolimbic Dopaminergic Neurons: Functional and Regulatory Roles), nucleus locus coeruleus (noradrenergic; see Pharmacology and Physiology of Central Noradrenergic Systems and Central Norepinephrine Neurons and Behavior), and brainstem raphe (serotonergic; see Anatomy, Cell Biology, and Maturation of the Serotonergic System: Neurotrophic Implications for the Actions of Psychotropic Drugs and Serotonin and Behavior: A General Hypothesis) and which send widespread projections to the cerebral cortex and thalamus probably also need to be included into this expanded ARAS. Each of these cholinergic and noncholinergic projections can exert a powerful influence on the information-processing state of the thalamus and cerebral cortex in ways that influence attentional, motivational, and arousal states. These ascending regulatory pathways provide the physiological matrix (or state) within which the discrete point-to-point projections that interconnect the cortex, thalamus, and basal ganglia set the vectors of complex behaviors.


I want to thank Leah Christie for expert secretarial assistance. This work was supported in part by a Javits Neuroscience Investigator Award (NS20285).

published 2000