Catecholaminergic-cholinergic interaction in the basal forebrain

L. Zaborszky¹, W.E. Cullinan², and V.N. Luine³

¹ Department of Neurology, University of Virginia, Charlottesville, VA.

² Mental Health Research Institute, University of Michigan, Ann Arbor, MI.

³ Department of Psychology, Hunter College of the City University of New York, NY.

Correspondence:

Lászlo Záborszky, M.D., Ph.D., D.Sc.

Associate Professor

Center for Molecular and Behavioral Neuroscience

Rutgers University

197 University Ave.

Newark, NJ 07102

Fax: (973) 353-1844

E-mail: zaborszky@axon.rutgers.edu

ABSTRACT

Dopamine-ß-hydroxylase (DBH) and tyrosine hydroxylase (TH)-containing axons and terminals are found in close proximity to forebrain corticopetal cholinergic projection neurons throughout extensive forebrain areas, including the nucleus of the horizontal limb of the diagonal band, sublenticular substantia innominata, ventral pallidum, and globus pallidus. Electron microscopic double-labeling studies confirmed synaptic contacts between DBH-positive terminals and cholinergic dendrites in the SI. DBH-positive boutons are usually large, containing clear and dense core vesicles. Synapses formed between DBH-positive terminals and cholinergic neurons were always of the asymmetric type with prominent postsynaptic subjunctional bodies. While the majority of basalocortical cholinergic cells appeared to receive a low number of synaptic contacts, a small population of cholinergic neurons was found to receive multiple synapses from the same axon in the form of climbing-type arrangements. Anterograde tracing studies using the PHA-L method suggested that at least a proportion of the DBH-positive varicosities represent noradrenaline containing terminals originating from the locus coeruleus. Cholinergic neurons located within the ventromedial globus pallidus and the region of the internal capsule were contacted by tyrosine hydroxylase-positive but not DBH-positive fibers, suggesting dopaminergic input to cholinergic neurons in these regions. In a complementary series of experiments, 6-hydroxydopamine lesions of the ascending catecholaminergic fiber systems resulted in reductions of choline acetyltransferase levels in forebrain areas containing cholinergic projection neurons, suggesting that catecholaminergic afferents modulate forebrain corticopetal cholinergic neurons.

Key words: locus coeruleus, dopamine-ß-hydroxylase, tyrosine hydroxylase, choline acetyltransferase, electron microscopic double labeling, biochemistry

Introduction

The basal forebrain cholinergic projection system (BFC) has been the focus of considerable attention as a result of evidence implicating it in a number of behavioral functions, including arousal, sensory processing, motivation, emotion, learning and memory (Bartus et al., l982; Deutch, l983; Rolls, l989; Steriade and McCarley, l990; Dykes et al., l991; Richardson and De Long, l991; Rasmusson, this volume). Cholinergic projection neurons are distributed across a number of classically defined regions of the basal forebrain and collectively project to the entire cortical mantle, including allocortical areas, such as the hippocampus, the amygdala, and the olfactory bulb (for ref. see Wainer and Mesulam, l990; Koliatsos and Price, l991; Zaborszky, l992). In spite of detailed information concerning the efferent projections of these neurons (e.g. Rye et al., 1984; Zaborszky et al., l986b), as well as significant recent progress in our understanding of the mechanisms of acetylcholine action in the cortex (McCormick, 1989; Stewart and Fox, l989; Halliwel, l990; Elaagounly et al., l991; Kunze et al., l992; see chapters of Krnjevic, McCormick, Steriade, Sillito and Woody in this volume), relatively little is known about the afferents which drive BFC neurons.

Data suggesting brainstem catecholaminergic cell groups as sources of input to BFC neurons have been advanced from pharmacological and anatomical studies. Pharmacological manipulations of the septohippocampal (Robinson et al., l979; Costa et al., l983; Robinson, l986, l989) or basalocortical cholinergic systems (Robinson, l986; Wood and McQuade, l986; Casamenti et al., l986; Pepeu et al., l990) suggested the possibility of catecholaminergic input to BFC neurons, although these studies remained open to alternative interpretations as to the site of catecholamine action due to a lack of selective interference of cholinergic neurons. Anatomical experiments using the glyoxylic acid fluorescence technique (Lindvall and Stenevi, l978; Lindvall et al., l978; Moore, l978; Björklund and Lindvall, l986), tyrosine hydroxylase (TH) and dopamine-ß-hydroxylase (DBH) immunocytochemistry (Hökfelt et al., 1977; Chang, l989), and tracing techniques either alone (Beckstead et al., l979; Fallon and Moore, l978; Haring and Wang, l986; Jones and Moore, l977; Jones and Yang, l985; Vertes, l988) or in combination with transmitter identification (Kaliwas et al., l985; Grove, l988; Martinez-Murillo et al., l988b; Semba et al., l988; Jones and Cuello, l989) demonstrated that, in addition to cortical projections, fibers from the ventral tegmental area (A10), locus coeruleus (A6), and the medullary catecholaminergic cell groups (A1-A2) project to the forebrain regions containing cholinergic projection neurons. However, the issue of whether these ascending fiber systems indeed contact cholinergic neurons remained unclear due to the anatomical complexity of the forebrain regions containing these cells: cholinergic neurons are dispersed and intermingled with several other chemically distinct neuronal populations, including GABAergic and peptidergic cells. Thus, a definitive determination of whether catecholaminergic axons contact BFC neurons requires a combined light-electron microscopic analysis, in which synaptic relations and the chemical identity of both the afferent fiber and the postsynaptic target are identified. This paper summarizes recent efforts to identify catecholaminergic inputs to BFC neurons in the rat using this approach. Additional biochemical studies tested the effect of specific removal of ascending brainstem catecholaminergic fibers on forebrain choline acetyltransferase (ChAT) activity. Portions of this work have been previously reported in abstract or review forms (Zaborszky et al., l986c; Zaborszky and Luine, l987; Zaborszky, 1989, 1992; Zaborszky et al., 1991).

Catecholaminergic input to basalocortical cholinergic neurons

Noradrenergic/adrenergic afferents

A light microscopic analysis of the distribution of dopamine-ß-hydroxylase (DBH)[1] -positive fibers and terminals in relation to BFC neurons (Figs. 1A, 2, 3A,B, 4A,B) suggested that cholinergic neurons might receive noradrenergic input. With the exception of those neurons of the globus pallidus and internal capsule, cholinergic neurons are approximated by DBH varicosites in most portions of the BFC system. As seen in the camera lucida drawing of Figure 2, a particularly prominent network of DBH-labeled fibers and terminals was observed in proximity to cholinergic neurons in the substantia innominata (SI), although scattered DBH-positive varicosities were also detected near cholinergic neurons in the horizontal limb of the diagonal band (HD) and magnocellular preoptic nucleus (MP) (Fig. 1A). A high magnification light microscopic screening revealed that an average of 1-3 DBH-positive varicosities per neuron could be detected in direct apposition to labeled cholinergic elements (Fig. 4A,B). These varicosities were randomly distributed around the dendrites, particularly the more distal portions, and were less frequently seen along the cell bodies.

Interestingly, a small population of BFC neurons appeared to receive multiple contacts from DBH-labeled terminals in the form of "climbing-type" arrangements. In such cases, DBH axons were seen to spiral around the cholinergic dendrites, distributing as many as 10-15 terminal boutons. Occasionally these varicosities were

arranged in a "cuff" about 150-200 µm from the cell body. Such "climbing-type" arrangements were most often detected in the SI (Figs. 3A and B), but were also occasionally seen in other basal forebrain regions.

Several types of DBH-positive fibers in the basal forebrain could be classified according to the characteristics of their varicosities and intervaricose segments. The vast majority of DBH terminals in close vicinity to BFC neurons were from axons having an assortment of terminal bouton sizes, although axons with uniformly large or small varicosities were also seen in proximity to cholinergic neurons. The camera lucida drawing of Fig. 2 and the micrographs of Fig. 3A and Fig. 3B illustrate the DBH innervation pattern in the SI. Parallel experiments at the electron microscopic level confirmed synaptic contacts between DBH-positive terminals and cholinergic dendrites. The identified DBH-positive boutons were usually large ( 1um), containing round clear and dense core vesicles, and the synapses were always of the asymmetric type with prominent postsynaptic subjunctional dense bodies (Fig. 5B). It remains to be established whether DBH-varicosities of the smaller type contact BFC neurons, since our selections for electron microscopic analyses were biased toward the larger varicosities.

Dopaminergic input

The distribution of tyrosine hydroxylase (TH)-positive fibers in relation to cholinergic projection neurons at the level of the SI is illustrated in Fig. 1B. At this forebrain level, the heavy TH-positive fiber bundles en route to the striatum are readily apparent ventromedial to and within the globus pallidus. The SI and adjacent lateral preoptic-hypothalamic areas are also pervaded by heavy TH fiber systems. A high magnification light microscopic screening of TH-positive terminals in direct approximation to BFC neurons is presented in Figs. 4C and D, which are from sections adjacent to those labeled for DBH/ChAT and depicted in Figs. 4A and B. As evident from these figures, the distribution pattern of putative TH/ChAT contacts observed was generally similar to the distribution of DBH/ChAT interactions in most forebrain areas. Notable exceptions to this were seen within the ventromedial portion of the globus pallidus, as well as within the caudal globus pallidus-internal capsule region, where putative contacts were observed between TH-positive terminals and cholinergic elements, whereas no such arrangements were detected between DBH-positive terminals and BFC neurons. Indeed, few DBH fibers/terminals were found in these regions. The majority of appositions between TH-labeled terminals and cholinergic neural elements were of the individual type, and were located adjacent to the cell body or proximal dendrite. In a number of cases the TH-positive terminals were apparently boutons 'en passant' from fibers en route to the striatum. Occasionally TH-labeled axons approximated cholinergic dendrites in climbing-type arrangements, such as shown in Fig. 3C.

Confirmation of synaptic contact between TH-positive terminals and cholinergic elements was obtained from electron microscopic experiments, an example of which is shown in Figure 6.

As the initial enzyme in catecholamine biosynthesis, the presence of TH in boutons contacting BFC neurons cannot be taken as evidence for dopaminergic nature. Indeed, the TH antibody used in our experiments labeled many fibers in the basal forebrain resembling DBH-positive fibers on morphological grounds. However, the differential distribution of TH- versus DBH-labeled axons in selected forebrain territories (Fig. 4), as well several distinct morphological characteristics observed at the ultrastructural level, suggested that a proportion of the TH-positive terminals in the globus pallidus and internal capsule represent dopaminergic inputs to BFC neurons.

The origin of catecholaminergic terminals in the basal forebrain

Noradrenaline

Experiments using the PHA-L anterograde tracing method suggest that at least part of the noradrenergic innervation of BFC neurons originates in the locus coeruleus (Zaborszky et al., in preparation). The results from one such case in which a PHA-L injection was made predominantly within the locus coeruleus is illustrated in Figures 7, 8, 9. PHA-L labeled varicosities were detected in direct apposition to cholinergic projection neurons in the SI, and HD/MP areas from this case (Figs. 7C, 8). In contrast, cholinergic neurons in peripallidal regions appeared to be in a position to receive few, if any, inputs from axons originating in the locus coeruleus (Fig. 7, inset). Preliminary experiments at the electron microscopic level (Fig. 9) indicate that corticopetal cholinergic neurons are indeed contacted by locus coeruleus axons, and that individual locus coeruleus fibers establish synaptic contacts both with cholinergic and non-cholinergic neuronal elements.

A high magnification light microscopic analysis of putative contact sites between basal forebrain cholinergic neurons and PHA-L labeled varicosities originating from the locus coeruleus revealed that the innervation of the BFC system from this region is rather diffuse (Fig 10B). A comparison of the distribution of these putative interaction sites and those between BFC neurons and DBH-positive varicosities (Fig. 10A) suggests contributions to the innervation of the BFC system from other noradrenergic cell groups as well, such as A1 and A2 (Zaborszky et al., l991). Regional variations noted in the type and density of catecholaminergic fibers in the basal forebrain lend further support to the idea that subpopulations of BFC neurons receive differential innervation from specific noradrenergic cell groups.

Dopamine

Retrograde tracer injections into the cholinergic rich region of the globus pallidus (Haring and Wang, l986; Hallanger and Wainer, l988; Martinez-Murillo et al., l988b; Semba et al., l988; Jones and Cuello, l989) resulted in a large number of backfilled neurons in the substantia nigra (A9 catecholaminergic cell group) and retrorubral field (RRF: A8 catecholaminergic cell group). Experiments combining retrograde tracing with TH immmunocytochemistry revealed that almost all retrograde neurons in the zona compacta of the substantia nigra were double labeled, while in the RRF 75% of the retrogradely labeled cells contained TH (Jones and Cuello, l989). Since many dopaminergic axons in the globus pallidus representing fibers of passage toward the striatum may have been retrogradely transported the tracer, further studies are needed to determine the origin and topographic distribution of putative dopaminergic input to BFC neurons.

Functional significance of noradrenergic/cholinergic interactions in the basal forebrain

Based on ultrastructural confirmation, three general classes of afferents to BFC neurons have been previously defined: a) inhibitory inputs from local GABAergic neurons (Zaborszky et al., l986b; Zaborszky and Cullinan, l992); b) peptidergic afferents from local and perhaps distant NPY-, somatostatin- (Zaborszky, l989, l992) substance P- (Bolam et al., l986) and enkephalin-containing neurons (Chang et al., l989; Martinez-Murillo et al., l988a), and c) presumably excitatory inputs from the amygdala, and to some extent, the hypothalamus (Zaborszky et al., l984; Zaborszky and Cullinan, l989; Cullinan and Zaborszky, l991). Both the long-range putative excitatory afferents and inhibitory inputs to basal forebrain cholinergic neurons show specific regional distribution patterns. In addition, ultrastructural evidence suggests that asymmeteric, presumably excitatory contacts occur mainly on dendrites, particularly the more distal segments, and that GABAergic and peptidergic synapses are primarily concentrated near the cell body and proximal dendritic elements (Zaborszky et al., l992). In contrast to these classes of afferents, noradrenergic inputs to BFC neurons appear to be diffusely distributed throughout widespread portions of the BFC system, and in general, are randomly established over the dendritic arbor, as well the cell bodies to some extent.

The postsynaptic effect of NE has been generally viewed as that of enhancement of the responsivity of the neurons to other strong inhibitory or excitatory inputs (Servan-Schreiber et al., 1990). These modulatory actions of NE on central neurons are mediated through different adrenoreceptor-coupled second messenger systems affecting various intracellular events, including conductance changes at voltage-gated ion channels (Foehring et al., l989; McCormick et al., l991; Harley, l991; Woodward et al., l991; Waterhouse et al., l991). Since the majority of BFC neurons apparently receive this type of NE input, it may be that their firing properties are tonically set according to the discharge pattern of locus coeruleus neurons which in turn is related to the level of vigilance, orienting and attending of the organism (Foote et al., l983, l991; Aston-Jones et al., l991). Noradrenergic inputs would thus gate, according to the behavioral state of the animal, the transmission of more "specific" sensory, or motivational information through the BFC neurons to the cortex. In other words, this noradrenergic innervation might be important to enable subpopulations of BFC neurons to participate in selective information processing driven by topographically organized excitatory or local inhibitory afferents. In the absence of these inputs this transmission would be expected to be disrupted, similar to the situation in the dentate gyrus, where high frequency stimulation of the perforant path can no longer elicit LTP in NE depleted slice preparations (Harley, l991).

An additional level of complexity is presented by the fact that a small proportion of the corticopetal cholinergic neurons receive a very dense noradrenergic input characterized by repetitive synaptic contacts by the same DBH-positive axon. Although these arrangements often occurred at considerable distances from the cell body, they appear to imply a powerful influence in shaping the output pattern of these cholinergic projection neurons. The source of this putative NE input is unclear. Hovewer, if these fibers would originate in the locus coeruleus, this mechanism may mediate the phasic control over the discharge of cholinergic neurons in alert state. Namely, it is well known that both locus coeruleus and BFC neurons show phasic activation related to sensory stimuli, particularly those which signal reinforcement contingencies (Richardson and De Long, l991; Sara and Segal, l991). Also unclear is whether these BFC neurons represent a predetermined (genetic or epigenetic) subpopulation of the forebrain cholinergic system with specialized cortical terminations and/or selective local connections in the forebrain.

Dopaminergic/cholinergic interaction in the basal forebrain

Although a definitive answer to the extent of direct dopaminergic/cholinergic interactions in the basal forebrain awaits double-labeling studies using antisera for ChAT and dopamine, our studies, and that of Milner (l991) using an antibody against TH, suggest that at least a subpopulation of corticopetal, and septohippocampal cholinergic neurons, respectively, may receive dopaminergic input. The effects of DA on its target neurons are complex and depend on the activation of different receptors (Gerfen et al., l990; Lindefors et al., l990). Striatal cholinergic interneurons, which are in many respect similar to BFC neurons (e.g. Alheid and Heimer, l988), express D2 DA receptors (LeMoine et al., l990), and it has been suggested that striatal acetylcholine release is under a tonic dopaminergic control through D2 receptors (Stoof et al., l992). In basal forebrain areas rich in corticopetal cholinergic neurons, both D1 and D2 DA receptors have been localized (Boyson et al., l986; Bouthenet et al., l987; Richfield et al., l987; Dawson et al., l988), although no direct data exist concerning the cellular localization of these receptors. In an electrophysiological study (Napier, l992), systemic administration of a selective D1 agonist increased firing rate in 83% of ventral pallidal neurons - an area containing corticopetal and amygdalopetal cholinergic neurons-, whereas selective D2 antagonist suppressed firing rate in 62% of the units. Although this study did not disclose the cellular site of action, the data suggest that the action of DA may be very complex and not simply excitatory or inhibitory to the postsynaptic neuron.

Regulation of ChAT activity in the basal forebrain cholinergic neurons

It is well known that levels of transmitters or associated enzymes may undergo considerable dynamic variation after axotomy (retrograde changes) or in response to anterograde and retrograde transneuronal regulation (Ip and Zigmond, l985; Joh and Baker, l988). This has been demonstrated for cholinergic neurons in the CNS and PNS, although the exact mechanism is not well understood (Wooten et al., 1978; Giacobini et al., l979; Helke et al., l983; Lams et al., l988; Collier, this volume). In the case of BFC neurons, it appears that gangliosides and target derived trophic factors, especially NGF, as well as NGF receptor expression in cholinergic neurons, are important in the regulation of ChAT activity (Ojika and Appel, l984; Stephens et al., 1985; Adler and Black, l986; Cuello et al., 1986 l989; Mufson and Kordower, l989; Woolf and Butcher, l989; see also chapters by Gage, Cuello, Hefti and Wainer in this volume). Data also suggest that basal forebrain cholinergic neurons which project to different target areas show a differential sensitivity to NGF, which may reflect differential regulatory mechanisms (Williams et al., l989).

While studies of neuronal development have long established a role for target tissue in the control of neuronal survival (Cowan, l973), recent evidence indicates that, in addition, the avalability of afferent supply is critically involved in the control of developmental neuronal death, both in the peripheral as well as in the central nervous system (Cunningham, l982; Okado and Oppenheim, l984; Furber et al., l987; Linden and Pinon, l987; Rubel et al., l990). Moreover, recent data suggest that transneuronal regulation of the postsynaptic neuron is also significant in the mature nervous system. Depending on the species, the system under investigation, and time after deafferentation, transneuronal influences may be manifested in changes in the transmitter synthetic capacity of the target neuron (Kawakami et al., l984; Young et al., l986; Joh and Baker, l988), altered expression of cytoskeletal proteins (Cotman et al., l990), increases or decreases of dendritic surface, as well as cell survival (Coleman and Flood, l986; Arendash et al., l989). For example, removal of the thalamostriatal fibers - a presumably glutamatergic input to striatal cholinergic neurons - resulted in decreased striatal levels of ChAT activity (Nieoullon et al., l985; Lapper and Bolam, in press).

In the present series of experiments, two weeks following 6-hydroxydopamine lesions of the ascending catecholaminergic pathways, ChAT activity was found to be significantly reduced in basal forebrain areas rich in cholinergic cell bodies (Fig. 11). The identity of the catecholamine(s) involved in affecting ChAT activity in the postsynaptic neuron in these studies is unclear, since both the ascending noradrenergic as well as dopaminergic axons were deliberately ablated in these studies. Interestingly, Consolo et al. (l990) reported a similar reduction of ChAT activity in the basal forebrain after electrolytic lesions in the mesopontine tegmentum. Since such lesions would inevitably disrupt the ascending fibers from the locus coeruleus or those fibers of the ventral noradrenergic bundle, this finding might support a facilitatory role for brainstem noradrenergic cell groups in regulating basal forebrain ChAT activity. Other evidence has shown that ascending midbrain dopaminergic fibers control ChAT activity in the forebrain, at least within the striatum, although in this case an inhibitory effect was suggested (Engberg et al., l991). Comparison of the neurochemical and morphological results presented here suggest that the extent of catecholaminergic influence on cholinergic cells may not be uniform in all areas, since ChAT activity was unchanged in the ventral part of the globus pallidus, whereas a significant reduction of ChAT activity in response to 6-OHDA administration occurred in the vertical and horizontal limbs of the diagonal band. A number of possibilities may account for these differences, including dependence on either NE alone, or on both NE and DA.

Concluding remarks

ChAT activity is decreased both in the basal forebrain and in cortical projection areas in Alzheimer's disease, and in Parkinson's disease with dementia (Mann, l988; Perry et al., l987; Ruberg and Agid, l988). It is suggested that the decline of ChAT activity in the basal forebrain in Alzheimer's disease reflects a down regulation of the production of this enzyme and that neuronal loss itself may be a secondary feature of this disease (Perry et al., l987). There is a consensus among many authors that the primary pathological events in Alzheimer's disease occur in the cortex, and that a down regulation of ChAT production in BFC neurons in this condition likely occurs secondarily through retrograde effects. Indeed, ample evidence indicates that BFC neurons can be retrogradely affected as discussed above (see also Pearson et al., l983; Sofroniew et al., l983, l986, l990), however, the possible contribution of anterograde transneuronal effects to the pathophysiology of Alzheimer's disease or other dementias with cholinergic deficit has received relatively little attention.

In aged rodents, nonhuman primates and humans, as well as in Alzheimer's and Parkinson's diseases, reductions in cell number have been reported in the locus coeruleus, in the substantia nigra, and in basal forebrain areas containing cholinergic corticopetal neurons, with concomitant changes in presynaptic markers for these transmitters (Brody, l976; Forno, l978; Arendt et al., 1983; McGeer et al., l984; Ichimaya et al., l986; Price et al., l986; Carlsson, 1987; Morgan et al., l987; Nordberg et al., 1987; Palmer et al., l987; Perry et al., l987; Reinikainen et al., l988; Jellinger, l990; Chain-Palay, l991). The extent to which reductions in catecholaminergic and cholinergic markers are independent or related processes is unclear. The extensive distribution of catecholaminergic terminals on cholinergic neurons in the basal forebrain together with the fact that 6-hydroxydopamine lesions of the ascending catecholaminergic pathways result in reductions in ChAT activity[2] in forebrain areas rich in cholinergic cell bodies suggests that deprivation of catecholaminergic afferents could be a factor in the metabolic deterioration of BFC neurons in Alzheimer's and Parkinson's diseases or aging. This notion is consistent with a recently proposed theory of transsynaptic systems degeneration in neurological diseases (Saper et al., l987) and with observations that perturbations of both the catecholaminergic and cholinergic systems results in behavioral deficits in animals which are similar to the cognitive impairments seen in patients with Alzheimer's disease (for review see Durkin, l989; Luine et al., l990; Decker and McGaugh, l991).

Acknowledgements

This paper is dedicated to Prof. T.H. Schiebler, on occasion of his 70th birthday. This work was supported by PHS grants NS23945, P01 30024 (L.Z.) AGO6384 and the American Federation for Aging Research (V.N.L.). The antibody against dopamine-ß-hydroxylase was a generous gift from Dr. R. Grzanna. Mr. Michael Forbes and Ms. Vickie Loeser have provided expert assistance.

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Legends

Fig. 1. Camera lucida drawings from alternate frontal sections stained for dopamine-ß-hydroxylase (DBH)/ChAT (A) and tyrosine hydroxylase (TH)/ChAT (B). Cholinergic cells are represented in red, DBH or TH-positive fibers/terminals in black. DBH or TH is detected with nickel-enhanced diaminobenzidine (NiDAB), and ChAT with diaminobenzidine tetrahydrochloride (DAB) according to the double immunolabeling protocol of Hsu and Soban (1982). CP, caudate putamen; f, fornix; GP, globus pallidus; ox, optic chiasm; sm, stria medullaris.

Fig. 2. Camera lucida drawing illustrating the relationship of cholinergic neurons and DBH fibers/terminals at the border between substantia innominata and globus pallidus. The box in the inset shows the approximate location of the drawing. Note that DBH-positive fibers show different morphology according to the size and shape of their varicosities and intervaricose segments.

Fig. 3. High magnification photomicrographs illustrate relationships between corticopetal cholinergic neurons and DBH (A,B) and TH-positive (C) fibers in the substantia innominata. A: a cholinergic neuron with cell body in the upper right corner of this micrograph is approximated by several DBH-positive varicosities, some of them marked with arrowheads. In the lower left part of the figure the cholinergic dendrite (marked between two arrowheads) is enwrapped by a DBH-positive axon bearing several varicosities. B: a distal cholinergic dendrite is surrounded by at least two axons bearing many varicosities. C: a cholinergic neuron is approached by several TH-positive varicosities. Some of the larger varicosities are indicated by arrowheads. Arrows in B and C point to fine caliber axons. Scale: 10 µm.

Fig. 4. Schematic drawing illustrating the pattern of DBH and TH terminals abutting cholinergic neurons. Alternate sections were processed for DBH/ChAT (A,B) or TH/ChAT (C,D) double-immunolabeling. Cholinergic cell bodies are represented by black dots. Zones of putative contacts between cholinergic profiles and catecholaminergic terminals are depicted as red (DBH) or green (TH) squares. Sections were screened using an ocular reticle (80 x 80 µm) at 63x and contact sites were marked on a camera lucida drawing of the corresponding section using a proportional grid. Putative contact was determined when a clearly identified DBH or TH-labeled terminal (including associated axon) directly abutted a labeled cholinergic profile in the same focal plane. Positive zones generally had 1-3 such arrangements, although occasionally greater number of putative contacts were identified. BSt, bed nucleus of the stria terminalis; f, fornix; GP, globus pallidus; HD: horizontal limb of the diagonal band; MP, magnocellular preoptic nucleus; SI, substantia innominata; ot, optic tract; ox; optic chiasm.

Fig. 5. A DBH-positive terminal contacts a cholinergic dendrite. A: Diagramatic reconstruction of a cholinergic neuron from the substantia innominata, receiving several varicosities (small arrows) all of which confirmed as synapses by electron microscopy. Large arrow in lower left points to the DBH-bouton shown in B. B: The dendrite of the cholinergic neuron identified by the flocculent DAB immunoprecipitate is contacted by a DBH terminal containing the heavy NiDAB deposit. Double labeling according to Hsu and Soban (l982). Arrowheads denote the postsynaptic thickening. Scale: 1 µm.

Fig. 6. Double labeling for TH and ChAT. A: Plastic section of the rostral forebrain labeled for ChAT and TH using the DAB/benzidine dihydrochloride (BDHC) technique according to Levey et al., (l986). The boxed area, which is enlarged in the inset, contains the identified neuron. B: a TH-positive varicosity (arrow) contacts a cholinergic dendrite; the cholinergic neuron (#1) is located among the myelinated fascicles in the ventral part of the globus pallidus (GP). As fiducial markers #2 denotes another unlabeled neuron and asterisk indicates a capillary. C: Low power electron micrograph rotated counterclockwise 45 degrees relative to (B) shows the same cholinergic neuron (N1= cell body); N2 and capillary (asterisk) serve for correlating (B) and (C). The framed area in (C) is shown at higher magnification in (D). D: The dendrite of the cholinergic neuron identified by the diffuse DAB precipitate is contacted by a TH-positive terminal containing the heavy BDHC deposit (arrowheads). Open arrows denote the postsynaptic site. For comparison see an unlabeled dendrite (d) and an unlabeled axon terminal (b). BSt, bed nucleus of the stria terminalis; f, fornix; ox, optic chiasm; SI, substantia innominata; sm, stria medullarisScale: A 0.5 mm, B: 10 µm, C and D: 1 µm.

Fig. 7. Tracing of axons from the locus coeruleus to basal forebrain corticopetal cholinergic cells. A: PHA-L injection site in the locus coeruleus. B: The same section immunostained with an antibody against DBH. Double fluorescence (FITC/RITC) technique. Note that all PHA-L labeled cell bodies are confined to the heavy DBH-positive area. Stars denote the same vessels. Scale: 100 µm. C: Camera lucida drawing showing the distribution of PHA-L labeled fibers in relation to forebrain cholinergic neurons following injection of the tracer into the locus coeruleus. Inset shows an adjacent section to (C) which was mapped at high-magnification light microscopy for the presence of putative contacts between PHA-L labeled varicosities and cholinergic neurons. Cholinergic cell bodies are represented by dots. Zone of putative contacts between cholinergic profiles and PHA-L labeled terminals are depicted as black squares (corresponding 80x80 µm in the section). GP, globus pallidus; f, fornix; ox, optic chiasm; sm, stria medullaris.

Fig. 8. Arborization of PHA-L labeled axons in the basal forebrain following the tracer injection into the locus coeruleus. A: Asterisk marks the location of picture in (B). B: Note that the majority of axon varicosities may contact non-cholinergic elements; however, a few of them are located adjacent to a cholinergic corticopetal neuron (arrowheads). CP, caudate putamen; f, fornix; GP: globus pallidus; HD, horizontal limb of the diagonal band; ic, internal capsule; MP, magnocellular preoptic nucleus; SI, substantia innominata; vhc, ventral hippocampal commissure. Scale: 10 µm.

Fig. 9. Correlated light and EM study to show that identified cholinergic neurons receive terminals from the locus coeruleus. A: The identified cholinergic neuron, which is located in the substantia innominata (SI), is approached by a PHA-L fiber bearing several varicosities (arrow in B). C: Low-power electron micrograph showing the cell body and part of a dendrite of this neuron. D: An enlargement of the boxed area from (C) showing that the PHA-L labeled terminal bouton makes an asymmetric synapse with an unlabeled dendrite (asterisk in C). Inset from an adjacent thin section demonstrates that the same PHA-L labeled terminal also contact the cholinergic dendrite. Arrows in D and in the inset point to the postsynaptic densities. Scale: in C and D: 1 µm.

(From Zaborszky and Heimer, 1989; with permission from the Publisher).

Fig. 10. Comparison of the distribution of putative interaction sites between DBH-positive terminals and cholinergic neurons (A) and those between BFC neurons and axon terminals originating in the locus coeruleus (B). These maps were composed from six camera lucida drawings (similar to that in Fig. 4 or Fig. 7 inset), which were aligned and superimposed to generate the final figure. Cholinergic cell bodies are represented by black dots. Zones of putative contacts between cholinergic elements and DBH or PHA-L-labeled terminals are depicted as red squares (corresponding 80 x 80 µm areas in the section).

Fig. 11. ChAT activity in forebrain regions 14 days after unilateral 6-OHDA injections (10 µg of 6-OHDA dissolved in 4 µl saline containing 1 mg/ml ascorbic acid) into the ascending catecholaminergic fiber bundle at the meso-diencephalic border. Values are expressed as percentage of the corresponding sham injections (vehicle only) where shaded columns are ipsilateral side and open columns are contralateral side. Maximal in vitro activity of ChAT was measured radiochemically using [³H]acetyl]-CoA (Luine and McEwen, l983). Protein was measured according to Bradford et al. (l976). ChAT activities reflect nmoles ACh produced/mg protein/hr and are mean + S.E.M. for determination in 7-8 rats/group from corresponding tissue punches. Data analyzed by two-way ANOVA and differences between all the groups tested by Newman-Keuls analysis. * group different from all other groups, at a level of p 0.05. VDB, vertical limb of the diagonal band; HDB, horizontal limb of the diagonal band; vGP, ventral part of the globus pallidus.