Direct Catecholaminergic-Cholinergic Interactions in the Basal Forebrain. I. Dopamine-ß-Hydroxylase- and Tyrosine Hydroxylase Input to Cholinergic Neurons

 

 

   L. ZABORSZKY* and W.E. CULLINAN

 

^{Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, NJ 07102 (L.Z.), and Department of Basic Health Sciences, Marquette University, Milwaukee, WI 53233 (W.E.C.)}

 

 

 

 

Running headline: Catecholaminergic-Cholinergic Interactions

 

 

 

Key words: correlated light and electron microscopy, double immunolabeling, Alzheimer’s disease

 

 

49 pages, 11 figures, 1 table

 

 

Associate Editor: P. E. Sawchenko

 

 

 

 

 

*Author for correspondence:

Laszlo Zaborszky, M.D.,Ph.D.

Center for Molecular and

Behavioral Neuroscience,

Rutgers University,

197 University Avenue,

Newark, NJ 07102, U.S.A.

TEL: 201-648-1080/Ext. 3181

FAX: 201-648-1272

ABSTRACT

            Immunocytochemical double-labeling techniques were used at the light and electron microscopic levels to investigate whether dopamine‑ß‑hydroxylase and tyrosine hydroxylase-containing axons contact basal forebrain cholinergic neurons.  Dopamine-ß-hydroxylase- and tyrosine hydroxylase-positive fibers and terminals were found in close proximity to cholinergic neurons throughout extensive basal forebrain areas, including the vertical and horizontal limb of the diagonal band nuclei, the sublenticular substantia innominata, bed nucleus of the stria terminalis, ventral pallidum, and ventrolateral globus pallidus.  Cholinergic cells in some aspects of the globus pallidus appeared to be contacted by tyrosine hydroxylase-positive but not dopamine-ß-hydroxylase-positive fibers, suggesting dopaminergic input to cholinergic neurons in these regions.  Direct evidence for the termination of dopamine-ß-hydroxylase and tyrosine hydroxylase-positive fibers on cholinergic neurons was obtained in electron microscopic double-immunolabeling studies.

            Using high magnification light microscopic screening, both qualitative and quantitative differences were noted in the catecholaminergic innervation of forebrain cholinergic neurons.  For example, while many cholinergic neurons were in close proximity to single dopamine-b-hydroxylase-positive varicosities, others, particularly those located in the substantia innominata-bed nucleus of the stria terminalis continuum, were apparently contacted by labeled fibers in repetitive fashion.  The findings of the present study, together with our preliminary biochemical experiments (Zaborszky et al., [1993] Prog. Brain Res. 98:31-49) suggest that catecholaminergic afferents can differentially modulate forebrain cholinergic neurons.  Such interactions may be important in learning and memory processes, and their perturbations may contribute to the cognitive decline seen in aging and in disorders such as Alzheimer's and Parkinson's diseases.

INTRODUCTION

            Cholinergic projection neurons are widely dispersed in the basal forebrain and provide the majority of the acetylcholine (ACh) found in the cerebral cortex and the amygdaloid body in the temporal lobe (for review see Geula and Mesulam, 1994).  Patients with Alzheimer’s disease (AD) have a significant decrease of ACh in the cortex and pathological changes in basal forebrain cells (Price et al., 1986). While the etiology of this disease is not well understood (Terry et al., 1994), it is reasonable to expect that a knowledge of the connections and regulatory mechanisms in specific basal forebrain circuits will contribute to our understanding of the deficiencies in  information processing that characterizes AD and related disorders. Indeed, the initial description of GABAergic terminals on basal forebrain cholinergic projection (BFC) neurons (Zaborszky et al., 1986b) prompted several experimental studies in animals and clinical studies in patients with AD. The results from these studies suggest that the impaired cognitive functions associated with cholinergic hypofunction may be ameliorated by transsynaptic modulation of the activity of cortically projecting cholinergic neurons in the basal forebrain (Sarter et al., 1988; Durkin, 1989; Decker and McGaugh, 1991; Durkin, 1992; Imperato et al., 1994; Sarter and Bruno, 1994).

            Such functional studies have been severely hampered until quite recently by a general lack of knowledge regarding the precise morphological and chemical characteristics of specific neural circuits, including afferents to BFC neurons.  The reasons for this are easily appreciated in view of the anatomical complexity of the basal forebrain, in which cholinergic neurons are intermingled among numerous non-cholinergic cells, and are distributed in close proximity to several major ascending and descending fiber systems (for review see Zaborszky et al., 1991, Zaborszky, 1992). Therefore the verification of actual synaptic contact between the afferent fiber system and the cholinergic projection neurons requires appropriate combinations of double labeling methods at the ultrastructural level, in which the afferent fiber system and the cholinergic nature of postsynaptic target can be unequivocally determined. The study of these inputs is further complicated by the morphological characteristics of these neurons. For example, several studies have suggested that the dendrites of BFC neurons extend for very long distances (Semba et al., 1987; Zaborszky et al., 1991), and that synaptic input to the cell bodies and proximal dendrites of these cells is notably sparse, whereas it increases on more distal dendritic segments (Ingham et al., 1985). Thus, a complete characterization of the afferents of the BFC neurons must take these factors into account. Such information is a prerequisite to the design of pharmacological and behavioral investigations of BFC function.

            Based on ultrastructural confirmation, three general classes of afferents to BFC neurons have been previously defined: a) inhibitory inputs from GABAergic neurons (Zaborszky et al., 1986b; Ingham et al., 1988; Zaborszky and Cullinan, 1992); b) peptidergic afferents from local and perhaps distant NPY-, somatostatin- (Zaborszky, 1989; 1992) substance P- (Bolam et al., 1986) and enkephalin-containing neurons (Martinez-Murillo et al., 1988b; Chang et al., 1987), and c) presumably excitatory inputs from the amygdala, and to some extent, the hypothalamus (Zaborszky et al., 1984; Zaborszky and Cullinan, 1989; Cullinan and Zaborszky, 1991).  Although ascending fibers originating in brainstem catecholaminergic cell groups project through forebrain areas containing BFC neurons (Swanson and Hartman, 1975; Hokfelt et al., 1977) and pharmacological manipulations of cholinergic systems (Robinson et al., 1979; Costa et al., 1983; Pepeu et al., 1986; Robinson, 1986a, 1986b; Wood and McQuade, 1986; Day and Fibiger, 1993; Robertson and Staines, 1994) have suggested the possibility of catecholaminergic input to BFC neurons, definitive morphological evidence on this issue has been lacking. Thus, in the present study a combined light-electron microscopic analysis was undertaken in which putative noradrenergic and dopaminergic axons were labeled by dopamine-b-hydroxylase and tyrosine hydroxylase, respectively, and cholinergic neurons were identified by immunoreactivity for choline acetyltransferase (ChAT). Part of this work has been reported earlier in a review paper (Zaborszky et al., 1993).

 

MATERIAL AND METHODS

Animals and perfusions

 

            In all experiments male Sprague-Dawley rats 275±25g (n=12) were used. Transcardial perfusions were performed under deep anesthesia (3.3 ml/kg Chloropent, Fort Dodge Lab., Fort Dodge IA) with 50 ml saline, followed by 350 ml of a fixative containing 4% paraformaldehyde, 0.1-0.2% glutaraldehyde, 15% saturated picric acid, in 0.1M phosphate buffer (PB) (Somogyi and Takagi, 1982). This was followed by 150 ml of the same fixative without glutaraldehyde.  Brains were removed immediately and post-fixed in the second fixative for 4-12 h, before cutting into 6 series of 40 mm coronal sections on an Oxford Vibratome.

 

Immunocytochemistry

 

            Light microscopy.  For immunohistochemical processing, sections were rinsed several times in ice cold PB prior to all steps. Antibodies were routinely diluted in a PB solution to which 0.5% triton X-100 and 0.25% carageenan (lambda type, Sigma) had been added.  All steps were carried out under gentle agitation, and except where noted, performed at room temperature.

            In order to reveal the distribution of noradrenergic/adrenergic fibers in relation to forebrain cholinergic projection neurons, we processed forebrain sections  using a double-labeling protocol whereby dopamine-ß-hydroxylase (DBH) is detected with nickel-enhanced diaminobenzidine (NiDAB), and ChAT with diaminobenzidine tetrahydrochloride (DAB), according to the technique of Hsu and Soban (1982).  Sections were incubated in an antiserum directed against DBH raised in guinea pig (Grzanna et al., 1978) at a dilution of 1:2000 for 36 h at 4˚C.  This was followed by incubation in a biotinylated anti-guinea pig IgG prepared in goat at 1:100 for 2 h, and the ABC complex (Vector Labs) at 1:500 for 2 h.  This was succeeded by the coupled oxidation reaction of Itoh et al. (1979) for 30-40 min. in a solution containing 50 mg DAB, 40 mg ammonium chloride, 0.4 mg glucose oxidase (Sigma, type VII), and 200 mg ß-D-glucose in 100 ml PB, to which 2 ml of a 0.05M solution of nickel ammonium sulfate was added.  The second immunohistochemical sequence involved incubation in a rat anti-ChAT monoclonal antibody (Eckenstein and Thoenen, 1982)  at 1:10 for 36 h at 4˚C, followed by goat anti-rat IgG (Sigma) at 1:100 for 2 h, rat monoclonal peroxidase-anti-peroxidase (PAP) (Sternberger-Meyer) at 1:100 for 1 h, and finally a DAB solution without the nickel ammonium sulfate solution.  Sections were then rinsed, dehydrated in a progressive series of alcohols, stored overnight in xylene, and coverslipped with DPX.

            To demonstrate putative dopaminergic fibers in relation to cholinergic neurons, a polyclonal antibody against tyrosine hydroxylase (TH) was used in conjunction with the monoclonal antibody against ChAT.  The DAB/NiDAB protocol was similar to that described above, except that the primary and secondary antibodies in the TH immunohistochemical sequence were rabbit anti-TH (Eugene Tech, Annandale, NJ, 1:1000, 36 h at 4˚C) and biotinylated goat anti-rabbit IgG, (1:100, 2h), respectively.  ChAT/TH double-labeling was also performed using a different immunohistochemical protocol involving distinct chromagens, (Levey et al., 1986) whereby ChAT was detected in the first immunolabeling sequence using DAB, and TH was labeled in the second sequence using benzidine dihydrochloride (BDHC).

            To control for the possibility of cross-reactivity between immunoreagents, or between secondary antibodies in the double labeling experiments, a separate series of sections was processed in the full immunohistochemical sequence with one or both primary antibodies deleted, anti-ChAT antibody replaced by PB and the anti-DBH or anti-TH antibody replaced with normal goat or PB. Evidence of cross-reactivity was never encountered in these experiments.  In addition, when examined individually, the pattern and distribution of each of the markers applied in the double-labeling experiments (DBH, TH, ChAT) completely corresponded to the results of experiments in which they were labeled alone, lending further support to the specificity of the double-labeling method.

            Electron microscopy.  Sections were processed for electron microscopic double-labeling similarly to those for light level analysis, except that incubation times and temperatures differed for the linking antibodies (4 h at 4˚C), and all antibodies were diluted in PB containing only 0.04% Triton X-100.  In addition, antisera penetration was facilitated by a freeze-thaw procedure performed prior to immunocytochemistry, whereby sections were floated in vials containing 10% sucrose PB until they sunk, and subsequently frozen in liquid nitrogen, thawed at room temperature, and rinsed in PB.  For DBH/ChAT double labeling the NiDAB/DAB technique was used, whereas for detecting putative dopaminergic axons on BFC neurons, both the NiDAB/DAB (TH/ChAT) and the DAB/BDHC (ChAT/TH) protocols were used. Following immunostaining, sections were post-fixed in 1% OsO₄ for 30 min., dehydrated in a series of alcohols, stained with uranyl acetate at the 70% ethanol stage, and flat embedded into Spurr-Epon (EMS) or Durcupan (Fluka) between glass slides and coverslips that had been treated with liquid release agent (EMS).  Selected embedded sections containing cholinergic cells in close apposition to labeled varicosities were subsequently photographed and mounted on cylindrical Araldite blocks.  Ultrathin sections were cut on a Reichert ultramicrotome, collected on single slot Formvar-coated nickel grids, and examined with a Zeiss EM 109 electron microscope.

Data analysis

 

            Camera lucida drawings were made at 25x from adjacent double-labeled sections at four basal forebrain levels. DBH or TH labeled fibers and ChAT-positive cell bodies and proximal dendritic segments were drawn, as illustrated in Figs. 1, 2, 3, and 4. To aid in the delineation of forebrain structures, coverslips were removed and sections were Nissl stained.  Relatively standard topographical terms have been used according to the atlas of Paxinos and Watson (1986).  In order to evaluate the distribution pattern of DBH or TH inputs in relation to the cholinergic forebrain projection system as a whole, zones of putative contact between DBH or TH terminals and labeled cholinergic elements were mapped.  ChAT-positive neurons from double-labeled sections were drawn (10x) from 6 forebrain levels.  Grids composed of 600-800 pixels, each measuring 80x80 mm, were placed over such drawings and pixels were screened individually at high magnification (63x) with the aid of an ocular reticule that precisely corresponded to the pixel dimensions of the drawings.  Pixels were then scored for the presence of putative contacts using the same criteria as those used to select cases for analysis at the EM level, i.e. a clearly labeled terminal (including associated axon) directly abutting a labeled cholinergic cell or dendrite in the same focal plane.  Drawings were then schematized as in Fig. 7.  Finally, in six sections stained for DBH/ChAT, basal forebrain areas were systematically scanned and putative contact sites as well as the cholinergic cell bodies associated with these contacts were marked using the Neurolucida computerized image analysis system (MicroBrightField, Inc.) connected to a Zeiss Axioplan microscope.

             

 

 

RESULTS

Mapping of DBH fibers in relation to cholinergic neurons

  

            Figures 1‑2 illustrate the distribution of DBH fibers in relation to basal forebrain cholinergic neurons at three rostral forebrain levels as seen at 25x. 

            At the level shown in Figure 1A, cholinergic projection neurons constitute a more or less continuous group of cells extending dorsally from the medial septum-vertical limb of the diagonal band complex (MS/VDB) to the ventral surface of the brain.  A small, pronounced DBH‑positive fiber/terminal system was found in the ventral part of the intermediate lateral septal nucleus, lateral to the cholinergic cells in the medial septum, although DBH-positive terminals were seen in more lateral and dorsal parts of the lateral septum as well.  Another prominent terminal network was seen in the rostral part of the bed nucleus of the stria terminalis (BSt), mainly in its ventromedial part.  A third notable collection of fibers/terminals was localized in the ventral part of the ventral pallidum (VP) along its border with the dorsal portion of the horizontal limb of the diagonal band (HDB), where a small group of cholinergic neurons was embedded in this terminal network.  This latter fiber/terminal system appeared to be continuous with a group of labeled fibers extending dorsally along the medial border of the BSt into the intermediate part of the lateral septal nucleus, and laterally across the ventral pallidum toward the dorsal part of the ventral pocket or fundus striati of the caudate nucleus.  Finally a small but very dense terminal network was seen at the ventral surface of the brain at the midline, and along the lateral aspect of the organum vasculosum of the laminae terminalis.   

            At the level of Figure 1B, cholinergic cells were seen primarily within the ventral part of the septum, ventral pallidum, HDB, and magnocellular preoptic nucleus.  Cholinergic cells along the ventral border of BSt were surrounded by a dense network of DBH-positive fibers and terminals.  Another prominent DBH-positive terminal network was noted in the ventral part of the ventral pallidum and beneath it, in a territory that appears to be a rostral continuation of the sublenticular substantia innominata (SI).

            Further caudal, at the level of the crossing of the anterior commissure (Fig. 2), cholinergic projection neurons are localized mainly in the ventral part of the globus pallidus, and in a large basal forebrain area beneath it, traditionally belonging to SI, HDB, and magnocellular preoptic area.  Prominent DBH-positive fiber/terminal arborizations were seen in the ventrolateral part of BSt, and in the SI, just beneath the globus pallidus.  A few cholinergic neurons were embedded in these terminal fields.

            Further caudal, about 1 mm posterior to the level shown in Figure 2, cholinergic projection neurons were seen within the same forebrain territories, and many were also found within the internal capsule.  A dense network of DBH-positive fibers and terminals was apparent in the SI, which extended to the dorsolateral part of the BSt.  Cholinergic neurons within these regions were surrounded by DBH labeled terminals.  Scattered DBH-positive fibers were also noted more ventrally and medially within the forebrain.

 

Mapping of TH fibers in relation to cholinergic neurons

 

            The distribution of TH-positive fibers in relation to cholinergic cells is illustrated at three rostral forebrain levels in Figures 3-4, which are from comparable levels to those shown for DBH/ChAT in Figures 1-2.  At the level of Figure 3A, a heavy TH innervation of the ventrolateral part of the lateral septum  is seen that appears to be in direct continuity with the nucleus accumbens.  Another network of TH labeled terminals was localized more medially, in the intermediate subdivision of the lateral septal nucleus, which partially overlapped with the location of cholinergic neurons in the lateral part of the medial septum.  Scattered TH-positive fibers and pericellular arrangements were often found in the more dorsal part of the septum.  A massive innervation by TH terminals was seen in the striatum (although only the ventral striatum is labeled here for simplicity) and several of the cell bridges of the ventral striatum (small arrows in Fig. 3A) were prominent due to their heavy TH-innervation.  The ventral pallidum was penetrated by a dense TH fiber network.     

            More caudal (Fig. 3B) the pattern of TH labeling clearly defined the ventral pallidum, which was pervaded by TH fibers.  The lateral division  of the BSt was heavily labeled, and a moderate to dense innervation of the intermediate and dorsal subdivisions of the lateral septal nucleus was apparent.

            Further caudal (Fig. 4) the heavy TH-positive bundles en route to the striatum were readily apparent ventromedial to and within the globus pallidus, as well as in the ventrolateral part of the BSt.  Especially at more caudal levels (not shown), the SI and adjacent lateral preoptic-hypothalamic areas were also densely labeled by TH fiber systems.

 

Arborization pattern of catecholaminergic axons

            Based on examination of axonal segements of distances between 100 and 300 mm, four types of DBH- and TH-positive fibers could be discerned, and are described below.

            DBH-positive fibers. Four types of DBH-positive fibers were distinguished in the basal forebrain (Fig. 5, left column) according to the dimensions of their varicosities and intervaricose segments:  1) axons with large varicosities (Fig. 5A), 2) axons with elongated or spindle-shaped varicosities (Fig. 5B), 3) axons with assorted varicosities (Fig. 5C), and 4) axons with small varicosities (Fig. 5D).  The first fiber ype (i.e. axons with large varicosities) was characterized by very large, uniform, spherical varicosities (2-3 mm diameter) with very thin intervaricose segments, resembling "beads on a string."  Although seen only occasionally, these fibers were typically encountered as individual axons which extended for relatively long distances (100-200 mm), and were found in most forebrain areas containing cholinergic neurons, particularly the SI and BSt.  Axons with elongated or spindle-shaped varicosities had intervaricose segments of between 1-5 mm.  These axons were most often seen in the medial septum where they coursed vertically, and may represent fibers of passage toward the cortex or hippocampus.  Axons with assorted varicosities were the most common DBH fiber type seen in the forebrain.  They were characterized by varicosities varying between 1-2 mm, with intervaricose segments of variable distance.  Axons with small varicosities had small, often fusiform varicosities, with very short, delicate intervaricose segments.  This category, however, was occasionally difficult to distinguish from type 3 fibers that were relatively small.

            In general, DBH terminals from all four types were found in close apposition to cholinergic neurons in most parts of the basal forebrain, although the vast majority (85%) of terminals were of the third type, and were frequently observed in "climbing" arrangements on more distal cholinergic dendrites. Such contacts were especially rich in the SI, and are illustrated in the color micrographs of Figure 6C and D.  Cholinergic cell bodies and most of their proximal dendritic portions were often found to be devoid of DBH-positive varicosities, but such putative contacts were often observed at some distance from the cell body.

            TH-positive fibers. Based on material labeled using the NiDAB method, four types of axons could be distinguished (Fig. 5, right column):  1) thick (0.7 mm diameter), smooth, fibers which passed through the VP and globus pallidus, and were often curved and most probably myelinated (Fig. 5E),  2) axons of medium size, having spindle-shaped varicosities of variable staining density, with long intervaricose segments (5-15 mm) (Fig. 5F), 3) axons of similar caliber to type 2, with varicosities of assorted dimensions (0.7-3 mm) and variable but relatively short intervaricose segments (1-5 mm) (Fig. 5G), and 4) very thin axons with very small, spherical varicosities that were densely stained, and intervaricose segments of 1.5-3 mm (Fig. 5H).

            In general, the majority of direct appositions between TH-labeled terminals and cholinergic neuronal elements were located adjacent to the cell body or proximal dendritic segments.  Approximately half of all such putative contacts involved the smallest caliber TH fiber type (type 4 in Fig. 5H).  This fiber type was often found in apposition to cholinergic neurons in the medial portion of the globus pallidus and adjacent forebrain regions.   Figure 6A demonstrates a cholinergic cell body from the ventromedial portion of the globus pallidus that is  approached by TH-positive axons possessing a number of varicosities.  Figure 6B demonstrates the most common type of association between TH-positive axons and cholinergic neurons: the cell body and proximal part of the dendrite of this neuron are approximated by a few small varicosities.

 

Comparison of DBH/ChAT and TH/ChAT putative interaction sites

 

            Figure 7 illustrates the distribution pattern of DBH- (A,C,E) and TH-positive (B,D,F) varicosities detected in close apposition to cholinergic neuronal elements, analyzed at three forebrain levels from alternate sections using high magnification light microscopic screening. Structural borders have been omitted from these figures to emphasize the fact that cholinergic projection neurons constitute a continuous system distributed across several distinct basal forebrain territories.  Cholinergic cell bodies are represented by black dots, and zones of putative contact between cholinergic profiles and DBH- or TH- positive terminals are depicted as green (one apposition), blue (two appositions) or red (three or more appositions) squares, each of which correspond to an 80x80 mm² area of the section.

             As is evident from these diagrams, the distribution pattern of DBH terminals in relation to cholinergic neurons was generally similar to the distribution of TH/ChAT interactions.  A notable exception was in the medial and caudal aspects of the globus pallidus where appositions were often observed between TH-positive terminals and cholinergic elements (Fig. 7D,F), but very few such interaction sites were detected in the DBH/ChAT material (Fig. 7C,E).  Indeed, only very few labeled DBH terminals were found in these regions (Fig. 2). Interestingly, the majority of these putative interactions involved the smallest caliber TH-positive fiber type (type 4 in Fig 5H), which is not present in the DBH material, but is identical to the fiber found to densely innervate the adjacent caudate putamen.  This fine caliber fiber type was also often (in approximately one-half of the cases) found in apposition to cholinergic elements in the lateral BST and SI. These obervations indirectly suggest that such putative contacts may be dopaminergic.  

            A more detailed description of potential DBH-cholinergic interaction sites in specific forebrain territories is included below.

            MS/VDB complex.  In the septum, DBH-positive varicosities were distributed diffusely in close approximation to cholinergic neurons in the dorsal portion of the MS/VDB complex.  A few putative contacts were also seen in the VDB, where it approaches the ventral surface of the brain.  In general, detected contacts were mainly from individual boutons, although occasionally dendrites received "climbing type" fibers bearing multiple boutons.

            Horizontal limb of the diagonal band.  DBH terminals were distributed rather homogeneously within the HDB at any given level, although putative contacts were detected with greatest frequency in the rostral HDB, declining at progressively more caudal levels. The fiber types seen in close association to cholinergic neuronal elements were similar to those found in the MS/VDB.   

            Ventral pallidum.  Two accumulations of putative contact sites were observed: one dorsomedially along the ventral border of the BSt, the other ventrolaterally toward the border with the SI.

            Bed nucleus of stria terminalis and substantia innominata. Based upon hodological and chemoarchitectural characteristics, certain parts of the amygdala, the bed nucleus of the stria terminalis (BSt) and an intervening part of the SI are thought to constitute a morphological and perhaps functional entity, as originally suggested by DeOlmos et al. (1985) and discussed more recently by Heimer et al. (1991).  Interestingly, a particularly rich distribution of apparent contact sites between DBH-labeled fibers (as well as TH-positive fibers) and cholinergic neurons was detected in this part of the basal forebrain.

            Although there are few cholinergic neurons in the BSt, relatively many putative contacts between DBH-positive terminals and cholinergic elements were seen, particularly within its ventral and lateral parts.  This can be explained by the fact that many of the apparent contacts occurred on the distal dendrites of cholinergic neurons located in the SI, which can be seen to course dorsomedially to the BSt, and then dorsally within the lateral aspect of this structure.

            DBH-positive terminals were also seen in direct apposition to cholinergic neurons throughout the rostro-caudal extent of the SI. At more caudal levels, where the SI becomes continuous with the amygdala laterally and merges with the lateral hypothalamic area medially, putative contacts with DBH-positive terminals were frequently observed.  Many "climbing" type arrangements were detected here, where DBH-labeled fibers were seen to spiral around distal cholinergic dendrites distributing as many as 10-15 terminals.  The number of putative contacts appeared to increase from rostral to caudal direction within the SI.

            Globus pallidus and internal capsule. A few DBH terminals were found in close proximity to cholinergic neurons located in the mot ventrolateral part of the globus pallidus, and in the more ventral part of the internal capsule.

            Table 1 summarizes our semi-quantitative data concerning DBH appositions on cholinergic neurons registered at the light microscopic (LM) level.  The majority of the putative DBH contacts were found in the BSt/SI continuum (43%) and in the HDB (27%).  The remainder was distributed in decreasing order as follows; VP/GP (19%), MS/VDB (9%) and internal capsule (2%).

            A further analysis of the DBH/ChAT material revealed that a large proportion of appositions on cholinergic neurons were of the single type (60%), the remaining ones were double (23%) or multiple (three or more: 17%) contacts. Our analysis also suggested that cholinergic profiles with single, double, or multiple contacts show a regionally heterogeneous distribution. Cholinergic profiles directly apposed by multiple DBH-labeled terminals were more often found in caudal areas of the basal forebrain than in more rostral sections. Double or multiple contacts showed the same distributional pattern as that of all appositions summarized in Table 1, however, when only multiple contacts are considered, the differences among the various forebrain regions are even larger: 55% of all multiple contacts were detected in the BSt/SI continuum, followed by the VP/ventral GP (22%) and HDB (16%).  Since DBH varicosities were usually apposed to distal cholinergic dendrites, whose cell bodies were not in the same section, only a small number of cell bodies with identified appositions on their dendrites were registered (n=89). Of these cell bodies, 38% were located in the BSt/SI continuum, followed by the HDB (35%) and VP/vGP (24%).

 

 

 

Electron microscopy

 

            DBH/ChAT double labeling. In correlated light-EM studies, the NiDAB/DAB technique was applied.  At the light microscopic level, DBH-labeled fibers and terminals were nickel-enhanced and appeared black (or bluish-black), and thus were easily differentiated from ChAT-positive elements, which appeared brown. This color difference persists after osmification and plastic embedding (Fig. 6C). At the electron microscopic (EM) level, DBH- and ChAT-positive profiles (confirmed at the light levels) could also be distinguished on the basis of density, the nickel-DAB typically appearing much denser compared to the flocculent DAB reaction product. A total of 12 cholinergic neurons surrounded by DBH-positive axon terminals were selected for serial thin sectioning. From these cases, 27 varicosities were identified under the light microscope, of which 12 could be confirmed at EM to synapse with the dendritic shaft of the cholinergic neuron. All the synapses appeared to be of the asymmetric type, often with subsynaptic dense bodies. From the 27 selected varicosities, 3 were adjacent to cell bodies, although none of these could be identified as synapses.  In other cases, identification of synapses was precluded by 1) the presence of dense immunoprecipitates at the contact sites, or 2) relatively poor ultrastrucural preservation caused by the addition of detergent required for antibody penetration.

            Figures 8-9 illustrate a cholinergic neuron from the caudal portion of the SI. A total of 11 varicosities were marked under the light microscope for ultrastructural identification from this cell. Five of these varicosities were found to be synaptic, two of which are displayed in Figures 8-9. These two synapses are located on dendritic shafts about 50 mm (Fig. 8C) and 100 mm (Fig. 9A) from the cell body, respectively, and the boutons have a diameter of about 1 mm.  Both synapses have prominent subsynaptic dense bodies. Figure 10 shows another cholinergic neuron located in the SI. A synaptic contact between a dendrite of this cholinergic neuron and a medium-sized DBH axon terminal is illustrated at high magnification in Figure 10D.  The synapse is of the asymmetric type, and a prominent postsynaptic density is associated with subjunctional bodies. 

            TH/ChAT double labeling. Figure 11 is an example from immunohistochemical double-labeling experiments designed to visualize ChAT neurons and TH terminals at the EM level.  The two cholinergic neurons in close apposition are located in the ventromedial part of the globus pallidus. In this case, the chromogen DAB was used to visualize the cholinergic neuron and NiDAB marks the TH-positive varicosities. The dendrite which is located on the left part in Figure 11A is approached by a small (less than 0.5 mm in diameter) NiDAB labeled bouton (arrowhead in Fig. 11A) which was found to be in synaptic contact with this neuron (Fig. 11C).  The immunoreactive products in the adjoining profiles prevented an unequivocal classification of the type (symmetric or asymmetric) of synaptic contact. In total, 5 cholinergic neurons apposed by 7 TH varicosities and located within the globus pallidus and ventral pallidum were reconstructed from 3 animals. Three synapses were confirmed on proximal dendrites, and one on the cell body.

 

 

DISCUSSION

 

            The results of this study demonstrate that catecholaminergic axons establish synapses with cholinergic neurons in the basal forebrain. Using the present dual strategy of mapping potential sites of contact under high resolution light microscopy, and subsequently identifying representative terminals under the electron microscope, our study suggests that noradrenergic afferents contact extensive portions of the basal forebrain cholinergic system. Furthermore, the differential topographic and cellular distributions of TH versus DBH varicosities in relation to cholinergic neurons suggests that some TH-positive terminals in the globus pallidus, and to some extent elsewhere in the basal forebrain  may represent dopaminergic input to BFC neurons.

 

Methodological  considerationss

            Double labeling method.  A number of dual-labeling methods are available for ultrastructural studies of synaptic interactions in the brain (Leranth and Pickel 1989; Pickel and Milner, 1989;  van den Pol and Decavel, 1990; Smith and Bolam, 1992). The DAB/NiDAB and the DAB/BDHC technique employ two contrasting immunomarkers. The advantage of these methods is that they can be used for correlated light and electron microscopic experiments: the light brown DAB reaction product is easily distinguished  from the black NiDAB or bluish-green BDHC deposit at the LM level. Under the electron microscope both the texture and the density of the NiDAB or BDHC product, respectively can be contrasted with the flocculent, diffuse DAB  precipitate. Although using the BDHC reaction we were able to observe many en passant TH-positive axons in direct apposition with cholinergic cell bodies or dendrites, the dense reaction products in both profiles precluded the unequivocal determination of these associations as synapses. Therefore, in most of our electron microscopic cases  we  used the DAB/NiDAB method. Since in material stained only for ChAT, cholinergic boutons in the basal forebrain have been reported to only rearely contact cholinergic elements (see for ref. Zaborszky, 1992), and more importantly, since the two profiles (varicose axon vs cell body or dendrite) were preselected at the light microscopic (LM) level and photographed in each sequential step of processing, there is little or no possibility of confusing the two reaction products at the EM level.

            Transmitter identification.  Since DBH occurs in noradrenaline and adrenaline containing neurons, it is likely that DBH-positive varicosities represent noradrenaline or adrenaline terminals. Considering the fact that the amount of adrenaline found in areas rich in cholinergic neurons is relatively low (Hökfelt et al., 1984; Palkovits and Brownstein, 1988; Chang and Kuo, 1989), it is likely that DBH-positive varicosities on cholinergic neurons represent primarily noradrenergic input.  The interpretation of data from TH immunolabeling requires more caution, since TH can be localized in all three types of catecholaminergic boutons. Immunocytochemical (Pickel et al., 1975; Hökfelt et al., 1977) and biochemical studies (Emson and Koob, 1978; Schmidt and Bhatnagar, 1979) in rodents have demonstrated that dopaminergic terminal areas contain greater amounts of TH than do noradrenergic areas.  In addition, previous immunocytochemical studies with anti-TH antisera strongly suggested that TH was detectable only in dopaminergic axons in rat cortex (Hökfelt et al., 1977).  Few studies, however, have addressed this question directly.  Noack and Lewis (1989) have shown in double-labeling experiments in monkey cortex that antisera directed against TH label noradrenergic axons to varying degrees depending on the antibody source.  We have confirmed (unpublished observations) that the TH antibody used in our experiments labels many fibers bearing medium-sized axonal varicosities in the basal forebrain that are also DBH positive. However, differences in the morphology and forebrain distribution patterns of TH/ChAT versus DBH/ChAT interactions suggest that at least a proportion of TH contacts on ChAT neurons represent dopaminergic terminals.

            Bridging the gap between LM and EM levels. It is well-known that in immunocytochemical studies the transition from light microscopic level to the ultrastructural level requires technical compromises.  In order to preserve reasonable ultrastructure, the detergents used to facilitate antibody penetration in EM preparations can be used only in low amounts, resulting in a dramatic decline of detectable axonal labeling.  Thus, it became clear that the assessment of catecholaminergic-cholinergic interactions in the forebrain would require an alternative approach.  Using material treated with relatively high levels of detergent to maximize labeling of fibers and terminals, we subsequently mapped the potential sites of contacts under high magnification light microscopy in an effort to establish the pattern of innervation.  In this case, identified terminals in direct apposition to cholinergic neuronal elements were recorded using the same criteria as that used to select cases from embedded sections in correlated LM/EM experiments.  It should be noted, however, that not all putative contacts identified in light microscopy are likely to represent true synapses on cholinergic cells, since at the EM level, labeled terminals abutting cholinergic processes were sometimes found to terminate on unlabeled elements. Furthermore, the heavy accumulation of reaction products within both pre- and postjunctional profiles precluded the unequivocal identification of some synaptic contacts.  Nevertheless, using our selection criteria, we could identify a synaptic relationship between a DBH-positive varicosity and the cholinergic profile in about 47% of the cases.  In addition, 40% of the putative contacts identified involved cholinergic elements that were abutted by two or more  catecholaminergic terminals, a pattern which usually is proven to be synaptic at the EM level (Toth et al., 1993).  Taken together, these data suggest that a substantial proportion of DBH-ChAT appositions recorded at the LM level indeed represent synaptic sites.  It should also be noted that despite the increased levels of detergent used in the material screened at high magnification LM, penetration of the DBH antibody was limited to only a few microns of the section, and together with the fact that the most distal cholinergic dendritic elements were likely to have remained unlabeled, many actual contacts are expected to have been undetected. 

            A limitation of our LM mapping method is that many cholinergic dendrites that received putative contacts could not traced back to their parent cell body.  Since cholinergic dendrites can extend for several hundred microns, it is unclear whether the contacts detected in a given region belong to neurons whose perikaryon is located in the same territory.  Interestingly, the greatest number of putative DBH contacts were detected in the BSt/SI continuum, an area which contained the highest number of cell bodies with identified terminals on their dendrites. Thus, it is likely that cholinergic cell bodies giving rise to dendritic branches receiving contacts were located within a range equivalent to the length of dendritic staining, which in the present experiments measured up to 200-300 microns.  Insight into this question may come from a better understanding of the dendritic organization of forebrain cholinergic neurons.

 

Catecholaminergic-cholinergic interaction in the basal forebrain

 

            Studies using the glyoxylic acid fluorescence technique (Lindvall and Stenevi, 1978; Moore, 1978), TH and DBH immunocytochemistry, (Swanson and Hartman, 1975; Hökfelt et al., 1976; 1977) and tracing techniques alone, (Jones and Moore, 1977; Fallon and Moore, 1978; Beckstead et al., 1979; Jones and Yang, 1985; Haring and Wang, 1986; Vertes, 1988) or in combination with transmitter identification (McKellar and Loewy, l982; Kaliwas et al., 1985; Grove, 1988; Semba et al., 1988; Jones and Cuello, 1989) have shown that catecholaminergic neurons from the ventral tegmental area, locus coeruleus and the medullary A1-A2 cell groups project to basal forebrain areas in a complicated, often overlapping fashion (Björklund and Lindvall, 1986).  In the present study, the presence of several morphologically distinct fiber types in the basal forebrain is compatible with the idea  that these axons originate in different brainstem catecholaminergic cell groups (Lindvall and Stenevi, 1978; Moore, 1978).  The question of a direct catecholaminergic-cholinergic interaction in the basal forebrain was previously addressed in a light microscopic study combining TH immunocytochemistry and acetylcholinesterase (AChE) histochemistry (Martinez-Murillo et al., 1988a), however, the lack of EM confirmation and unreliability of AChE as a definitive cholinergic marker precluded firm conclusions. Another previous study combined TH and ChAT immunocytochemistry (Milner, 1991b) at the EM level, however this report focused upon those cholinergic neurons located within the septal region.

            Noradrenergic input.  To our knowledge only one electron microscopic study has used DBH immunocytochemistry to determine whether catecholaminergic, presumably noradrenergic (NE), axons terminate in the substantia innominata (Chang, 1989).  This single labeling study, however, concluded that cholinergic neurons in the SI were unlikely to receive this input.  In contrast, we were able to follow cholinergic dendrites for several hundred microns, and found that more distal portions of such dendrites were often encompassed by DBH-positive axons/terminals in spiraling arrays.  Indeed, a major finding of the present EM study is that DBH-containing, presumably noradrenergic terminals are found in synaptic contact with cholinergic neurons, primarily on their distal dendrites.  In spite of the heavy immunostaining on both sides of the synapse, the contacts appear to be characterized by asymmetric membrane specializations, which is consistent with studies using the same antibody in several NE terminal fields (Olschowka et al., 1981) as well as, with a commercially available antiserum (Chang, 1989). It _{should be noted, however, that at the EM level many DBH-positive boutons were} ^{found to contact unlabeled, presumably non-cholinergic structures in all forebrain regions examined.}     

            Although the present study suggests that the forebrain cholinergic projection system receives diffuse noradrenergic input, this innervation is apparently not uniform; regional variations were evident.  For example, cholinergic neurons in the caudal SI appear to receive repetitive contacts in the form of "climbing" fiber arrangements that are likely to represent a powerful noradrenergic influence on these neurons.  In contrast, certain cholinergic neurons, most notably the majority of those located in the globus pallidus and internal capsule, do not appear to receive this input.

            In a preliminary PHA-L study which traced the axonal projections of the locus coeruleus (Zaborszky et al., 1993), labeled varicosities were detected in direct apposition to cholinergic projection neurons in extensive basal forebrain areas, including the MS/VDB, HDB and SI. A comparison of the distribution of the putative interaction sites between PHA-L labeled varicosities and cholinergic neurons with that of DBH-ChAT interactions suggests that the majority of DBH-positive contacts found in the present study may indeed represent noradrenergic input from the locus coeruleus (Zaborszky et al., 1993, Fig. 10). The regional variations noted in the type and density of DBH-positive varicosities in the basal forebrain may also suggest contributions from other  noradrenergic cell groups, such as A1 and A2 (Zaborszky et al., 1991).

            Though limited in number, the location of identified cholinergic cell bodies whose dendrites receive DBH positive boutons suggests that these neurons are mainly located in more ventral parts of the basal forebrain, and are thus likely to project primarily to allo- and mesocortical areas, and the amygdala (Rye et al., 1984; Carlsen et al., 1985; Zaborszky et al., 1986a; Luiten et al., 1987; Grove, 1988; Gaykema et al., 1990).  Interestingly, these are the cortical areas which in rat show high amounts of NE content and ChAT activity, and high densities of cholinergic and noradrenergic terminals (Palkovits et al., 1979; Lysakowski et al., 1988). Since NE fibers of the cerebral cortex are almost exclusively of locus coeruleus origin (Jones and Moore, 1977), and much of the noradrenergic input to BFC cells may also originate in the locus coeruleus, the noradrenergic/cholinergic interaction in the basal forebrain may tune ACh release in the cortex synergistically with locus coeruleus activation, which in turn is related to the level of vigilance of the organism (Foote et al., 1983).

            Dopaminergic input.  Several data from the present study indirectly suggest that dopaminergic-cholinergic interactions may occur in the basal forebrain.  For example, in the rostral, ventromedial globus pallidus, as well as in more caudal aspects of the globus pallidus, a comparison of the distribution patterns of DBH/ChAT with TH/ChAT interaction sites suggests that these contacts may be primarily dopaminergic, since TH but not DBH positive terminals were seen to approximate cholinergic neuronal elements (compare Figs. 7C-D and E-F).  Indeed, few DBH-positive varicosities were seen in this region.  The EM double-labeling experiments from these areas confirmed the presence of direct associations between TH-positive axons and cell bodies or proximal dendritic portions of cholinergic neurons.  The possible dopaminergic nature of such interactions is also supported by the fact that the afferent TH fiber was often of a type not represented in the DBH material, and very silimar to the TH fibers which profusely innervate the adjacent striatum.   Many (approximately one-half) of the TH-ChAT putative contacts in the lateral BST and SI also involved this fiber type (fine caliber axons with uniformly small varicosities and relatively long intervaricose segments). Previous evidence has also indicated the presence of dopaminergic axons in these regions (Lindvall and Stenevi, 1978; Moore, 1978), and 6-OHDA lesions of the substantia nigra resulted in a significant decrease of dopamine in the SI (Geula and Slevin, 1989).  In addition, electrophysiological studies have shown that local applications of dopamine alter the neuronal activity in the VP/SI (Napier and Potter, 1989; Napier, 1992).

            The situation in the MS/VDB area appears to be more complicated.  For example,  fluorescent histochemical studies indicated an absence of dopaminergic terminals in this area (Lindvall and Stenevi, 1978; Moore, l978). However, biochemical studies (Brownstein et al., l974; Versteeg et al., l976) detected high amounts of dopamine in the nucleus of the diagonal band of Broca, and TH-positive axons have been localized in the MS/VDB complex (Gall and Moore, 1984; Milner 1991a). In addition, Milner (1991b) in a study employing TH/ChAT  double immunolabeling at the EM level, suggested that a proportion of TH appositions on ChAT cells in this region indeed may represent dopaminergic input.  A more detailed discussion  of this topic can be found in the companion paper of this series (Gaykema and Zaborszky, 1996).

 

Concluding remarks

 

            The decline of ChAT activity found in areas containing cholinergic projection neurons after catecholamine depletion (Zaborszky et al., 1993), together with present ultrastructural evidence of direct catecholaminergic-cholinergic interactions, suggests that catecholamine afferents may transsynaptically affect ChAT levels in the postsynaptic neuron. These findings may have significance in relation to learning and memory functions.  Interestingly, catecholaminergic-cholinergic interactions have been implicated in memory functions from pharmacological and behavioral studies.  For example, depletion of norepinephrine enhances the disruptive effect of muscarinic blockers on spatial memory performance (Decker and Gallagher, 1987), and prevents the ability of cholinomimetics to improve memory deficits resulting from forebrain cholinergic lesions (Haroutunian et al., 1990).  Dopaminergic-cholinergic interactions in spatial memory performance have also been shown (Galey et al., 1985; Levin et al., 1990; McGurk et al., 1988; 1989).  

            The findings of this report may also be relevant to age-related decline in cognitive function. In aged rodents, nonhuman primates and humans, catecholaminergic neurons show reduced levels of transmitter and synthesizing enzymes in their target areas (Carlsson, 1987; Morgan et al., 1987).  Moreover, age- dependent decreases in cell number have been reported in the substantia nigra and locus coeruleus (Brody, 1976; McGeer et al., 1977; Tomlinson et al., 1981; Iversen et al., 1983; German et al., 1988; Chan-Palay and Asan, 1989).  Similarly, age-dependent decreases have been reported in forebrain presynaptic cholinergic markers, and in cholinergic cell numbers in the nucleus basalis (McGeer et al., 1984; Decker, 1987; Fisher et al., 1989; de Lacalle, et al., 1991).  The extent to which reductions in catecholaminergic and cholinergic markers are independent or related processes is unclear.  Recent behavioral studies, however, indicate that cognitive deficits of aged rats are correlated with activity of both the catecholaminergic and cholinergic systems (Luine  et al., 1990).

            The present findings may also have important implications for the neuropathology of age-related diseases (e.g. Alzheimer's and Parkinson's diseases).  Monoaminergic presynaptic markers in the forebrain are reduced in patients suffering from Alzheimer's disease (Price et al., 1986; Shimohama et al., 1986; van Hoesen and Damasio, 1987; Palmer et al., 1987; Davison, 1988) and the loss of neurons in the locus coeruleus or the ventral tegmental area in early onset cases is usually severe and may precede the loss of cholinergic basal forebrain projection neurons (Forno, 1978; Ichimiya et al., 1986; Nordberg et al., 1987; Mann, 1988).  The frequent association of Parkinson's disease with dementia (Jellinger, 1986; Ruberg and Agid, 1988) and cell loss in the locus coeruleus (Chan-Palay and Asan, 1989) and in the nucleus basalis (Jellinger 1986) suggests the possibility that a common neuropathological mechanism may underlie some of the symptoms found in these diseases (Rossor, 1981).

            Taken together with the aforementioned behavioral and neuropathological data, our findings suggest that deprivation of catecholaminergic afferents could be a factor in the metabolic deterioration of the forebrain cholinergic projection neurons in Alzheimer's disease, Parkinson's diseases, or aging, a notion consistent with a previously proposed theory of transsynaptic systems degeneration in neurological diseases (Saper et al., 1987).

ACKNOWLEDGMENTS

            We  thank Prof. L. Heimer whose interest and initial support (USPHS grant NS 17743) greatly facilitated our work. Special thanks are due to Mr. F.L. Snavely and Vinessa Alones for invaluable help with the electron microscopy.   Support was provided by USPHS grants NS 23945 and 30024.  The antibody against DBH was a generous gift from Dr. R. Grzanna.

 

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LEGENDS

 

Fig. 1.  Camera lucida drawings (25x) from frontal sections labeled for DBH/ChAT, using the NiDAB/DAB technique. Cholinergic cells are represented in red, DBH-positive fibers/terminals in black. A is rostral, B is caudal. 

 

Fig. 2.  Camera lucida drawing (25x) from a frontal section (at level caudal to Fig. 1) labeled for DBH/ChAT, using the NiDAB/DAB technique. Cholinergic cells are represented in red, DBH-positive fibers/terminals in black.

 

Figs. 3.  Camera lucida drawings from frontal sections adjacent to those of Fig. 1. which were immunolabeled for TH (black fibers/terminals) and ChAT (red cells), using the DAB/BDHC technique.  A, rostral, B, caudal.

 

Fig. 4.  Camera lucida drawing from a frontal section adjacent to those of Fig. 2. which were immunolabeled for TH (black fibers/terminals) and ChAT (red cells),   stained with the DAB/BDHC technique.

 

Fig. 5.  Drawing illustrates the morphology of catecholaminergic fibers labeled with antisera directed against dopamine-ß-hydroxylase (DBH) or tyrosine-hydroxylase (TH).  These drawings were prepared from a collection of high magnification photomicrographs from material stained for DBH/ChAT or TH/ChAT. Scale bar: 10 mm.

 

Fig. 6.  Collection of high magnification color photomicrographs illustrate relationships between cholinergic neurons and TH-positive (A-B) or DBH-positive (C-D) fibers in the globus pallidus (A) and substantia innominata (B-D).  A: A TH positive axon (arrowheads) with several varicosities (arrow) approaching a cholinergic neuron.  B: A cholinergic neuron is apposed by several varicosities from type 3 and 4 TH-positive fibers. Arrowheads point to fine caliber axons. Small arrows denote smaller varicosities, the large arrow indicates two larger varicosities.     C:  DBH-positive varicosities (arrows) around a distal cholinergic dendrite. This neuron was photographed from a plastic embedded section processed for electron microscopy (see Figs. 8-9). D: Distal dendrites of cholinergic neurons in the lateral part of the substantia innominata apparently innervated by DBH-positive varicosities (some of which are indicated by arrows) from type 3 fiber.  Arrowhead denotes axon. Photomicrographs in (B-D) were taken from sections using DAB/NiDAB method.  Picture (A) is from a section processed with the DAB/BDHC technique. Scale bar (in C, for A-D): 10 mm.

 

Fig. 7.  Schematic drawing illustrates the pattern of DBH- and TH-positive terminals detected in direct apposition to cholinergic neurons.  Cholinergic neurons are represented by black dots.  Zones of putative contacts between cholinergic profiles and catecholaminergic terminals are depicted as green (one apposition), blue (2 apposition) or red (three or more) squares.  Sections were screened using an ocular reticle (80x80 mm) at 63X, and contact sites were marked on camera lucida drawing of the corresponding section using a proportional grid. Sections A,C,E are from material stained for DBH/ChAT, sections B,D,F  are processed for TH/ChAT. Parts (C-F) are modified from Zaborszky et al.  (Catecholaminergic-cholinergic interaction in the basal forebrain, 1993, p. 4) with kind permission from Elsevier Science.

 

Fig.8.  DBH-positive terminal contacts a dendrite of a cholinergic neuron located in the caudal part of the substantia innominata. A: Low magnification electron micrograph with two dendritic segments. Box in lower left is enlarged in (D). B: Light microscopic view of the neuron. (Note that part of this neuron is depicted in the color micrograph of Fig. 6C). Asterisks in (B) and (C) mark the cell body. Arrowhead points to the DBH varicosity identified in (D). C: Low magnification view of the section under the electron microscope. Boxed area is shown in (A). D: Enlarged view of the synapse. The DBH-positive bouton is densely labeled. Arrows denote postsynaptic density. Note the prominent subsynaptic dense bodies. Scale: 100 mm in B;  10 mm in A and C; 1 mm in D. An electron micrograph of the same bouton taken from an adjacent thin section was published earlier in Zaborszky et al. (Catecholaminergic-cholinergic interaction in the basal forebrain, 1993, p. 37)  with kind permission from Elsevier Science.

 

Fig. 9.  An identified DBH axodendritic synapse on the same cholinergic neuron shown in Fig. 8.  A: Low power view of the section under the electron microscope. Note the location of the cell body which is indicated by an asterisk. Box is enlarged in (B). B: Two cell bodies, several portions of the dendritic tree of the cholinergic neuron and DBH-positive varicosities can be identified as fiducial markers in this micrograph. Boxed area is enlarged in (C). C: Note the synaptic cleft and the strong subsynaptic density (arrows). Scale: 10 mm in A and B; 1 mm in C.

 

Fig. 10. A DBH-positive terminal contacts a cholinergic dendrite. A: Location of the  cholinergic neuron in the substantia innominata is shown in box. B: Enlarged view of the boxed area in (A). Arrow indicates a medium sized DBH-positive varicosity. C: Low power electron micrograph of the identified neuron. 1,2, 3 label neurons, and asterisks marks the same capillary in (B) and (C). D: High magnification view of the contact site. The dendrite of the cholinergic neuron contains diffuse DAB precipitate which is distinguishable from the heavy metal intensified axon terminal. Arrowheads denote the postsynaptic thickening and subjunctional bodies. Scale bar A: 100 mm; B,C: 10 mm; D: 1 mm.

 

Fig. 11.  TH/ChAT interaction using the NiDAB/DAB method. A: Arrohead denotes a TH-positive varicosity identified under the electron microscope in (C). B: Low power electron micrograph of the same area as (A).  Box is enlarged in (C). C: The heavily stained small TH-positive varicosity is separated from the cholinergic dendrite by the synaptic cleft. Arrows point to the thin postsynaptic density. Scale: 10 mm in A and B; 1 mm in  C.

 

 

 

Abbreviations

 

3V                   third ventricle

ac                    anterior commissure

Acb                 accumbens nucleus

AHA               anterior hypothalamic area

BL                   basolateral amygdaloid nucleus

BSt                  bed nucleus stria terminalis

CPu                caudate putamen

f                       fornix

FSt                   fundus striati

GP                   globus pallidus

HDB               nucleus horizontal limb diagonal band

ic                     internal capsule

lo                     lateral olfactory tract

LOT                nucleus lateral olfactory tract

LPO                lateral preoptic area

LSd                 lateral septal nucleus, dorsal

LSi                  lateral septal nucleus, intermediate

LSv                 lateral septal nucleus, ventral

LV                   lateral venrticle

MCP               magnocellular preoptic nucleus

MP                  medial preoptic nucleus

MPA               medial preoptic area

MS                  medial septal nucleus

ot                     optic tract

ox                    optic chiasm

SI                     sublenticular substantia innominata

sm                   stria medullaris

SO                   supraoptic nucleus

VDB                nucleus vertical limb diagonal band

VP                   ventral pallidum

 

 

 

 

 

 

Table 1.  Distribution of DBH-Appositions on Cholinergic Neurons in the

Basal Forebrain

 

 

	MS/VDB	HDB	VP/GP	BSt/SI	ic
% of all ChAT cells bodies*	16	28	25	20	11
% of all DBH appositions**	9	27	19	43	2
DBH app./ChAT cell body***	0.3	0.3	0.6	1.4	0.1

 

 

*  ChAT cell bodies = 2,482

**DBH appositions on ChAT dendrites and perikarya = 1,260

***# of all DBH varicosities divided by # of all cholinergic cell bodies

Data were collected from 13 sections in two brains