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The distribution of m4 muscarinic acetylcholine receptors in the islands of Calleja and striatum of rats and cynomolgus monkeys
Journal of Chemical Neuroanatomy 28 (2004) 107–116
The distribution of m4 muscarinic acetylcholine receptors in the islands of Calleja and striatum of rats and cynomolgus monkeys David Wirtshafter∗ , Catherine V. Osborn Laboratory of Integrative Neuroscience, Department of Psychology, M/C 285, University of Illinois at Chicago, 1007 W. Harrison St., Chicago, IL 60607-7137, USA Received 26 November 2003; received in revised form 9 April 2004; accepted 21 May 2004 Available online 18 August 2004
Abstract The distribution of m4 muscarinic acetylcholine receptors, and their relation to a number other markers, was examined using immunocytochemical techniques. Staining in the dorsal striatum tended to be more pronounced in the striosomal than the matrix compartment of both rats and cynomolgus monkeys. Within the ventral striatum, immunoreactivity was more pronounced within the olfactory tubercle and the shell region of the nucleus accumbens than in the nucleus accumbens core and was especially marked within the lateral striatal stripe. Modest staining was also seen in the external plexiform layer of the olfactory bulb. By far, the most intense staining in the forebrain of both rats and cynomolgus monkeys was found in islands of Calleja, where it appeared to be a selective marker for the core or hilus regions of the islands, or an analogous region found adjacent to them. The core regions of different islands appear to be continuous with each other so as to form a complex three-dimensional structure, which is largely encased by layers of granule cells. The neuronal elements in the islands of Calleja, which express m4 receptors, remain to be identified, but it is unlikely that cholinergic neurons are a major locus of these receptors. Although there are certain similarities between the islands of Calleja and other components of the striatal complex, the current studies emphasize the extent to which the islands are unique in terms of their architecture and chemical anatomy. © 2004 Elsevier B.V. All rights reserved. Keywords: Islands of Calleja; Muscarinic receptors; Cholinergic receptors; Olfactory tubercle; Nucleus accumbens; Shell; Core; Striatum; Olfactory bulb; Cell clusters; Lateral stripe
1. Introduction The m4 muscarinic acetylcholine receptor is expressed at high levels in several regions of the basal forebrain including the olfactory tubercle, the nucleus accumbens and the dorsal striatum (Levey et al., 1991; Weiner et al., 1990). Within these regions m4 receptors are distributed in a highly heterogeneous fashion, but the relation between irregularities in the distribution of m4 receptors and the known anatomical features of these regions has not been thoroughly investigated. The olfactory tubercle is among the most cytoarchitecturally complex regions of the forebrain and has been re-
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ported to contain patches of extremely intense m4 receptor expression, which were presumed to represent the islands of Calleja (Levey et al., 1991; Weiner et al., 1990). The islands of Calleja are densely packed clusters of granule cell which are present in all mammals studied to date, and are especially well developed in humans (Meyer et al., 1989). The islands show dramatic staining with the NADPH-diaphorase (NADPH-D) histochemical technique (Vincent and Kimura, 1992) and many islands have a core, or deeply indented hilus, which is devoid of granule cells but contains a number of loosely packed medium or large sized neurons (Fallon et al., 1978; Millhouse, 1987). In pilot studies on the immunocytochemical distribution of m4 receptors, we confirmed that patches of dense m4-like immunoreactivity could be observed within the olfactory tubercle; the size of these patches, however, appeared to be considerably smaller that that of the islands of Calleja. Examination of
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published figures also suggested a similar size mismatch (Levey et al., 1991; Weiner et al., 1990). These observations raised questions as to whether it was really the islands of Calleja that displayed intense m4 expression or some other cell grouping. It should be noted that the olfactory tubercle contains prominent cell clusters in addition to the islands of Calleja (Cajal, 1995; Fallon et al., 1978; Furuta et al., 2002; Phelps and Vaughn, 1986); indeed, the structures originally described by Calleja are apparently not those to which his name is now generally applied (Millhouse, 1987). The first goal of the current studies was to use a variety of multiple labeling techniques to examine the precise localization of m4 immunoreactive elements in the olfactory tubercle and islands of Calleja. We studied both rats and cynomolgus monkeys, in which the islands tend to have a somewhat different structure. We also extended our investigations to the olfactory bulb in order to investigate possible similarities between this structure and the islands of Calleja (Berezhnaia et al., 1998; Hosoya, 1973). High levels of m4 receptors have also been reported to be present in the dorsal striatum and the nucleus accumbens (Levey et al., 1991; Vilaro et al., 1991; Weiner et al., 1990). The dorsal striatum is composed of two major compartments, the striosomes and the matrix, which differ from each other with respect to a number of parameters including levels of mu-opiate receptors and of the calcium binding protein calbindin (Graybiel, 1990). A number of cholinergic markers are also organized with respect to compartmental boundaries (Graybiel et al., 1986; Herkenham and Pert, 1981; Nastuk and Graybiel, 1988, 1989), but the extent to which m4 receptors are compartmentally organized, has not yet been determined. Several neurochemically and connectionally defined subregions are also present within the territory of the nucleus accumbens (Zahm and Brog, 1982), but again, little is known about the relative expression of m4 receptors within them. A second goal of the current experiments was thus to describe the distribution of m4 receptors and their relation to other neurochemical markers in the dorsal and ventral striatum of rats and in monkeys.
2. Methods 2.1. Subjects Data were obtained from 11 adult, male, Sprague–Dawley derived rats obtained from a colony maintained by the Psychology Department of the University of Illinois at Chicago. Additional studies were conducted on tissue obtained from four adult female cynomolgus monkeys (Macaca fascicularis) obtained from Charles River (Huston, TX) and housed at Abbott Laboratories (Abbott Park, IL) prior to their use here. The current experiments were conducted in accordance with the N.I.H. Guide for the Care and Use of Laboratory Animals and were approved by the animal use
and care committees of the University of Illinois at Chicago or Abbott Laboratories. 2.2. Perfusion and tissue preparation Rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with normal saline followed by 500 ml of 10% formalin prepared in phosphate buffer. Brains were post-fixed in the same fixative for 1–2 h after which they were placed in a 20% solution of sucrose in phosphate buffered saline (PBS) for 24–48 h until they were cut. The monkeys were originally used in studies of the effects of dopaminergic drugs on Fos expression (Asin and Wirtshafter, 1997; Wirtshafter and Asin, 1999), and received injections of dopamine agonists or antagonists 2 h prior to sacrifice. It is extremely unlikely that these pretreatments would have affected staining for the compounds examined here; additionally, similar patterns of staining were seen in the islands of Calleja of all of the monkeys despite the fact that they received different drugs. The monkeys were perfused under deep sodium pentobarbital anesthesia with saline followed by phosphate buffered formalin using a protocol we have described in detail elsewhere (Asin et al., 1996). The brains were blocked into several pieces and post-fixed for 2 h and then stored for 48 h prior to sectioning in PBS containing 20% sucrose. Serial cryostat sections were taken through the olfactory tubercle and, in some of the rats, the olfactory bulb, thalamus and substantia nigra, at a thickness of 35 um (rats) or 30 um (primates). Tissue was stored in a cryoprotectant solution (Watson et al., 1986) at −20 ◦ C until staining which appeared to result in excellent preservation of immunoreactivity. 2.3. Histochemistry and immunocytochemistry In addition to routine staining with cresyl violet, some tissue sections were histochemically stained for the enzyme NADPH-diaphorase, while others were immunocytochemically stained for a variety of antigens. The NADPH-D staining technique we used was a slight modification of a method we have employed previously (Pitzer and Wirtshafter, 1997). Free floating sections were first rinsed thoroughly in PBS and then placed for 2 h at 36 ◦ C in a reaction mixture containing 0.04% NADPH, and 0.04% nitro blue tetrazolium in PBS (pH 7.2) containing 0.3% Triton X-100. All reagents were obtained from the Sigma Chemical Company (St. Louis, MO). The tissue was then rinsed in PBS, mounted and coverslipped with Eukitt (Calibrated Instruments, Hawthorne, NY). Immunohistochemical localization of m4-like immunoreactivity was accomplished using a monoclonal antibody raised against the i3 loop of the human m4 receptor (Chemicon, Temecula, CA, MAB1578, 2000×). According to information provided by the manufacturer, this antibody does not react with other forms of the muscarinic receptor. As a control for specificity we examined m4-like immunoreac-
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tivity in a number of regions which have been reported to contain low levels of m4 binding sites, including the thalamus, cortex and substantia nigra (Levey et al., 1991). Staining in all of these regions was extremely light compared to that observed in the striatal complex, despite the fact that high levels of other muscarinic subtypes have been reported to be present within these regions (Levey et al., 1991). The following primary antibodies were additionally employed: a rabbit anti-mu-opiate receptor antibody (Immunostar, Hudson, WI, #24216, 16,000×), a goat anti-choline acetyltransferase (ChAT) antibody (Chemicon, AB144P, 1200×), and a mouse anti-calbindin antibody (Sigma, St. Louis, C8666, 50,000×). For single labeling studies, free floating sections were rinsed and then incubated for 48 h at 4 ◦ C in the appropriate antibody prepared in Tris buffered saline containing 2% blocking serum, 0.3% Triton X-100, and 0.05% sodium azide. Sections were then rinsed in PBS, incubated for 2 h in appropriate secondary antibodies and, after further rinsing, developed using a Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) with nickel intensified diaminobenzadine (DAB) serving as the chromogen. Some tissue was counterstained with cresyl violet; in these sections unintensified DAB was used, as it yielded better contrast with the Nissl stain, and the DAB was only lightly developed so as to prevent the obscuring of tissue details. In control materials, omission of the primary antibodies eliminated specific staining. Some of the tissue was examined using a fluorescent double labeling technique to allow for the simultaneous detection of m4-like and ChAT-like immunoreactivity. These sections were incubated for 48 h in a cocktail containing both the anti-m4 and the anti-ChAT antibodies and then, after rinsing, were placed for 2 h at 36 ◦ C in a cocktail of Cy3 labeled donkey anti-goat serum and FITC labeled donkey anti-mouse serum (Jackson ImmunoResearch, West Grove, PA, 300× and 200×, respectively). Sections were then rinsed, mounted, coverslipped with VectaMount (Vector) and examined through a Leitz epifluorescence microscope. No bleed through between the Cy3 and FITC filter sets could be detected, and control tissue indicated that there was no detectable cross-reactivity between the secondary antibodies and the primary antibodies against which they were not directed.
3. Results 3.1. General description of m4-like immunoreactivity in the rostral forebrain of the rat Very little m4-like staining was seen in the cortex or globus pallidus, but moderate levels were seen within the dorsal and, to a greater extent, the ventral striatum. Within the dorsal striatum, staining tended to be more pronounced laterally than medially and faint patches of slightly intensified staining, resembling striosomes, as detected by other
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Fig. 1. Correspondence between regions of intensified m4-like immunoreactivity (m4-LI) (left hand column, panels A, C, and E) and striosomal markers (right hand column, panels B, D, and F) on near adjacent sections from rat (top two rows) and monkey (bottom row). The top row shows the alignment between a patch of enhanced m4 staining (panel A) and mu-opiate receptor-like expression (panel B). The second row shows a similar correspondence in the ventrolateral portion of the “subcallosal stripe” just medial to the external capsule. The bottom row shows patches of m4-LI in the primate caudate nucleus (panel E) which are aligned with regions displaying weak calbindin-like immunoreactivity (panel F). Scale bar = 250 um.
methods, could occasionally be seen within various regions of the striatum. Somewhat more consistent and distinct staining could also be seen within ventral portions of the subcallosal streak. In both cases, examination of adjacent sections indicated that the zones of intensified m4-like immunoreactivity were aligned with patches of mu-opiate receptor-like immunoreactivity (Fig. 1A–D), indicating that they corresponded to striosomes. Staining tended to be more pronounced in the shell than the core of the nucleus accumbens: at far rostral levels this tendency was apparent in both the lateral and, to a lesser extent, the intermediate and medial portions of the shell (Fig. 2A). At slightly more caudal levels (Fig. 2B) only the lateral shell showed intensified staining relative to the core of the accumbens. The region of the extreme ventrolateral striatal complex which has been referred to as the “lateral striatal stripe” (Phelps et al., 1985; Phelps and Vaughn, 1986; Zahm and Brog, 1982), showed markedly intensified staining which clearly demarcated it from surrounding regions (Fig. 2). At rostral levels, the lat-
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Fig. 3. Panels A–C display three consecutive sections through the olfactory tubercle of the rat at a level similar to that shown in Fig. 2B. Note that m4-like immunoreactivity (panel A) is aligned with histochemical staining for NADPH-diaphorase (panel C) but not with patches of intense mu-opiate receptor-like immunoreactivity (arrows in panel B) which mark ventral extensions of the cell clusters found in the basal portion of the nucleus accumbens. Panel D displays a section through the olfactory bulb. In this panel, g, glomerular layer; e, external plexiform layer; gr, granule cell layer. Scale bars = 250 um.
of more rostral tissue indicated that moderate staining was also seen within the external plexiform layers of both the main (Fig. 3D) and accessory olfactory bulb. The intensity of staining in these regions appeared similar to that seen in the nucleus accumbens shell, but did not approach the intensity of that present in the patches found within the olfactory tubercle. Fig. 2. Photomontages showing m4-like immunoreactivity at rostral (A) and caudal (B) levels of the nucleus accumbens and olfactory tubercle. Inserts indicate the location of the imaged region on camera lucida tracings of the sections from which the images were taken. Medial is to the right. White lines indicate the border between shell and core regions, as determined from adjacent sections stained for calbindin-like immunoreactivity. Note that m4-like staining is especially pronounced in the lateral portion of the shell, a tendency which is more pronounced at the more caudal level. White arrowheads indicate patches of intense m4-like immunoreactivity which correspond to the cores of the islands of Calleja. Black arrows indicate the lateral striatal stripe. Scale bar = 500 um.
eral striatal stripe extended ventrally into the most lateral portions of the olfactory tubercle and staining in this structure could be traced caudally as far as the amygdala. Staining in the olfactory tubercle itself was generally similar to that in the overlying shell from which it was largely separated by regions of lighter staining, corresponding to the rostral portions of the ventral pallidum and fascicles of the medial forebrain bundle. Embedded within the olfactory tubercle proper were small patches of extremely intense immunoreactivity (Figs. 2 and 3A) which far surpassed the level of staining seen anywhere else in the forebrain. Examination
3.2. Comparison of m4 staining with other markers in the olfactory tubercle In two animals, serial sections were prepared through the rostral forebrain and every third section was stained with antibodies directed either against the m4 receptor or the mu-opiate receptor, or by the histochemical procedure for NADPH-D. Identical results were obtained at all levels of both subjects and a representative series of three consecutive sections is shown in Fig. 3, panels A–C. Fig. 3C illustrates the intense NADPH-D staining characteristic of the islands of Calleja, with no obvious distinction between the core and the granule cell portions of the island being present. Scattered NADPH-D positive neurons are also seen in the olfactory tubercle surrounding the islands. Comparison of Fig. 3A and C, shows that the patches of intense m4 staining seen in Fig. 3A correspond perfectly in location to the islands of Calleja, but are much smaller than the islands themselves. Staining in the remaining portions of the islands did not appear to be more intense than that seen in the surrounding portions of the olfactory tubercle. Although on any individual section, the patches of intense m4 staining appeared to
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Fig. 5. Photomicrographs of islands of Calleja in the rat processed for m4-like immunoreactivity and cresyl violet. In panel A, the m4 immunoreactive cell sparse core is almost surrounded by granule cells whereas in panel B, the cores of two of the islands are partially “exposed”. The color balance of the image was slightly adjusted using Adobe Photoshop to enhance color separation between the two types of staining. Note that typical medium sized neurons stained with cresyl violet can be visualized within the core. Scale bar = 50 um.
Fig. 4. A sequential rostral to caudal series of eight 35 um sections through the olfactory tubercle spaced 70 um apart and stained for m4-like immunoreactivity. Note that regions of intensified staining can be traced from one section to another and that stained patches which appear discrete on any given section (numbered 1–4) can be seen to be interconnected on other sections (arrowheads) so as to form a single, connected, three-dimensional structure. Scale bar = 250 um.
be discrete and disconnected, careful examination of consecutive sections indicated that in virtually every case connections could be demonstrated between adjacent patches at other levels (Fig. 4). Fig. 3B illustrates the small patches of intense mu-opiate staining present in the olfactory tubercle, which are likely to correspond to ventral extensions of the cell clusters described by Herkenham et al. (1984). In sections from more rostral levels, the patches of intense mu-opiate receptor staining in the olfactory tubercle could be seen to be continuous with similar patches located along the ventral border of the nucleus accumbens (not shown). Careful comparison of adjacent sections indicated that opiate receptor staining was most frequently found in regions immediately adjacent to the islands of Calleja, but never overlapped with the zones of intense m4 staining. There was also
no strong association between m4 staining and the patches of opiate binding found in the ventral accumbens. In other sections we attempted to clarify the relation between the patches of m4 staining and the islands of Calleja by first processing the tissue for m4-like immunoreactivity and then counterstaining it with cresyl violet. In using this double staining method, processing in the chromogen diaminobenzadine (DAB) was done for a shorter time than normal to prevent the DAB reaction product from obscuring the cresyl violet staining. In this tissue, it could be clearly seen that the regions of intense m4 staining corresponded precisely to the cell sparse cores of the islands of Calleja (Fig. 5). In most cases, the immunochemically stained cores were completely surrounded by granule cells (e.g., Fig. 5A). In other cases, the intensely stained regions were situated in a deep cleft or hilus within the granule cell clusters (Fig. 5B). It is striking that even in these latter cases, the borders of the regions of intense m4 staining were still relatively sharply demarcated from the neuropil of the adjacent olfactory tubercle. In three additional rats we compared the distribution of m4-like and ChAT-like immunoreactivity using a fluorescent double labeling method. Although the granule cells themselves were always unstained, the neuropil between them displayed somewhat more ChAT-like immunoreactivity than did the surrounding tubercle, so that the outlines of the islands could be clearly visualized. Large, ChAT immunoreactive neurons could be visualized in the cores of the islands of Calleja and in the regions surrounding them and, less frequently, interstitial to the granule cells of the islands. ChAT cells or processes, found outside the cores never displayed m4-like immunoreactivity, and, in general, no correspondence could be seen between ChAT staining and the intense m4-like immunoreactivity within the core. ChAT immunoreactive neurons sometimes appeared to be located in small lacunae in the heavily m4 stained core and, in a some cases (Fig. 6), presumptive cholinergic neurons located at the bor-
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Fig. 6. Fluorescent images of a single section through an island of Calleja in the rat taken through filter sets for choline acetyltransferase (ChAT)-like immunofluorescence (panel A) and m4-like immunofluorescence (panel B). Note that ChAT immunoreactive cells do not display m4 staining and that, in several instances, they appear to occupy shallow depressions on the surface of the region of intense m4-like immunoreactivity (arrows). Scale bar =100 um.
der between the core and granule cell portions of the island appeared to occupy shallow “depressions” on the surface of the region of m4 staining. These appearances suggest that many cholinergic neurons within the islands of Calleja do not display prominent m4 receptor-like immunoreactivity. 3.3. Observations in primates In four cynomolgus monkeys, several pairs of adjacent, or near adjacent, sections through the olfactory tubercle were stained for m4-like immunoreactivity, and for either calbindin-like immunoreactivity or for cell bodies using cresyl violet. Staining for m4 receptors in the material we studied was in general lighter than that seen in rats, which made it difficult to study the pattern of immunoreactivity in regions showing only modest staining. Staining in the accumbens shell tended to be more pronounced than in the
Fig. 7. Images of near adjacent sections through islands of Calleja in the monkey stained for m4-like immunoreactivity (left hand column, panels A and A ) and cresyl violet (panels B and B ). Panels A and B display the insula magna, and it can be seen that m4 staining occupies the cell-sparse core of the island. Panels A and B show an ordinary island in which the granule cells form a sheet closely applied to the pial surface of the brain (panel B ). In this case, a region of intense m4-like staining is present immediately internal (i.e., dorsomedial) to the granule cell component of the island. Scale bar = 200 um.
core, but the difference was less marked than seen in rats. Faint patches resembling striosomes could be seen in the caudate nucleus of three of the four monkeys and in all of these cases examination of nearby sections indicated that these corresponded to calbindin-poor striosomes (Fig. 1E and F). Intense patches of m4-like immunoreactivity were, however, clearly visible in the olfactory tubercle of all subjects and, as was the case in rats, a strong and selective relation was apparent between these patches and the islands of Calleja. In the insula magna, and in some of the ordinary islands, the situation appeared identical to that seen in rats in that the m4 staining was precisely localized to cell sparse cores found within the islands (Fig. 7A and B). Many islands in the monkey, however, differ from the type most commonly seen in rats in that the granule cells frequently form solid masses or plates, which are closely applied to the pial surface of the tubercle so that an obvious core region is lacking. In these cases, a layer of intense m4-like immunoreactivity could be seen closely applied to the dorsal (internal) surface of the clusters (Fig. 7A and B ). Clusters of small to medium sized neurons could also be occasionally observed in Nissl sections within the nucleus accum-
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bens itself, but these were never associated with obvious m4 staining.
4. Discussion The current immunocytochemical studies are consistent with previous reports that m4 acetylcholine receptors are more concentrated in striatal structures than in the overlying cortex, a pattern opposite to that reported for m1 receptors (Levey et al., 1991; Weiner et al., 1990). Within the striatal complex, m4-like immunoreactivity was distributed in a highly heterogeneous fashion with by far the most intense staining seen in the islands of Calleja. Many authors have shown that the islands of Calleja in the rat consist of a peripheral region of densely packed granule cells surrounding a cell-sparse central core or hilus region in which a moderate number of medium to large sized neurons are located (Fallon et al., 1978; Millhouse, 1987). The current results demonstrate a marked neurochemical difference between the two components of the islands of Calleja in that the cores of the islands stain intensely for the m4 muscarinic receptor whereas only light staining is seen in the peripheral zones. Equivalent patterns are seen both in the insula magna, and in the so-called ordinary islands. Relatively light staining, similar to that seen in the periphery of the islands, is present in the surrounding regions of the olfactory tubercle with the result that the core regions stand out dramatically from adjacent structures and can be clearly visualized even at low magnification. The zones of intense m4-like immunoreactivity were sharply demarcated from surrounding regions of the olfactory tubercle even in cases in which they were not completely surrounded by granule cells. This pattern of staining suggests that the cores of the islands of Calleja constitute unique, neurochemically specialized regions. Staining for several other neurochemical markers has also been reported to differ between the core and periphery of the islands of Calleja (de Vente et al., 1998; Fallon et al., 1978; Loopuijt, 1989; Wahle and Meyer, 1986; Weiner et al., 1990), although these differences, in general, do not seem as pronounced as those observed here. Patches of intense m4-like immunoreactivity in the olfactory tubercle could be easily traced from one section to the next and examination of series of sections indicated that connections could almost always be found between adjacent regions of intense labeling. These observations suggest that the regions of intense m4-like immunoreactivity may actually form a single structure whose complex shape accounts for the fact that several apparently disconnected portions of it can be visualized on any individual section. Earlier authors, based on partial three-dimensional reconstructions of Nissl stained material, had suggested that adjacent granule cell clusters also frequently fuse to form complex, interconnected structures (Fox, 1940; Popoff and Popoff, 1929). This conclusion was convincingly demonstrated in a more recent study which examined material
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stained for NADPH-diaphorase, which labels both the core and granule cell regions of the islands (de Vente et al., 2001). Considering these results together, it appears likely that the zones of intense m4 staining form a connected skeleton-like framework which is largely, but not entirely, “coated over” by granule cells. In most regions, the m4 stained zone is completely covered by granule cells generating the appearance in cross sections of a hollow granule cell island surrounding a cell sparse core, like that shown in Fig. 5A. At other locations, the granule cells may not entirely enwrap the core, generating a situation like that shown in Fig. 5B where the intensely m4 immunoreactive region is continuous with, but still demarcated from, the “extrainsular” regions of the olfactory tubercle. Intense m4-like staining was also closely associated with the islands of Calleja in cynomolgus monkeys. The presence of similar distributions of m4 receptors in members of two distantly related orders suggests that this association is likely to have developed at an early stage of mammalian evolution, and that the m4 enriched zones are likely to form a fundamental component of the island of Calleja complexes. The insula magna of monkeys is similar to that of rats in that it consists of an intensely m4 immunoreactive core surrounded by a mass of granule cells. Many of the ordinary islands of the monkey, however, consist of a relatively thin layer of granule cells closely applied to the ventral or ventromedial surface of the brain which do not enclose an obvious core region. This type of island is rarely seen in the rat, but is common in a number of other animals including cats and humans (Meyer et al., 1989; Meyer and Wahle, 1986; Talbot et al., 1988). It is tempting to speculate that these structural differences may reflect the fact that lamination is much better developed in the olfactory tubercle of rodents than it is in carnivores or primates (Meyer et al., 1989). At any account, although the morphology of these granule cell clusters in monkeys is different from that seen in rodents, a close association between them and zones of intense m4 staining was still present. Thus, in the monkeys, a thin band of intense m4-like immunoreactivity was found immediately internal to each subpial granule cell cluster which we observed, and it is highly likely that these zones are homologous to the core regions of rodents. These findings further support the view that the intense m4 staining is a marker for a special region of the island complexes and that the presence of these regions is likely to play an essential role in the functioning of the islands in a number of species. The findings in monkeys again illustrate the fact that the regions of intense m4-like staining do not have to be surrounded by granule cells in order to be sharply set off from adjacent regions of the olfactory tubercle. It would be interesting to determine whether the granule cell and core regions form connected masses in primates, as they do in rats. Despite the anatomical specificity of the m4 immunostaining, it is not clear exactly which tissue elements were being labeled. Since mRNA for m4 receptors has been identified in the islands of Calleja (Weiner et al., 1990), it is likely that
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staining is present primarily in local cell bodies or dendrites. An obvious candidate is the cholinergic neurons which are found within the islands (Mesulam et al., 1984; Phelps and Vaughn, 1986), but the current results provide no support for this possibility. The majority of ChAT immunoreactive neurons we observed within the cores of the islands occurred against a background of such intense m4 staining that it was impossible to tell whether or not they were double labeled. In many cases, however, ChAT immunoreactive neurons were found to occupy small lacunae within, or at the borders of, the regions of intense m4 staining. Again, cholinergic neurons are extremely common adjacent to the islands (Ichitani et al., 1993; Mesulam et al., 1984), and are occasionally seen within the granule cell zones of the islands, but these cells were never double labeled, even though they were very similar in appearance to those seen in the cores. Clear ChAT-like immunoreactivity could also be observed in the sparse neuropil found between the granule cells, as reported by previous authors (Phelps and Vaughn, 1986), but m4-like staining was again very weak in these regions. These results indicate that many cholinergic neurons do not contain detectable m4-like immunoreactivity. Although these findings do not rule out the possibility that a specialized subset of cholinergic neurons found within the islands of Calleja may express m4 receptors, it seems more likely that local cholinergic neurons provide the source of acetylcholine which acts at receptors located on an as yet to be identified population of cells. Detectable m4-like immunoreactivity was also found in several other regions of the basal forebrain. Clear, but modest, staining was present within the external plexiform layers of the main and accessory olfactory bulbs. The marked contrast between the intensity of this staining and that seen in the islands of Calleja provides little support for the concept that mitral cells in the olfactory bulb are analogous to the medium sized cells of the islands of Calleja (Berezhnaia et al., 1998; Hosoya, 1973). Relatively light staining was also seen throughout the dorsal striatum, and occasional “patches” of intensified staining were seen in both rats and monkeys. Similar observations had been previously reported in rats (Levey et al., 1991; Ince et al., 1997). We were able to extend these observations by demonstrating, in both rats and monkeys, that the patches of intensified m4-like immunoreactivity correspond to striosomes identified by mu-opiate receptor-like or calbindin-like immunoreactivity. Staining for m4 receptors appears to be a relatively weak marker for striosomes and in both species many striosomes could not be visualized in m4 stained material. Previous studies using autoradiographic methods have shown that binding of both the nonselective muscarinic ligand propylbenzilylcholine mustard (Nastuk and Graybiel, 1985) and the “M1” ligand pirenzepine (Nastuk and Graybiel, 1988, 1989) is greater in striosomes than matrix. As pirenzepine has substantial affinity for both the m1 and m4 receptors (Moriya et al., 1999), it is possible that the compartmental selectivity of the binding of this compound is due in part to an action
at m4 receptors. Staining for m4 receptors was also more pronounced in the shell than the core of the nucleus accumbens and would thus appear to be another marker capable of distinguishing between these two regions of the ventral striatum (Zahm and Brog, 1982). Immunoreactivity was much more pronounced in the lateral than the medial portions of the accumbens shell, a finding consistent with other results suggesting that there may be important neurochemical differences between these two subregions (Jongen-Relo et al., 1994). Substantially more intense m4-like staining was seen in the so-called “lateral stripe” found in the ventrolateral portions of the striatum extending ventrally into the lateral olfactory tubercle (Phelps et al., 1985; Phelps and Vaughn, 1986; Zahm and Brog, 1982). The intensity of staining in this slender region was second only to that seen in the cores of the islands of Calleja, and this finding supports the suggestion that the striatal stripe may represent a neurochemically specialized region of the striatal complex (Phelps et al., 1985; Phelps and Vaughn, 1986; Zahm and Brog, 1982). Phelps and Vaughn (1986) have suggested that the lateral stripe may be related to clusters of medium sized neurons found in the extrainsular olfactory tubercle, a conclusion also supported by the work of Furuta et al. (2002), but the current results raise the possibility that it may also resemble the cores of the islands of Calleja. Indeed, if the cell clusters observed by Phelps and Vaughn correspond to the mu-opiate receptor rich clusters identified by Herkenham et al. (1984) our results would demonstrate a difference in the chemical anatomy of these regions and the striatal stripe since we did not observe a marked intensification of m4-like immunoreactivity in the cell cluster regions. Many authors currently agree that the olfactory tubercle is part of a larger “basal ganglia complex” and there have been a number of attempts to demonstrate how the islands of Calleja, which receive a dense dopaminergic innervation (Fallon et al., 1978), might fit into such a scheme. It has been suggested, for example, that the granule cells of the islands might be analogous to medium sized spiny striatal neurons, while the cells concentrated in the cores of the islands might be analogous to pallidal output neurons (Fallon, 1983; Fallon et al., 1983). While not refuting these sorts of theories, the current results do serve to emphasize the extent to which the chemical structure of the islands of Calleja is unlike that seen in other parts of basal ganglia. Indeed, there are many unique aspects of the islands of Calleja, ranging from the size and packing density of the neurons found within them, to the their close association with blood vessels (Fallon et al., 1983; Meyer et al., 1989, 1994), to the way in which the granule cells of the islands respond to various drugs (MacGibbon et al., 1994; Vahid-Ansari and Robertson, 1996; Wirtshafter, 1998). Likewise, although there are certain similarities between the cells of the islands and those of the olfactory bulb (Berezhnaia et al., 1998; Hosoya, 1973), there are, as the current study demonstrates, important differences as well. It may be that progress in understanding the function of the islands of Calleja will come more from a
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consideration of their many unique attributes than from an attempt to find similarities between them and other nearby structures in the basal forebrain.
Acknowledgements The studies were supported by NIH grant NS33992. The authors thank Dr. Karen Asin for her many helpful comments on the manuscript, and Robert Canavan for his help with photography and imaging in this, and many other, studies.
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The distribution of m4 muscarinic acetylcholine receptors in the islands of Calleja and striatum of rats and cynomolgus monkeys David Wirtshafter∗ , Catherine V. Osborn Laboratory of Integrative Neuroscience, Department of Psychology, M/C 285, University of Illinois at Chicago, 1007 W. Harrison St., Chicago, IL 60607-7137, USA Received 26 November 2003; received in revised form 9 April 2004; accepted 21 May 2004 Available online 18 August 2004
Abstract The distribution of m4 muscarinic acetylcholine receptors, and their relation to a number other markers, was examined using immunocytochemical techniques. Staining in the dorsal striatum tended to be more pronounced in the striosomal than the matrix compartment of both rats and cynomolgus monkeys. Within the ventral striatum, immunoreactivity was more pronounced within the olfactory tubercle and the shell region of the nucleus accumbens than in the nucleus accumbens core and was especially marked within the lateral striatal stripe. Modest staining was also seen in the external plexiform layer of the olfactory bulb. By far, the most intense staining in the forebrain of both rats and cynomolgus monkeys was found in islands of Calleja, where it appeared to be a selective marker for the core or hilus regions of the islands, or an analogous region found adjacent to them. The core regions of different islands appear to be continuous with each other so as to form a complex three-dimensional structure, which is largely encased by layers of granule cells. The neuronal elements in the islands of Calleja, which express m4 receptors, remain to be identified, but it is unlikely that cholinergic neurons are a major locus of these receptors. Although there are certain similarities between the islands of Calleja and other components of the striatal complex, the current studies emphasize the extent to which the islands are unique in terms of their architecture and chemical anatomy. © 2004 Elsevier B.V. All rights reserved. Keywords: Islands of Calleja; Muscarinic receptors; Cholinergic receptors; Olfactory tubercle; Nucleus accumbens; Shell; Core; Striatum; Olfactory bulb; Cell clusters; Lateral stripe
1. Introduction The m4 muscarinic acetylcholine receptor is expressed at high levels in several regions of the basal forebrain including the olfactory tubercle, the nucleus accumbens and the dorsal striatum (Levey et al., 1991; Weiner et al., 1990). Within these regions m4 receptors are distributed in a highly heterogeneous fashion, but the relation between irregularities in the distribution of m4 receptors and the known anatomical features of these regions has not been thoroughly investigated. The olfactory tubercle is among the most cytoarchitecturally complex regions of the forebrain and has been re-
∗
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ported to contain patches of extremely intense m4 receptor expression, which were presumed to represent the islands of Calleja (Levey et al., 1991; Weiner et al., 1990). The islands of Calleja are densely packed clusters of granule cell which are present in all mammals studied to date, and are especially well developed in humans (Meyer et al., 1989). The islands show dramatic staining with the NADPH-diaphorase (NADPH-D) histochemical technique (Vincent and Kimura, 1992) and many islands have a core, or deeply indented hilus, which is devoid of granule cells but contains a number of loosely packed medium or large sized neurons (Fallon et al., 1978; Millhouse, 1987). In pilot studies on the immunocytochemical distribution of m4 receptors, we confirmed that patches of dense m4-like immunoreactivity could be observed within the olfactory tubercle; the size of these patches, however, appeared to be considerably smaller that that of the islands of Calleja. Examination of
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published figures also suggested a similar size mismatch (Levey et al., 1991; Weiner et al., 1990). These observations raised questions as to whether it was really the islands of Calleja that displayed intense m4 expression or some other cell grouping. It should be noted that the olfactory tubercle contains prominent cell clusters in addition to the islands of Calleja (Cajal, 1995; Fallon et al., 1978; Furuta et al., 2002; Phelps and Vaughn, 1986); indeed, the structures originally described by Calleja are apparently not those to which his name is now generally applied (Millhouse, 1987). The first goal of the current studies was to use a variety of multiple labeling techniques to examine the precise localization of m4 immunoreactive elements in the olfactory tubercle and islands of Calleja. We studied both rats and cynomolgus monkeys, in which the islands tend to have a somewhat different structure. We also extended our investigations to the olfactory bulb in order to investigate possible similarities between this structure and the islands of Calleja (Berezhnaia et al., 1998; Hosoya, 1973). High levels of m4 receptors have also been reported to be present in the dorsal striatum and the nucleus accumbens (Levey et al., 1991; Vilaro et al., 1991; Weiner et al., 1990). The dorsal striatum is composed of two major compartments, the striosomes and the matrix, which differ from each other with respect to a number of parameters including levels of mu-opiate receptors and of the calcium binding protein calbindin (Graybiel, 1990). A number of cholinergic markers are also organized with respect to compartmental boundaries (Graybiel et al., 1986; Herkenham and Pert, 1981; Nastuk and Graybiel, 1988, 1989), but the extent to which m4 receptors are compartmentally organized, has not yet been determined. Several neurochemically and connectionally defined subregions are also present within the territory of the nucleus accumbens (Zahm and Brog, 1982), but again, little is known about the relative expression of m4 receptors within them. A second goal of the current experiments was thus to describe the distribution of m4 receptors and their relation to other neurochemical markers in the dorsal and ventral striatum of rats and in monkeys.
2. Methods 2.1. Subjects Data were obtained from 11 adult, male, Sprague–Dawley derived rats obtained from a colony maintained by the Psychology Department of the University of Illinois at Chicago. Additional studies were conducted on tissue obtained from four adult female cynomolgus monkeys (Macaca fascicularis) obtained from Charles River (Huston, TX) and housed at Abbott Laboratories (Abbott Park, IL) prior to their use here. The current experiments were conducted in accordance with the N.I.H. Guide for the Care and Use of Laboratory Animals and were approved by the animal use
and care committees of the University of Illinois at Chicago or Abbott Laboratories. 2.2. Perfusion and tissue preparation Rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with normal saline followed by 500 ml of 10% formalin prepared in phosphate buffer. Brains were post-fixed in the same fixative for 1–2 h after which they were placed in a 20% solution of sucrose in phosphate buffered saline (PBS) for 24–48 h until they were cut. The monkeys were originally used in studies of the effects of dopaminergic drugs on Fos expression (Asin and Wirtshafter, 1997; Wirtshafter and Asin, 1999), and received injections of dopamine agonists or antagonists 2 h prior to sacrifice. It is extremely unlikely that these pretreatments would have affected staining for the compounds examined here; additionally, similar patterns of staining were seen in the islands of Calleja of all of the monkeys despite the fact that they received different drugs. The monkeys were perfused under deep sodium pentobarbital anesthesia with saline followed by phosphate buffered formalin using a protocol we have described in detail elsewhere (Asin et al., 1996). The brains were blocked into several pieces and post-fixed for 2 h and then stored for 48 h prior to sectioning in PBS containing 20% sucrose. Serial cryostat sections were taken through the olfactory tubercle and, in some of the rats, the olfactory bulb, thalamus and substantia nigra, at a thickness of 35 um (rats) or 30 um (primates). Tissue was stored in a cryoprotectant solution (Watson et al., 1986) at −20 ◦ C until staining which appeared to result in excellent preservation of immunoreactivity. 2.3. Histochemistry and immunocytochemistry In addition to routine staining with cresyl violet, some tissue sections were histochemically stained for the enzyme NADPH-diaphorase, while others were immunocytochemically stained for a variety of antigens. The NADPH-D staining technique we used was a slight modification of a method we have employed previously (Pitzer and Wirtshafter, 1997). Free floating sections were first rinsed thoroughly in PBS and then placed for 2 h at 36 ◦ C in a reaction mixture containing 0.04% NADPH, and 0.04% nitro blue tetrazolium in PBS (pH 7.2) containing 0.3% Triton X-100. All reagents were obtained from the Sigma Chemical Company (St. Louis, MO). The tissue was then rinsed in PBS, mounted and coverslipped with Eukitt (Calibrated Instruments, Hawthorne, NY). Immunohistochemical localization of m4-like immunoreactivity was accomplished using a monoclonal antibody raised against the i3 loop of the human m4 receptor (Chemicon, Temecula, CA, MAB1578, 2000×). According to information provided by the manufacturer, this antibody does not react with other forms of the muscarinic receptor. As a control for specificity we examined m4-like immunoreac-
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tivity in a number of regions which have been reported to contain low levels of m4 binding sites, including the thalamus, cortex and substantia nigra (Levey et al., 1991). Staining in all of these regions was extremely light compared to that observed in the striatal complex, despite the fact that high levels of other muscarinic subtypes have been reported to be present within these regions (Levey et al., 1991). The following primary antibodies were additionally employed: a rabbit anti-mu-opiate receptor antibody (Immunostar, Hudson, WI, #24216, 16,000×), a goat anti-choline acetyltransferase (ChAT) antibody (Chemicon, AB144P, 1200×), and a mouse anti-calbindin antibody (Sigma, St. Louis, C8666, 50,000×). For single labeling studies, free floating sections were rinsed and then incubated for 48 h at 4 ◦ C in the appropriate antibody prepared in Tris buffered saline containing 2% blocking serum, 0.3% Triton X-100, and 0.05% sodium azide. Sections were then rinsed in PBS, incubated for 2 h in appropriate secondary antibodies and, after further rinsing, developed using a Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) with nickel intensified diaminobenzadine (DAB) serving as the chromogen. Some tissue was counterstained with cresyl violet; in these sections unintensified DAB was used, as it yielded better contrast with the Nissl stain, and the DAB was only lightly developed so as to prevent the obscuring of tissue details. In control materials, omission of the primary antibodies eliminated specific staining. Some of the tissue was examined using a fluorescent double labeling technique to allow for the simultaneous detection of m4-like and ChAT-like immunoreactivity. These sections were incubated for 48 h in a cocktail containing both the anti-m4 and the anti-ChAT antibodies and then, after rinsing, were placed for 2 h at 36 ◦ C in a cocktail of Cy3 labeled donkey anti-goat serum and FITC labeled donkey anti-mouse serum (Jackson ImmunoResearch, West Grove, PA, 300× and 200×, respectively). Sections were then rinsed, mounted, coverslipped with VectaMount (Vector) and examined through a Leitz epifluorescence microscope. No bleed through between the Cy3 and FITC filter sets could be detected, and control tissue indicated that there was no detectable cross-reactivity between the secondary antibodies and the primary antibodies against which they were not directed.
3. Results 3.1. General description of m4-like immunoreactivity in the rostral forebrain of the rat Very little m4-like staining was seen in the cortex or globus pallidus, but moderate levels were seen within the dorsal and, to a greater extent, the ventral striatum. Within the dorsal striatum, staining tended to be more pronounced laterally than medially and faint patches of slightly intensified staining, resembling striosomes, as detected by other
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Fig. 1. Correspondence between regions of intensified m4-like immunoreactivity (m4-LI) (left hand column, panels A, C, and E) and striosomal markers (right hand column, panels B, D, and F) on near adjacent sections from rat (top two rows) and monkey (bottom row). The top row shows the alignment between a patch of enhanced m4 staining (panel A) and mu-opiate receptor-like expression (panel B). The second row shows a similar correspondence in the ventrolateral portion of the “subcallosal stripe” just medial to the external capsule. The bottom row shows patches of m4-LI in the primate caudate nucleus (panel E) which are aligned with regions displaying weak calbindin-like immunoreactivity (panel F). Scale bar = 250 um.
methods, could occasionally be seen within various regions of the striatum. Somewhat more consistent and distinct staining could also be seen within ventral portions of the subcallosal streak. In both cases, examination of adjacent sections indicated that the zones of intensified m4-like immunoreactivity were aligned with patches of mu-opiate receptor-like immunoreactivity (Fig. 1A–D), indicating that they corresponded to striosomes. Staining tended to be more pronounced in the shell than the core of the nucleus accumbens: at far rostral levels this tendency was apparent in both the lateral and, to a lesser extent, the intermediate and medial portions of the shell (Fig. 2A). At slightly more caudal levels (Fig. 2B) only the lateral shell showed intensified staining relative to the core of the accumbens. The region of the extreme ventrolateral striatal complex which has been referred to as the “lateral striatal stripe” (Phelps et al., 1985; Phelps and Vaughn, 1986; Zahm and Brog, 1982), showed markedly intensified staining which clearly demarcated it from surrounding regions (Fig. 2). At rostral levels, the lat-
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Fig. 3. Panels A–C display three consecutive sections through the olfactory tubercle of the rat at a level similar to that shown in Fig. 2B. Note that m4-like immunoreactivity (panel A) is aligned with histochemical staining for NADPH-diaphorase (panel C) but not with patches of intense mu-opiate receptor-like immunoreactivity (arrows in panel B) which mark ventral extensions of the cell clusters found in the basal portion of the nucleus accumbens. Panel D displays a section through the olfactory bulb. In this panel, g, glomerular layer; e, external plexiform layer; gr, granule cell layer. Scale bars = 250 um.
of more rostral tissue indicated that moderate staining was also seen within the external plexiform layers of both the main (Fig. 3D) and accessory olfactory bulb. The intensity of staining in these regions appeared similar to that seen in the nucleus accumbens shell, but did not approach the intensity of that present in the patches found within the olfactory tubercle. Fig. 2. Photomontages showing m4-like immunoreactivity at rostral (A) and caudal (B) levels of the nucleus accumbens and olfactory tubercle. Inserts indicate the location of the imaged region on camera lucida tracings of the sections from which the images were taken. Medial is to the right. White lines indicate the border between shell and core regions, as determined from adjacent sections stained for calbindin-like immunoreactivity. Note that m4-like staining is especially pronounced in the lateral portion of the shell, a tendency which is more pronounced at the more caudal level. White arrowheads indicate patches of intense m4-like immunoreactivity which correspond to the cores of the islands of Calleja. Black arrows indicate the lateral striatal stripe. Scale bar = 500 um.
eral striatal stripe extended ventrally into the most lateral portions of the olfactory tubercle and staining in this structure could be traced caudally as far as the amygdala. Staining in the olfactory tubercle itself was generally similar to that in the overlying shell from which it was largely separated by regions of lighter staining, corresponding to the rostral portions of the ventral pallidum and fascicles of the medial forebrain bundle. Embedded within the olfactory tubercle proper were small patches of extremely intense immunoreactivity (Figs. 2 and 3A) which far surpassed the level of staining seen anywhere else in the forebrain. Examination
3.2. Comparison of m4 staining with other markers in the olfactory tubercle In two animals, serial sections were prepared through the rostral forebrain and every third section was stained with antibodies directed either against the m4 receptor or the mu-opiate receptor, or by the histochemical procedure for NADPH-D. Identical results were obtained at all levels of both subjects and a representative series of three consecutive sections is shown in Fig. 3, panels A–C. Fig. 3C illustrates the intense NADPH-D staining characteristic of the islands of Calleja, with no obvious distinction between the core and the granule cell portions of the island being present. Scattered NADPH-D positive neurons are also seen in the olfactory tubercle surrounding the islands. Comparison of Fig. 3A and C, shows that the patches of intense m4 staining seen in Fig. 3A correspond perfectly in location to the islands of Calleja, but are much smaller than the islands themselves. Staining in the remaining portions of the islands did not appear to be more intense than that seen in the surrounding portions of the olfactory tubercle. Although on any individual section, the patches of intense m4 staining appeared to
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Fig. 5. Photomicrographs of islands of Calleja in the rat processed for m4-like immunoreactivity and cresyl violet. In panel A, the m4 immunoreactive cell sparse core is almost surrounded by granule cells whereas in panel B, the cores of two of the islands are partially “exposed”. The color balance of the image was slightly adjusted using Adobe Photoshop to enhance color separation between the two types of staining. Note that typical medium sized neurons stained with cresyl violet can be visualized within the core. Scale bar = 50 um.
Fig. 4. A sequential rostral to caudal series of eight 35 um sections through the olfactory tubercle spaced 70 um apart and stained for m4-like immunoreactivity. Note that regions of intensified staining can be traced from one section to another and that stained patches which appear discrete on any given section (numbered 1–4) can be seen to be interconnected on other sections (arrowheads) so as to form a single, connected, three-dimensional structure. Scale bar = 250 um.
be discrete and disconnected, careful examination of consecutive sections indicated that in virtually every case connections could be demonstrated between adjacent patches at other levels (Fig. 4). Fig. 3B illustrates the small patches of intense mu-opiate staining present in the olfactory tubercle, which are likely to correspond to ventral extensions of the cell clusters described by Herkenham et al. (1984). In sections from more rostral levels, the patches of intense mu-opiate receptor staining in the olfactory tubercle could be seen to be continuous with similar patches located along the ventral border of the nucleus accumbens (not shown). Careful comparison of adjacent sections indicated that opiate receptor staining was most frequently found in regions immediately adjacent to the islands of Calleja, but never overlapped with the zones of intense m4 staining. There was also
no strong association between m4 staining and the patches of opiate binding found in the ventral accumbens. In other sections we attempted to clarify the relation between the patches of m4 staining and the islands of Calleja by first processing the tissue for m4-like immunoreactivity and then counterstaining it with cresyl violet. In using this double staining method, processing in the chromogen diaminobenzadine (DAB) was done for a shorter time than normal to prevent the DAB reaction product from obscuring the cresyl violet staining. In this tissue, it could be clearly seen that the regions of intense m4 staining corresponded precisely to the cell sparse cores of the islands of Calleja (Fig. 5). In most cases, the immunochemically stained cores were completely surrounded by granule cells (e.g., Fig. 5A). In other cases, the intensely stained regions were situated in a deep cleft or hilus within the granule cell clusters (Fig. 5B). It is striking that even in these latter cases, the borders of the regions of intense m4 staining were still relatively sharply demarcated from the neuropil of the adjacent olfactory tubercle. In three additional rats we compared the distribution of m4-like and ChAT-like immunoreactivity using a fluorescent double labeling method. Although the granule cells themselves were always unstained, the neuropil between them displayed somewhat more ChAT-like immunoreactivity than did the surrounding tubercle, so that the outlines of the islands could be clearly visualized. Large, ChAT immunoreactive neurons could be visualized in the cores of the islands of Calleja and in the regions surrounding them and, less frequently, interstitial to the granule cells of the islands. ChAT cells or processes, found outside the cores never displayed m4-like immunoreactivity, and, in general, no correspondence could be seen between ChAT staining and the intense m4-like immunoreactivity within the core. ChAT immunoreactive neurons sometimes appeared to be located in small lacunae in the heavily m4 stained core and, in a some cases (Fig. 6), presumptive cholinergic neurons located at the bor-
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Fig. 6. Fluorescent images of a single section through an island of Calleja in the rat taken through filter sets for choline acetyltransferase (ChAT)-like immunofluorescence (panel A) and m4-like immunofluorescence (panel B). Note that ChAT immunoreactive cells do not display m4 staining and that, in several instances, they appear to occupy shallow depressions on the surface of the region of intense m4-like immunoreactivity (arrows). Scale bar =100 um.
der between the core and granule cell portions of the island appeared to occupy shallow “depressions” on the surface of the region of m4 staining. These appearances suggest that many cholinergic neurons within the islands of Calleja do not display prominent m4 receptor-like immunoreactivity. 3.3. Observations in primates In four cynomolgus monkeys, several pairs of adjacent, or near adjacent, sections through the olfactory tubercle were stained for m4-like immunoreactivity, and for either calbindin-like immunoreactivity or for cell bodies using cresyl violet. Staining for m4 receptors in the material we studied was in general lighter than that seen in rats, which made it difficult to study the pattern of immunoreactivity in regions showing only modest staining. Staining in the accumbens shell tended to be more pronounced than in the
Fig. 7. Images of near adjacent sections through islands of Calleja in the monkey stained for m4-like immunoreactivity (left hand column, panels A and A ) and cresyl violet (panels B and B ). Panels A and B display the insula magna, and it can be seen that m4 staining occupies the cell-sparse core of the island. Panels A and B show an ordinary island in which the granule cells form a sheet closely applied to the pial surface of the brain (panel B ). In this case, a region of intense m4-like staining is present immediately internal (i.e., dorsomedial) to the granule cell component of the island. Scale bar = 200 um.
core, but the difference was less marked than seen in rats. Faint patches resembling striosomes could be seen in the caudate nucleus of three of the four monkeys and in all of these cases examination of nearby sections indicated that these corresponded to calbindin-poor striosomes (Fig. 1E and F). Intense patches of m4-like immunoreactivity were, however, clearly visible in the olfactory tubercle of all subjects and, as was the case in rats, a strong and selective relation was apparent between these patches and the islands of Calleja. In the insula magna, and in some of the ordinary islands, the situation appeared identical to that seen in rats in that the m4 staining was precisely localized to cell sparse cores found within the islands (Fig. 7A and B). Many islands in the monkey, however, differ from the type most commonly seen in rats in that the granule cells frequently form solid masses or plates, which are closely applied to the pial surface of the tubercle so that an obvious core region is lacking. In these cases, a layer of intense m4-like immunoreactivity could be seen closely applied to the dorsal (internal) surface of the clusters (Fig. 7A and B ). Clusters of small to medium sized neurons could also be occasionally observed in Nissl sections within the nucleus accum-
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bens itself, but these were never associated with obvious m4 staining.
4. Discussion The current immunocytochemical studies are consistent with previous reports that m4 acetylcholine receptors are more concentrated in striatal structures than in the overlying cortex, a pattern opposite to that reported for m1 receptors (Levey et al., 1991; Weiner et al., 1990). Within the striatal complex, m4-like immunoreactivity was distributed in a highly heterogeneous fashion with by far the most intense staining seen in the islands of Calleja. Many authors have shown that the islands of Calleja in the rat consist of a peripheral region of densely packed granule cells surrounding a cell-sparse central core or hilus region in which a moderate number of medium to large sized neurons are located (Fallon et al., 1978; Millhouse, 1987). The current results demonstrate a marked neurochemical difference between the two components of the islands of Calleja in that the cores of the islands stain intensely for the m4 muscarinic receptor whereas only light staining is seen in the peripheral zones. Equivalent patterns are seen both in the insula magna, and in the so-called ordinary islands. Relatively light staining, similar to that seen in the periphery of the islands, is present in the surrounding regions of the olfactory tubercle with the result that the core regions stand out dramatically from adjacent structures and can be clearly visualized even at low magnification. The zones of intense m4-like immunoreactivity were sharply demarcated from surrounding regions of the olfactory tubercle even in cases in which they were not completely surrounded by granule cells. This pattern of staining suggests that the cores of the islands of Calleja constitute unique, neurochemically specialized regions. Staining for several other neurochemical markers has also been reported to differ between the core and periphery of the islands of Calleja (de Vente et al., 1998; Fallon et al., 1978; Loopuijt, 1989; Wahle and Meyer, 1986; Weiner et al., 1990), although these differences, in general, do not seem as pronounced as those observed here. Patches of intense m4-like immunoreactivity in the olfactory tubercle could be easily traced from one section to the next and examination of series of sections indicated that connections could almost always be found between adjacent regions of intense labeling. These observations suggest that the regions of intense m4-like immunoreactivity may actually form a single structure whose complex shape accounts for the fact that several apparently disconnected portions of it can be visualized on any individual section. Earlier authors, based on partial three-dimensional reconstructions of Nissl stained material, had suggested that adjacent granule cell clusters also frequently fuse to form complex, interconnected structures (Fox, 1940; Popoff and Popoff, 1929). This conclusion was convincingly demonstrated in a more recent study which examined material
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stained for NADPH-diaphorase, which labels both the core and granule cell regions of the islands (de Vente et al., 2001). Considering these results together, it appears likely that the zones of intense m4 staining form a connected skeleton-like framework which is largely, but not entirely, “coated over” by granule cells. In most regions, the m4 stained zone is completely covered by granule cells generating the appearance in cross sections of a hollow granule cell island surrounding a cell sparse core, like that shown in Fig. 5A. At other locations, the granule cells may not entirely enwrap the core, generating a situation like that shown in Fig. 5B where the intensely m4 immunoreactive region is continuous with, but still demarcated from, the “extrainsular” regions of the olfactory tubercle. Intense m4-like staining was also closely associated with the islands of Calleja in cynomolgus monkeys. The presence of similar distributions of m4 receptors in members of two distantly related orders suggests that this association is likely to have developed at an early stage of mammalian evolution, and that the m4 enriched zones are likely to form a fundamental component of the island of Calleja complexes. The insula magna of monkeys is similar to that of rats in that it consists of an intensely m4 immunoreactive core surrounded by a mass of granule cells. Many of the ordinary islands of the monkey, however, consist of a relatively thin layer of granule cells closely applied to the ventral or ventromedial surface of the brain which do not enclose an obvious core region. This type of island is rarely seen in the rat, but is common in a number of other animals including cats and humans (Meyer et al., 1989; Meyer and Wahle, 1986; Talbot et al., 1988). It is tempting to speculate that these structural differences may reflect the fact that lamination is much better developed in the olfactory tubercle of rodents than it is in carnivores or primates (Meyer et al., 1989). At any account, although the morphology of these granule cell clusters in monkeys is different from that seen in rodents, a close association between them and zones of intense m4 staining was still present. Thus, in the monkeys, a thin band of intense m4-like immunoreactivity was found immediately internal to each subpial granule cell cluster which we observed, and it is highly likely that these zones are homologous to the core regions of rodents. These findings further support the view that the intense m4 staining is a marker for a special region of the island complexes and that the presence of these regions is likely to play an essential role in the functioning of the islands in a number of species. The findings in monkeys again illustrate the fact that the regions of intense m4-like staining do not have to be surrounded by granule cells in order to be sharply set off from adjacent regions of the olfactory tubercle. It would be interesting to determine whether the granule cell and core regions form connected masses in primates, as they do in rats. Despite the anatomical specificity of the m4 immunostaining, it is not clear exactly which tissue elements were being labeled. Since mRNA for m4 receptors has been identified in the islands of Calleja (Weiner et al., 1990), it is likely that
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staining is present primarily in local cell bodies or dendrites. An obvious candidate is the cholinergic neurons which are found within the islands (Mesulam et al., 1984; Phelps and Vaughn, 1986), but the current results provide no support for this possibility. The majority of ChAT immunoreactive neurons we observed within the cores of the islands occurred against a background of such intense m4 staining that it was impossible to tell whether or not they were double labeled. In many cases, however, ChAT immunoreactive neurons were found to occupy small lacunae within, or at the borders of, the regions of intense m4 staining. Again, cholinergic neurons are extremely common adjacent to the islands (Ichitani et al., 1993; Mesulam et al., 1984), and are occasionally seen within the granule cell zones of the islands, but these cells were never double labeled, even though they were very similar in appearance to those seen in the cores. Clear ChAT-like immunoreactivity could also be observed in the sparse neuropil found between the granule cells, as reported by previous authors (Phelps and Vaughn, 1986), but m4-like staining was again very weak in these regions. These results indicate that many cholinergic neurons do not contain detectable m4-like immunoreactivity. Although these findings do not rule out the possibility that a specialized subset of cholinergic neurons found within the islands of Calleja may express m4 receptors, it seems more likely that local cholinergic neurons provide the source of acetylcholine which acts at receptors located on an as yet to be identified population of cells. Detectable m4-like immunoreactivity was also found in several other regions of the basal forebrain. Clear, but modest, staining was present within the external plexiform layers of the main and accessory olfactory bulbs. The marked contrast between the intensity of this staining and that seen in the islands of Calleja provides little support for the concept that mitral cells in the olfactory bulb are analogous to the medium sized cells of the islands of Calleja (Berezhnaia et al., 1998; Hosoya, 1973). Relatively light staining was also seen throughout the dorsal striatum, and occasional “patches” of intensified staining were seen in both rats and monkeys. Similar observations had been previously reported in rats (Levey et al., 1991; Ince et al., 1997). We were able to extend these observations by demonstrating, in both rats and monkeys, that the patches of intensified m4-like immunoreactivity correspond to striosomes identified by mu-opiate receptor-like or calbindin-like immunoreactivity. Staining for m4 receptors appears to be a relatively weak marker for striosomes and in both species many striosomes could not be visualized in m4 stained material. Previous studies using autoradiographic methods have shown that binding of both the nonselective muscarinic ligand propylbenzilylcholine mustard (Nastuk and Graybiel, 1985) and the “M1” ligand pirenzepine (Nastuk and Graybiel, 1988, 1989) is greater in striosomes than matrix. As pirenzepine has substantial affinity for both the m1 and m4 receptors (Moriya et al., 1999), it is possible that the compartmental selectivity of the binding of this compound is due in part to an action
at m4 receptors. Staining for m4 receptors was also more pronounced in the shell than the core of the nucleus accumbens and would thus appear to be another marker capable of distinguishing between these two regions of the ventral striatum (Zahm and Brog, 1982). Immunoreactivity was much more pronounced in the lateral than the medial portions of the accumbens shell, a finding consistent with other results suggesting that there may be important neurochemical differences between these two subregions (Jongen-Relo et al., 1994). Substantially more intense m4-like staining was seen in the so-called “lateral stripe” found in the ventrolateral portions of the striatum extending ventrally into the lateral olfactory tubercle (Phelps et al., 1985; Phelps and Vaughn, 1986; Zahm and Brog, 1982). The intensity of staining in this slender region was second only to that seen in the cores of the islands of Calleja, and this finding supports the suggestion that the striatal stripe may represent a neurochemically specialized region of the striatal complex (Phelps et al., 1985; Phelps and Vaughn, 1986; Zahm and Brog, 1982). Phelps and Vaughn (1986) have suggested that the lateral stripe may be related to clusters of medium sized neurons found in the extrainsular olfactory tubercle, a conclusion also supported by the work of Furuta et al. (2002), but the current results raise the possibility that it may also resemble the cores of the islands of Calleja. Indeed, if the cell clusters observed by Phelps and Vaughn correspond to the mu-opiate receptor rich clusters identified by Herkenham et al. (1984) our results would demonstrate a difference in the chemical anatomy of these regions and the striatal stripe since we did not observe a marked intensification of m4-like immunoreactivity in the cell cluster regions. Many authors currently agree that the olfactory tubercle is part of a larger “basal ganglia complex” and there have been a number of attempts to demonstrate how the islands of Calleja, which receive a dense dopaminergic innervation (Fallon et al., 1978), might fit into such a scheme. It has been suggested, for example, that the granule cells of the islands might be analogous to medium sized spiny striatal neurons, while the cells concentrated in the cores of the islands might be analogous to pallidal output neurons (Fallon, 1983; Fallon et al., 1983). While not refuting these sorts of theories, the current results do serve to emphasize the extent to which the chemical structure of the islands of Calleja is unlike that seen in other parts of basal ganglia. Indeed, there are many unique aspects of the islands of Calleja, ranging from the size and packing density of the neurons found within them, to the their close association with blood vessels (Fallon et al., 1983; Meyer et al., 1989, 1994), to the way in which the granule cells of the islands respond to various drugs (MacGibbon et al., 1994; Vahid-Ansari and Robertson, 1996; Wirtshafter, 1998). Likewise, although there are certain similarities between the cells of the islands and those of the olfactory bulb (Berezhnaia et al., 1998; Hosoya, 1973), there are, as the current study demonstrates, important differences as well. It may be that progress in understanding the function of the islands of Calleja will come more from a
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consideration of their many unique attributes than from an attempt to find similarities between them and other nearby structures in the basal forebrain.
Acknowledgements The studies were supported by NIH grant NS33992. The authors thank Dr. Karen Asin for her many helpful comments on the manuscript, and Robert Canavan for his help with photography and imaging in this, and many other, studies.
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