THE JOURNAL OF COMPARATIVE NEUROLOGY 297~165-181(1990)

Distribution of Two Morphologically Distinct Subsets of Serotoninergic Axons in the Cerebral Cortex of the Marmoset JEAN-PIERRE HORNUNG, JEAN-MARC FRITSCHY, AND ISTVAN TdRK Institute of Anatomy, Rue du Bugnon 9,1005 Lausanne, Switzerland (J.-P.H., J.-M.F.), and School of Anatomy, New South Wales University, Kensington, Sydney, NSW 2033, Australia (I.T.)

ABSTRACT The serotoninergic innervation of the marmoset (New World monkey, Callithrix jacchus) cerebral cortex has beefi analyzed by using immunocytochemistry. The use of a sensitive monoclonal antibody against serotonin allowed the visualization of the fine morphology of individual axons. Two types of terminal axons were demonstrated: one has sparse, small, ovoid varicosities (dia. less than 1pm), and the other has large, spheroidal varicosities (up to 5 pm in dia.), which are more densely clustered. The first type of axon is distributed through all cortical layers, with a characteristic laminar distribution that varies from area to area. The second type of axons was distributed sparsely in all regions but was markedly denser in the frontal and anterior parietal lobes, and in the hippocampal formation. Axons with large varicosities typically surrounded certain cell bodies and proximal dendrites, forming pericellular arrays, or baskets. These morphological specializations were most frequent in the frontal and anterior parietal cortex, where they were found around stellate and horizontal cells in layer I and around stellate and bipolar cells in layer I1 and 111. Similar baskets were also found in the hippocampal formation, mainly along the border between the hilus and the granule cell layer of the dentate gyrus, across the CA4 field, and at each side of the pyramidal cell layer of the CA3 regions. The distribution and cellular morphology of the cell surrounded by the 5-HT basket fibres were suggestive of a subpopulation of interneurons, possibly GABAergic and/or peptidergic. I n agreement with previous reports on the innervation of the cerebral cortex of other mammalian species, the marmoset cerebral cortex is innervated by two separate subsystems of serotoninergic axons. One of these may have a strong and specific influence on the cortical inhibitory circuitry, via relay through cortical interneurons. Key words: immunocytochemistry, dual innervation, baskets, interneurons, primates, monoamines

The serotoninergic innervation of the mammalian cerebral cortex originates from the median and dorsal raphe nuclei of the mesencephalon (Anden et al., '66; Ungerstedt, '71; Bobillier et al., '76; O'Hearn and Molliver, '84). The distribution of 5-HT axons in the cortex, as revealed a t first with histofluorescence (Fuxe, '65) or autoradiography (Parent et al., '81), was reported to be sparse. In contrast, immunocytochemical detection of serotonin revealed a dense cortical innervation (Lidov e t al., '80; Steinbusch, '81; Morrison et al., '82; Kosofsky et al., '84). The cortical 5-HT axons are mostly thin with small, ovoid varicosities. However, coarse serotoninergic axons with large beaded varicosities have been noticed in numerous studies (e.g., Kohler et al., '81; Takeuchi and Sano, '83; Foote and Morrison, '84; Kosofsky et al., '84; Wilson et al., '89). o 1 9 9 0 WILEY-LISS, INC.

Growing evidence has been collected over the past decade suggesting that the serotoninergic projection to the cerebral cortex is comprised of two distinct components. This concept stems from tracing studies revealing the distinct organization of the serotoninergic projections of the dorsal and median raphe nuclei (Azmitia and Segal, '78; Kohler e t al., '80; O'Hearn and Molliver, '84; Waterhouse et al., '86) and from morphological characterization of two populations of 5-HT axons in the cortex (Kohler et al., '80; Kosofsky and Accepted March 7,1990. Address reprint requests to Dr. J.P. Hornung, Institute of Anatomy, Rue du Bugnon 9,1005 Lausanne, Switzerland. J.-P. Fritschy is currently at the Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

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SEROTONINERGIC AXONS I N MARMOSET CEREBRAL CORTEX Molliver, '87; Mulligan and Tork, '87, '88; Wilson et al., '89). Mulligan and Tork ('87, '88) demonstrated that in the cat cerebral cortex serotoninergic axons with large varicosities form complex pericellular arrays that surround the soma and proximal dendrites of presumably nonpyramidal neurons. In the rat cerebral cortex, fine and coarse 5-HT axons have a distinct distribution (Blue and Molliver, '88). Fine axons form a relatively uniform plexus of terminal branches throughout the cortex, with an overall greater density in the frontal pole. Large varicose 5-HT have a restricted areal and laminar distribution and are mostly found in clusters. They have not been observed to form such elaborate axonal terminal arborizations as the pericellular arrays described in the cat (Kosofsky and Molliver, '87). Further evidence that fine and coarse 5-HT axons constitute two functionally distinct subsystems has been presented recently, based on pharmacological evidence. Molliver and his collaborators demonstrated that fine 5-HT axons, which originate in the dorsal raphe nucleus, are selectively vulnerable to the neurotoxic effects of amphetamine derivatives, whereas coarse axons, which originate in the median raphe nucleus, are spared following treatment with these drugs (Mamounas and Molliver, '88; O'Hearn et al., '88; Wilson et al., '89; see Molliver, '88 for review). In the primate cortex, the distribution of 5-HT axons is characterized by marked laminar variations in different areas (Morrison et al., '82; Kosofsky e t al., '84; Campbell e t al., '87; Wilson et al., '89). Several studies noticed the presence of a minor component of axons with large varicosities but did not detect the presence of complex terminal arrangements such as pericellular arrays. The existence of a dual serotoninergic innervation of the macaque cerebral cortex has been demonstrated recently by Wilson et al. ('89). These authors documented the existence of two morphologically distinct types of axons, which, as in the rat, have a unique distribution pattern in various cortical areas and are differentially vulnerable to the neurotoxic effects of 3,4methylenedioxymethamphetamine (MDMA). The aim of the present study was to provide further evidence for the existence of a dual 5-HT innervation of the primate cortex and to establish whether complex pericellular arrays, comparable to those described in the cat cortex, can be identified. Using 5-HT immunocytochemistry, we conducted a comprehensive analysis of the serotoninergic innervation of the marmoset cerebral cortex, including the hippocampal formation. The results demonstrate the existence of two morphologically distinct populations of 5-HTimmunoreactive (5-HT-IR) axons. Numerous complex pericellular arrays were observed, confined to restricted areas of the cortex.

MATERIALS AND METHODS Nine marmosets (Callithrir jacchus),aged from 3 months to over 10 years, were deeply anaesthetized with an intraperi-

Fig. 1. Brightfield photomicrographs illustrating the fine morphology of 5-HT-IR axons in the marmoset neocortex. A and B. Thick nonvaricose fibre (presumably preterminal axon); compare, in A, with large and fine varicose fibres. C and D. Fine varicose fibres in layer IVc of t.he primary visual cortex; notice the high density of these fibres and their random orientation. E,F, and G. Large varicose axons; notice the spherical shape of the varicosities, which are closely spaced and connected by very thin intervaricose segments. Scale bars = 20 pm.

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toneal injection of Nembutal (60 mg/kg) and perfused through the ascending aorta with a saline solution followed by 1 litre of fixative. The fixatives used were either: (1)49; paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4), or ( 2 ) the same solution in which 15% of saturated picric acid was added, or (3) 4 % paraformaldehyde and 0.5% zinc salicylate (pH = 6.7) (Mugnaini and Dahl, '83). After 1hour in the skull a t 4OC, the brain was dissected out and postfixed for 1hour in 4 % paraformaldehyde. For frozen sections, the brain was kept overnight in a 30% sucrose solution. Fifty micron-thick sections were cut in either the frontal or the horizontal plane, using a Vibratome (Oxford Instruments) or a freezing microtome. Every fourth (or every eighth) section was processed for immunohistochemistry and its adjacent section Nissl-stained. For immunocytochemical staining, sections collected in phosphate buffer were preincubated for 1 hour in phosphate-buffered saline (PBS) containing 3 to 5 % normal rabbit serum and 0.1 c/o Triton-X 100. Sections were then transferred to a solution containing the primary antiserum (rat monoclonal anti-5-HT, SeraLab, England) diluted 1:250 in PBS. After 48 hours of incubation a t 4 O C , sections were washed three times in PBS and incubated in biotinylated rabbit antirat serum (1:200, Vector Laboratories, Burlingame, CA) for 1hour (Hsu e t al., '81). Following another wash, the sections were incubated in the avidin-HRP complex (1:100, Vector Laboratories) for 1 hour, rinsed in 0.1 M phosphate buffer for 10 minutes, in 0.1 M Tris buffer (pH = 7.6) for 5 minutes, and in 0.2 70 nickel ammonium sulfate in Tris buffer for 10 minutes. The DAB (Sigma, grade 11, 0.05% in the latter solution) reaction was carried out, with 0.01% hydrogen peroxide, for 3 to 5 minutes. Sections were then rinsed, mounted on glass slides, and coverslipped. The distribution and morphology of 5-HT-IR axons were studied by brightfield light microscopy. Maps were drawn with the help of a camera lucida. The specificity of the antibody was characterized previously (Consolazione et al., '81). In addition, no reaction was found in the cortex when the primary antibody was replaced by normal serum. Finally, the same antibody stained selectively the 5-HT cell bodies in the raphe nuclei of the brainstem of the same animals (Hornung and Fritschy, '88).

RESULTS Immunohistochemical staining of 5-HT fibres with the monoclonal antibody against 5-HT and the nickel intensification resulted in a contrasty and delicate labeling. Serotoninergic axons could be analyzed in brightfield and a t high magnification, thus providing excellent resolution of their fine morphology (Figs. 1-2). The main fibre type that was visualized in all regions of the cerebral cortex was characterized by small, ovoid, sparse varicosities (dia. of less than 1 wm) interconnected by thin intervaricose segments (Fig. lC,D). A second fibre type could be readily distinguished by the presence of larger and more closely spaced varicosities (1 to 5 Hm in diameter) (Fig. IE-G). Typically, these fibres closely surrounded the cell bodies and proximal dendrites of certain neurons. This second class of fibre was mainly found in the supragranular layers. In some regions of the cortex its density was exceptionally high. A third type of axon, present in small numbers throughout the cortex, was characterized by the absence of varicosities and a diameter larger than

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SEROTONINERGIC AXONS I N MARMOSET CEREBRAL CORTEX that of the previous classes (around 1 pm in diameter) (Fig. lA,B). The present study focuses on the distribution of the two classes of varicose axons, both in the neocortex and in the hippocampus, and on the morphology of the specialized terminal arbors formed by the large varicose axons (pericellular arrays).

Distribution of small varicose axons Numerous small varicose axons were observed throughout the marmoset cerebral cortex. Their distribution was not uniform but was characterized by abrupt changes in density across the cortical layers. Thus distinct innervation patterns could be recognized in various cortical areas (Figs. 2-4). Nissl-stained sections adjacent to sections processed for 5-HT immunocytochemistry were used to identify architectonic areas, according to the criteria of Brodmann ('09) and Peden and von Bonin ('47). The following general observations were made about the overall organization of the serotoninergic innervation of the neocortex: (1) the highest density of 5-HT-IR axons was confined to layer IV in primary sensory and association cortex; in areas, such as anterior cingulate, entorhinal, or motor cortex, where layer IV was missing or dramatically reduced, this tier of denser innervation was missing; (2) in layer I, which is in general the second densest innervated tier, the amount of 5-HT-IR fibres decreased progressively from rostra1 to caudal; and (3) layers 11,111, and VI had an intermediate density of innervation, whereas layer V was in general the most sparsely innervated tier. The serotoninergic innervation of the hippocampal formation had a distinct organization (see below). In the occipital cortex, area 17 was readily distinguishable by a double band of very high density of 5-HT-IR axonal branches bordering the stria of Gennari, located in layer IVb (Fig. 2). The deeper band corresponded to layer IVc; the upper band extended from layer IVa into the bottom of layer I11 (Lund, '73). The density of 5-HT-IR axons in layer 11 was particularly low, compared to all the other areas investigated. The high fibre density in layer IV dropped drastically at the 17/18 border, outlining precisely the limits of the striate cortex. In area 18 (Fig. a), layer I1 received a stronger innervation as compared to area 17. In the posterior parietal cortex, the pattern of the 5-HT-IR axon distribution was transitory between area 18 and the somatosensory cortex, and the density of 5-HT-IR fibres was low. In the somatosensory cortex, the density of 5-HT-IR fibres in layer IV and in the supragranular layers increased substantially (Fig. 2), whereas the innervation of the infragranular layer remained very sparse. Further anteriorly, the motor cortex contrasted by its rather dense and homogenous distribution of 5-HT-IR axons throughout all layers (Fig. 2). The major characteristic feature of this area is the absence of a band of dense innervation in the middle of the cortex, where layer IV was reduced to a tier of transition between layers I11 and V. The same pattern of serotoninergic innervation was found in the premotor cortex, which is identified cytoarchitectonically by the absence of the large Betz cells in layer V, which are confined to the motor cortex (Fig. 2). Density of 5-HT-IR axons reached its maximum in the prefrontal cortex, characterized also by the presence of a conspicuous layer IV. On the medial aspect of the hemisphere, the cingulate cortex could be divided in an anterior part made of mesocortex (i.e., lacking completely a granular layer), and a poste-

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rior part, which had all the cytoarchitectonic features of neocortex (Fig. 3). In both parts, the serotoninergic innervation was dense, with a slight preponderance in the anterior cingulate cortex. This region, lacking a granular layer, was devoid of a middle tier of dense 5-HT-IR axons, like the motor and premotor cortex. On the contrary, the posterior cingulate cortex presented this typical middle tier of dense serotoninergic innervation, corresponding to its layer IV. The lateral aspect of the temporal lobe (Fig. 3) was occupied by a typical granular cortex, which displayed a pattern of serotoninergic innervation characteristic for all homotypical cortices (i.e., a dense band 5-HT-IR axons covering layer IV and a predominance of the innervation in the supragranular layers as compared to the infragranular layers). In the inferomedial aspects of the temporal lobe (Fig. 4), there was a drastic transition. The entorhinal cortex, progressively losing its six-layered structure, also lost the denser middle band of 5-HT-IR axons, as layer IV became less and less distinct. In the hippocampus, there was a diffuse 5-HT innervation across all layers, with acondensation in a thin band located a t the transition between the stratum radiatum and moleculare (Fig. 4). In the dentate gyrus, there was a denser serotoninergic innervation of the polymorphic layer and of the outer part of the molecular layer (Fig. 4).

Morphology of the large varicose fibres and pericellular arrays Large varicose fibres (Fig. 1E-G) could be differentiated without ambiguity from small varicose fibres on three grounds: (1)the diameter (1to 5 pm) of their varicosities was significantly larger than that of the small varicose fibres (less than 1 pm), (2) the shape of the varicosities was round rather than fusiform, and (3) the spacing between large varicosities was shorter than for small varicosities, giving a beaded appearance to large varicose fibres. Although large varicose fibers could be observed in isolation, they often formed pericellular arrays (or baskets), which constituted a conspicuous morphological feature of this type of 5-HT-IR axons (Fig. 5). Pericellular arrays were typically made only of axons with large varicosities surrounding a single cell body and its proximal dendrites. The complexity of the arborization forming such an array could vary from one to several axonal branches converging around a neuron. Simple baskets were made of small clusters of 5 or more varicosities closely apposed to a neuronal soma (Fig. 5). Baskets located a t a surface of the section could be partially cut and thus appeared as a poorly developed baskets. The most complex baskets surrounded cells located in the middle of the sections, with both the cell body and the proximal dendrites (up to 150 to 200 pm) closely apposed by numerous large varicose axons (Figs. 5-7). An axonal branch that entered the array would border the neuron, intermingled with other branches, and finally leave the basket a t some distance as an isolated branch, which finally disappeared as it leaved the plane of the section. As the most elaborate baskets surrounded a substantial part of the neuron, the shape of this cell could be appreciated from the thick outline made by the immunoreactive terminals and branches (Figs. 6-9). In layer I, neurons surrounded by a basket had dendrites oriented in any direction, but predominantly, for the longer distances, parallel to the pial surface. This morphology was reminiscent of the horizontal cells of layer I (Marin-Padilla, '84) (Fig. 7). In layers I1 and 111, the predominant orientation of the cells surrounded by basket fibres was vertical,

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Fig. 3. Pairs of Nissl-stained (left) and 5-HT immunostained sections from areas of the lateral hemisphere (above) and from areas of the medial hemisphere (below). The location of the selected areas is indicated by a dot on the drawing of the hemisphere. Scale bar = 0.5 mm.

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SEROTONINERGIC AXONS I N MARMOSET CEREBRAL CORTEX with only a few primary dendrites leaving the apical and basal poles of the neuronal soma (Figs. 5-7). The morphology of these neurons resembled that of the bipolar or bitufted neurons (Fairen et al., '84). The baskets found in the hippocampus had the same morphological features as those of the neocortex (Figs. 8-9). They were also made by large varicose 5-HT-IR axons, which in restricted regions would closely surround the cell body and dendrites of certain neurons. The most common type of neuron surrounded by a basket was a multipolar cell with long sparsely ramified dendrites. I t was common that dendrites surrounded by 5-HT-IR terminals crossed the dense granule cell layer of the dentate gyrus or the pyramidal cell layer of the hippocampus, t o reach the molecular layer (Fig. 8).

Distribution of the large varicose fibres and pericellular arrays Axons with large varicosities were found mainly in the supragranular layers of all cortical areas (Fig. 5D,E). They were very sparse in the occipital, the posterior parietal, and most of the superolateral temporal cortices. In the anterior parietal and inferior temporal cortices, the number of these axons increased markedly. Large varicose axons were most numerous in the frontal, cingulate, entorhinal cortices, and in the hippocampal formation. The distribution of the 5-HT-IR baskets paralleled the distribution of the densest innervation of large varicose fibres (Fig. 10). In the neocortex, baskets were located almost exclusively through layers I to 111, and their distribution across cortical areas was not homogenous. The highest density of baskets was observed in the lateral aspect of the frontal lobe a t the junction between the premotor and the prefrontal cortex. A slightly sparser distribution of baskets extended posteriorly in the motor, somatosensory, and a restricted area of the superior temporal cortex. Anteriorly, a similar distribution continued in the prefrontal cortex and then further caudally on the medial aspect of the hemisphere in the cingulate cortex. A separate cluster of baskets was seen in the hippocampal formation (Fig. 10).Again, it paralleled a dense innervation of the region by large varicose fibres. Most baskets were located in the dentate gyrus at the interface between the granule cell layer and the polymorphic layer, and in its molecular layer, in its inner half (Fig. 9). In the hippocampus, several baskets were also found dispersed in the CA4 region (the hilus of the dentate gyrus), and in the CA3 region, at the inner and outer borders of the pyramidal cell layer.

DISCUSSION The present immunocytochemical study demonstrates the existence of two distinct populations of 5-HT terminal axons in the marmoset cerebral cortex. These two subsets can be distinguished on the basis of their morphology, distribution, and terminal arborization. Axons with small elongated varicosities form an extensive plexus across the entire cortex, including the hippocampus. In contrast, large varicose axons are much fewer and have a highly restricted

Fig. 5. Brightfield photomicrographs illustrating the distribution of pericellular arrays relative to fine varicose axons in the superficial layers of cingulate (A),premotor (B and C), and prefrontal cortex (D and E). In A to C, the baskets are located in layers 11-111,and in D and E, a t the interface between layers I and 11. Scale bars = 50 pm.

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distribution. These axons typically form conspicuous terminal arborizations that closely surround the cell body and proximal dendrites of certain neurons. This study provides the first detailed account of the morphology and distribution of these pericellular arrays of serotoninergic axons in the primate cortex. These pericellular arrays, or baskets, are most numerous in the frontal cortex and hippocampal formation. A small number of nonvaricose 5-HT-IR axons, about 1 pm thick, was also observed; these fibres are believed to be the stem fibres of the large varicose terminal axons (Mulligan and Tork, '88). Previous knowledge of the organization of the serotoninergic innervation of the primate cortex was derived mostly from studies in the macaque, an Old World monkey, and the squirrel monkey, a New World monkey. Area 17 has been the only cortical region in which detailed comparative studies of the serotoninergic innervation are available (Morrison e t al., '82; Kosofsky et al., '84; Takeuchi and Sano, '84; Morrison and Foote, '86). Although the overall distribution of 5-HT fibres in both species is similar, two differences have been pointed out: (1) the contrast between the dense innervation of layer IV and that of the infragranular layers is least in the macaque brain, where the relative innervation of layer V is richer, as compared to the squirrel monkey brain, and (2) sublaminar variations in the density of innervation within layer IV are more obvious in the macaque than in the squirrel monkey (Kosofsky et al., '84). These differences in serotoninergic innervation have been attributed to the more advanced evolutionary development of the brain structures devoted to the visual system in Old World monkeys (Kosofsky et al., '84; Morrison and Foote, '86). The results of studies of the serotoninergic innervation of the macaque cortex in cortical areas other than primary visual cortex (Campbell et al., '87; DeFelipe and Jones, '88; Wilson et al., '89) do not reveal any prominent differences in the distribution of 5-HT axons between the marmoset and the macaque. The only consistent difference, in all areas compared, is the weaker innervation of the infragranular layers of the New World primate cortex. The organization of nuclei of origin of the ascending serotoninergic system in both Old World and New World monkeys shares the characteristic features of primate brains (Hubbard and DiCarlo, '74; Felten and Sladek, '83; Azmitia and Gannon, '86; Hornung and Fritschy, '88). Although regional and laminar variations in the relative density of thick or thin fibers have been observed in comparing several species of monkeys, the striking similarities in the overall organization of cortical 5-HT projections among all species studied indicate that the marmoset brain is a suitable model for the investigation of the serotoninergic system in primates. The existence of two populations of 5-HT varicose axons in the cerebral cortex has been documented in several mammalian species. In the rat, it was demonstrated by anterograde tracing that 5-HT neurons in the median raphe and in the dorsal raphe nuclei give raise to morphologically distinct axons innervating the cortex, large varicose (beaded) axons and fine varicose axons, respectively (Kosofsky and Molliver, '87). It was further shown that these two types of axons differ in their distribution across the cortex (Blue and Molliver, '88). The same conclusion was reached independently about the serotoninergic innervation of the cat cortex, in which two morphological types of terminal axons have a distinct laminar and areal distribution (Mulligan and Tork, '88). In monkeys, several authors have reported the presence of different morphological types of terminal axons

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SEROTONINERGIC AXONS I N MARMOSET CEREBRAL CORTEX (Morrison et al., '82; Takeuchi and Sano, '84; Kosofsky et al., '84; Foote and Morrison, '84; DeLima et al., '88; Wilson e t al., '89). Our results extend these observations in primates by demonstrating the presence of fine and large varicose 5-HT axons in the marmoset cortex. Since the serotoninergic innervation of the primate cortex also originates from the dorsal and median raphe nuclei (Porrino and GoldmanRakic, '82; Tigges et al., '82, '83),it is likely that a segregation in the origin of fine and beaded axons exists, similar to that observed in the rat (Kosofsky and Molliver, '87; Mamounas and Molliver, '88). Recently, Wilson e t al. ('89) demonstrated the existence of two morphologically distinct types of 5-HT axons in the macaque cortex, which are differentially vulnerable to the neurotoxic drug effects of the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA). As in the rat (see Molliver, '88 for review), fine varicose axons are eliminated following systemic administration of this drug, whereas large varicose 5-HT axons appear unaffected. A major finding of the present study is the observation, as in the macaque (Wilson et al., '89), that specific areas of the marmoset cerebral cortex are innervated differentially by large varicose 5-HT-IR axons. Moreover, we report that large varicose fibers form pericellular arrays of similar morphology and complexity to those observed in cat (Mulligan and Tork, '88). Serotoninergic baskets are characterized by the same preferential distribution than isolated large varicose axons, which suggests that possibly all beaded axons contribute to the formation of pericellular arrays. In rodents, beaded axons form a conspicuous subset of 5-HT axons, whose distribution is more diffuse than in both the cat and marmoset (Kosofsky and Molliver, '87; Blue and Molliver, '88). Furthermore, baskets do not seem to be present in the rat brain. It is noteworthy that the number of 5-HT neurons in the median raphe, where the beaded fibers originate, is significantly increased in the cat (Jacobs et al., '84) and in primates (Sladek e t al., '82; Azmitia and Gannon, '86; Hornung and Fritschy, '88), compared to rodents (Steinbusch, '81; Tork, '85). Thus there seems to be a correlation between the relative expansion of the median raphe and the extent of innervation of the cerebral cortex by large varicose axons. The present demonstration of the existence of pericellular arrays formed by beaded 5-HT axons in the marmoset cortex suggests that this morphological specialization is not merely a peculiarity of the cat brain but might be present in a number of mammalian species. In the macaque cortex, 5-HT pericellular arrays have been observed only occasionally (Foote and Morrison, '84; Wilson et al., 'S9), and they appear less elaborate than in the cat or in the marmoset. The discrepancy between the present results and the apparent paucity of baskets in the macaque cortex could be explained in part by the fact that areas rich in 5-HT baskets in the marmoset cortex have been least investigated in the macaque cortex. Further studies of the macaque brain should clarify this point. In the cat, 5-HT baskets are distributed in small numbers throughout the cortex and in higher numbers in the supra-

Fig. 6. Four micrographs of pericellular arrays (baskets) of different morphologies made by thick varicose 5-HT-IR axons in layers II/III of the neocortex. The part of the arrays that appear to surround a cell body is indicated by an arrow. Scale bars = 50 pm.

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sylvian, auditory, and entorhinal cortices, and in the hippocampal formation (Mulligan and Tork, '88). In the marmoset cortex, the 5-HT baskets are mostly concentrated in two regions: (1)the frontal, anterior parietal, cingulate, and part of the superior temporal cortices, and (2) the hippocampal formation. Unlike in the cat cortex, baskets in the marmoset monkey were virtually absent outside the region of high density distribution. Although there are similarities in the distribution of the 5-HT baskets between the two species, there are also singularities in the denser innervation of the cat auditory fields and the marmoset frontal lobe. These areas, in the cat and marmoset, are involved in different and apparently unrelated functions. One can assume from these observations that large varicose 5-HT axons forming baskets innervate regions of the cerebral cortex that subserve different functions in diverse species. The dense pericellular arrays, closely surrounding the soma and dendrites of the cortical neurons, make the morphology of these cells apparent. In layer I, the cortical neurons densely innervated by 5-HT axons have the morphology of horizontal and stellate nonpyramidal neurons (Marin-Padilla, '84). In layers I1 and 111, the majority of the target neurons have the morphology of bitufted or bipolar neurons (Fairen et al., '84; Peters, '84). These cell types belong to the group of nonpyramidal neurons. Similarly in the cat cortex, all pericellular arrays made by large varicose axons selectively surround nonpyramidal neurons (Mulligan and Tork, '88). It was further demonstrated in the cat that nonpyramidal neurons enveloped by a 5-HT basket were immunoreactive for the GABA synthetic enzyme glutamate acid decarboxylase and for GABA itself (Tork et al., '88). It is likely that neurons surrounded by a basket in the marmoset cortex are also GABAergic, since the distribution and morphology of 5-HT baskets overlap that of GABAergic neurons found in primates (Fitzpatrick et al., '87; Hendry et al., '87). Therefore, as suggested for the cat serotoninergic system, the marmoset serotoninergic innervation might, in part, influence cortical function by acting directly on inhibitory cortical interneurons. It has already been suggested that GABAergic neurons in the macaque cortex might receive a serotoninergic input (DeFelipe and Jones, '88). The existence of a true chemical synapse at the terminals of the 5-HT axons has been debated for many years. Early ultrastructural autoradiographic studies (Descarries et al., '75; Beaudet and Descarries, '76) reported that 5-HT terminals in the cerebral cortex lack synaptic membrane specializations, thus supporting further the concept of a diffuse, rather unspecific monoaminergic innervation. More recently, several ultrastructural immunocytochemical studies have convincingly identified synaptic specialization a t 5-HT terminals of the cerebral cortex (Molliver et al., '82; Papadopoulos et al., '87; DeLima et al., '88), but were not confirmed by others (DeFelipe and Jones, '88), who found only rare incidences of chemical synapses a t 5-HT varicosities. In the cat auditory cortex, 5-HT large varicose axons forming pericellular arrays were analyzed in serial ultrathin sections (Tork et al., '86). At each varicosity, one or several synapses with clear pre- and postsynaptic specialization were observed. The similarities between the 5-HT baskets in the cat and marmoset cerebral cortex suggest that a similar direct chemical synaptic transmission takes place between the large varicose 5-HT afferents and a subpopulation of nonpyramidal neurons of the marmoset brain. The serotoninergic innervation of the marmoset cerebral cortex, like that of other mammalian species, is made of two

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Fig. 8. Micrographs of 5-HT-IR baskets in the hippocampal formation. A: A pericellular array located at the hilar side of the granule cell layer ( g r ) . B: A pericellular array in the stratum radiatum of the CA3 region. Notice the different morphology of the fine varicose fibers indicated by arrowheads. The part of the arrays that appear to surround a cellbody is indicated by an arrow. Scale bars = 50 pm.

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Fig. 10. Drawings of nine representative levels of a series of parallel horizontal sections through the hemisphere incubated with 5-HT antibodies. The location of each complex 5-HT-IR periceliular array is represented by a dot. Scale bar = 10 mm.

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180 dit€erent populations of terminal axons, originating in two distinct raphe nuclei of the midbrain. These two nuclei and their ascending pathways form two separate channels between brainstem and forebrain (Azmitia and Segal, '78; O'Hearn and Molliver, '87). Each channel has its specific and independent infiuence on cortical activity. Small varicose fibres are distributed over the entire cortex, with predominant laminar distribution varying from area to area, and terminate mainly on distal dendrites of two types (spiny and nonspiny) of neurons (Papadopoulos et al., '87; DeLima et al., '88; DeFelipe and Jones, '88). Large varicose fibres are preferentially distributed in restricted areas, where they terminate on the cell body and proximal dendrites of certain inhibitory nonpyramidal neurons (Mulligan and Tork, '88; Tork et al., '86, '88). These two serotoninergic pathways are present in the various mammalian species studied so far, but with relative development and distribution that vary from species to species. The complexity of the organization of the serotoninergic projections to the cerebral cortex suggests that they may be associated with the specific activation (or suppression) of several sets of cortical neurons, with distinct functions, rather than having a diffuse influence on all cortical neurons.

ACKNOWLEDGMENTS We are grateful to S. Daldoss and M. Birchen for the illustrations, and to M.C. Cruz and C. Blagov for histology. We thank Dr. M.E. Molliver for helpful comments and for providing the equipment used to prepare some of the illustrations (NIH grant NS-21001). This work was supported by Swiss NSF grants 3.158 and 3.3230.86, and by a grant of NH and MRC of Australia (to I.T.).

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