electron-microscopic study of dopaminergic structures in the medial subdivision of the monkey...

Exp Brain Res (1996) 111:41-50 �9 Springer-Verlag 1996

K e i k o I k e m o t o �9 Kei j i Sa to h �9 K u n i o K i t a h a m a M i c h e l Gef fard �9 Tosh ih iro M a e d a

Electron-microscopic study of dopaminergic structures in the medial subdivision of the monkey nucleus accumbens

Received: 28 December 1995 /Accepted: 13 March 1996

A b s t r a c t The medial subdivision of the monkey nucleus accumbens (NAC) is rich in dopamine (DA) and pep- tides. In the present investigation the mode of DA transmission in the medial subdivision was studied morpho- logically by light- and electron-microscopic immunocytochemistry using a monoclonal antibody raised against dopamine. The medial subdivision showed extremely dense accumulation of thick DA-immunoreactive varicose fibers. Electron-microscopic observation of single sections revealed that DA afferents had a relatively high incidence (33.2%) of asymmetric junctions in this area. Approximately 50% of the targets were dendritic shafts, 44.2% dendritic spines, and 5.1% somata. Some DA axons showed terminal profiles en passant within the synaptic complex, some of which showed synaptic triads. The unique ultrastructural features of DA terminals in the medial NAC indicate the existence of specific styles of DA transmission in the limbic structure.

K ey words Immunocytochemistry - Dopamine �9 Nucleus accumbens �9 Ultrastructure �9 Monkey

Introduction

Recent studies have shown that the nucleus accumbens (NAC) in the primate is a distinct anatomical region within the ventral striatum (Martin et al. 1991, 1993; Ikemoto et al. 1995). The nucleus accumbens is heavily

K. Ikemoto - K. Satoh (~) Department of Psychiatry, Shiga University of Medical Science, Seta Tsukinowacho, Otsu, 520-21, Japan

K. Kitahama D6partement de Mddecine Exp6rimentale, Universit6 Claude Bernard, Lyon, France

M. Geffard Laboratoire d' Immunologie, Universit6 de Bordeaux II, Bordeaux, France

T. Maeda Department of Anatomy, Shiga University of Medical Science, Seta Tsukinowacho, Otsu, 520-21, Japan

innervated by midbrain dopaminergic neurons. The nucleus is also composed of neurons containing various neuropeptides, including cholecystokinin (CCK), enkephalin (ENK), substance P (SP), and neurotensin (NT), in various mammalian species (Haber and Elde 1982; Beach and McGeer 1984; Bouras et al. 1984; Groenewegen and Russchen 1984; Beckstead and Kersey 1985; Haber and Watson 1985; Inagaki and Parent 1985; Zfiborszky et al. 1985; Mai et al. 1986, 1987; Voorn et al. 1989; Martin et al. 1991; Ikemoto et al. 1995). There is evidence indicating that dopamine (DA) and noradrenaline (NA) influence the neuronal activity within the NAC (Nemeroff et al. 1983; Kalivas and Miller 1984; Drum- heller et al. 1986; Merchant et al. 1988; Blaha et al. 1990; Beauregard et al. 1992; Zahm 1992). It has been proposed that the NAC is involved in the pathogenesis of schizophrenia (Bird et al. 1977; Lee and Seeman 1978, 1980; Mackey et al. 1982) and in the therapeutic action of antipsychotic drugs (Merchant et al. 1992).

The compartmental organization of the NAC is different from that of the corpus striatum, which can be divided into patch and matrix regions (Graybiel and Ragsdale 1983; Zahm and Brog 1992). According to recent reviews, the rat NAC can be divided into three subterritories, i.e., shell, core, and rostral pole (Zahm and Brog 1992; Zahm and Heimer 1993). These three subterritories have been shown to have different afferent and efferent projections in the rat (Domesick 1981; Heimer et al. 1991; Berendse et al. 1992; Brog at al. 1993; for reviews see Koobs et al. 1993). In contrast, as previously described in our recent study, the primate NAC is more complex and can be subdivided into three subterritories, i.e., medial, dorsolateral, and ventral subdivisions even in the caudal level (Ikemoto et al. 1995). The medial subdivision is rich in DA- and peptide (SP and NT)-immunoreactive (IR) fibers, and the dorsolateral subdivision possesses many NT-IR cell bodies and low to moderate densities of DA-IR fibers and neuropeptide (SP, NT, ENK)-containing fibers. The ventral subdivision shows low to moderate densities of DA- and neuropeptide (SR NT, ENK)-IR fibers. Fos immunohistochemis-

42

try fol lowing administration of haloperidol, a DA antag- onist, made it possible to visualize neuroleptic sensitive neurons in the monkey NAC, particularly in the medial subdivision (Ikemoto et al. 1995). These data might indicate that the medial subdivision plays an important role in the manifestat ion o f the antipsychotic action.

In the rat, studies using an antibody raised against DA demonstrated that the medial NAC exhibits an intense

DA immunoreact ivi ty (Voorn et al. 1986, 1989). Ultra- structural observation of the rat medial NAC revealed that dopaminergic terminals form symmetr ic synapses most ly with dendritic spines and shafts (Voorn et al. 1986). As previously indicated (Ikemoto et al. 1995), the morphological characteristics o f the NAC appear to be more highly differentiated in the monkey than in the rat. Thus, it is possible that the ultrastructural features o f DA

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Fig. 1A-C Diagrammatic representation of DA-containing structures in three subterritories of the macaque NAC (Ikemoto et al. 1995). The distribution of DA-IR fibers is more homogenous in rostral NAC (A) than in the middle (B) and caudal (C) levels of NAC. The dorsolateral subdivision (DL) is distinguishable by the compactly arranged small cells and is encircled by the broken lines. The DL contains DA-IR fibers in low to moderate quantities. Some of the DA-IR field expands ventrally into the ventral subdi-

vision (V). The medial subdivision (M) is most heavily innervated by DA-IR fibers at the middle (B) and caudal (C) levels. DA immunoreactivity in the ventral subdivision is low to moderate in density. Arrowheads indicate EP-lines. High-power magnification of the areas indicated by small white arrow and large white arrow are shown in Fig. 2A, B, respectively (cn caudate nucleus, ic inter- nal capsule, mC major island of Calleja, S septum) Bar 2 mm

Fig. 2A, B Dopamine-containing fibers in the macaque NAC. A Characteristic dense accumulation of DA-IR varicose fibers in the medial subdivision. B Region with relatively fewer DA-containing fibers in the medial subdivision. Low amount of the DA-IR varicose fibers and moderate amount of DA-IR puncta are observed. Bars 50 gm

terminals in the primate NAC might also be more complex.

The present study was conducted to clarify the morphological characteristics of DA-containing structures in the medial subdivision of the monkey NAC by means of electron microscopy and with a DA monoclonal antibody (Geffard et al. 1984).

Fig. 3 Dopamine-containing fibers in the thalamus of the macaque monkey. The nucleus ventralis anterior thalami (VA) and the nucleus ventralis lateralis thalami (VL) are sparsely innervated by DA-containing fibers. In the Forel H r (H1), there are many DA-IR fibers of passage. Concentration of DA antibody is same as that for the NAC. The section is counterstained with neutral red. Bar 100 gm

Fig. 4A, B Electron micrographs of DA-IR structures in the macaque NAC. A Small DA-IR terminal in the medial subdivision. Many small, clear and round vesicles (30-40 nm in diameter) are found in the DA-IR terminal. B In addition to the small, round and clear vesicles, a large vesicle (arrow, 70-100 nm in diameter) is observed within the DA-IR terminal. Bars 0.5 gm

43

Materials and methods

Animals

Three adult Japanese monkeys, Macaca fuscata, of both sexes, 6 8 years old and weighing 6-9 kg, were used. The animals were housed and fed for several months in the Institute for Experimen- tal Animals, in Shiga University of Medical Science. The study was conducted in strict adherence to our institution's Standards of Animal Experiment and Animal Care.

Immunocytochemistry

Each monkey was deeply anesthetized with ketamine hydrochlo- ride (Ketalar, Sankyo, 25 mg/kg, i.m.) and sodium pentobarbital (Nembutal, Dainippon Pharmaceuticals, 10 mg/kg, i.v.) and per- fused through the ascending and descending aorta with 1 1 of 1% sodium pyrosulfite (MBS) in 0.01 M phosphate-buffered saline (pH 7.4), followed by the fixative containing 5% glutaraldehyde (GA) and 1% MBS, pH 7.5 (Van Eden et al. 1987). After perfu- sion for 30 min, the brain was removed from the cranium and cut into 4-mm-thick blocks, followed by postfixation for 3 h in the solution containing 2% GA and 1% MBS. The blocks were then stored in a 0.1 M phosphate buffer (PB) solution containing 15% sucrose, 1% MBS and 0.1% sodium azide at 4~ (pH 7.4) for more than 3 weeks (for details see Wakabayashi et al. 1993; Mae- da et al. 1995). Coronal sections 30 and 60 gm thick were cut using a cryostat for light- and electron-microscopic studies, respectively. These sections were stored in the same PB-sucrose-MBS solution. The sections for light microscopy were rinsed in 0.3% Triton X-100 in phosphate-buffered saline (0.1 M PBS, pH 7.4, 0.9% NaC1) for at least 3 days, and sections for electron microscopy were treated with 0.005% trypsin for 5 min at 20~ before pro- ceeding to DA immunohistochemistry. The brain sections were in- cubated in the primary antibody raised against DA (dilution of ll:ll0,000, Geffard et al. 1984) for 1 week at 4~ then treated with biotinylated goat antimouse IgG (Vector, diluted 11:11000) followed by the ABC procedure (Hsu et al. 1981), and finally re- acted with 3,3'-diaminobenzidine-hydrogen peroxide solution containing 1% nickel ammonium sulfate (Tago et al. 1986). The specificity of the immunoreactivity was examined by an absorption test. No immunoreactivity was observed. The specificity was also confirmed by immunostaining (see Fig. 3). After immunostaining, the sections for light microscopy were mounted on gelatin-coated glass slides and some were counterstained with neutral red. Some of the adjacent sections were stained with cresylecht violet. The sections for electron microscopy were postfixed in 1% OsO4, de- hydrated and flat-embedded in Luveak-812 between silicon-coated slide glass and coverslip. Regions in the NAC medial subdivision

44

Fig. SA-D Electron micrographs of DA-IR structures in the macaque NAC. A Dopamine-IR terminal showing an asymmetric synapse (large arrow) with a dendritic shaft (D). A large vesicle (small arrow) is observed. B Dopamine-IR terminal forming an asymmetric synapse (arrow) with a dendritic spine (S). C Dopa- mine-IR terminal which synapses with a soma (star) forming an asymmetric synapse (arrow). D Dopamine-IR terminal containing many vesicles, which synapses with a dendritic shaft (D) forming a symmetric synapse (arrow). Heterogenous DA immunoreactivity is observed. Bars 0.5 gm

were dissected and the section was remounted on an Epon stage and cut with an ultramicrotome. Ultrathin sections of the medial subdivision of the NAC (for example see Fig. 1B, small white arrow) were studied using Hitachi H-500 and H-7100 electron mi- croscopes. Sampling was randomly performed.

Analysis

The areas of the DA-IR terminals were measured using a computer-assisted image analyzer (Nexus 6400, Tokyo, Japan) with a

high-resolution color video camera as described previously (Tohy- ama et al. 1988). In measurement of the areas of post-synaptic unlabeled dendrites of DA-IR terminals, the shortest diameters were estimated as the diameters of the unlabeled dendrites and the areas were calculated. The mean areas of terminals and targets were ex- pressed as means+_SD. The frequency of junctions of DA-IR terminals was calculated from electron-microscopic photographs of single sections. Data were analyzed statistically using the t-test.

Results

DA-IR fibers were extremely dense and unevenly distrib- uted in the medial subdivision of the NAC (Figs. 1, 2A). The delineation between the medial subdivision and the caudate nucleus was readily discernible in the DA immu- nostained sections in its dorsal part, since the medial subdivision showed a higher density of DA-IR fibers (Fig. 1). A characteristic dense accumulat ion of DA-IR varicose fibers was observed in this subdivision (Fig. 2A). There

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Table 1 Target structures and types of synaptic junctions of dopamine-immunoreactive terminals (n=217)

Asymmetric Symmetric junctions junctions

Dendritic shafts 50.7% 40% 60% Dendritic spines 44.2% 24% 76% Somata 5.1% 45% 55%

100%

25

2o

15

10

0.2

A r e a s o f D 0 p a m i n e r g i c T e r m i n a l s in N A C

E o

o

o em

o

m=0.33 e 0.27 ~ m a) n=311

0.4 0.6 0.8 1 1.2 1.4 1.6

c r o s s - s e c t i o n a l a r ea (/zm 2)

i i

1.8

found with a frequency of 66.8% (n=145), and asymmetric junctions (i.e. post-synaptic precipitation of cyto- plasm in junction; Fig. 5A-C) with a frequency of 33.2% (n=72). The width (20-30 nm) of the synaptic clefts between the junctional membranes was the same in the asymmetric and in the symmetric junctions.

Approximately half (50.7%) of DA-IR terminals with recognizable junctions were detected on the dendritic shafts (Fig. 5A, D), 44.2% on the dendritic spines (Fig. 5B), and 5.t % on the somata (Fig. 5C, Table 1).

Dendritic shafts

The size of the target dendrites varied. They were both distal and proximal dendrites. Of the total number of synaptic contacts with dendritic shafts, 40% were asymmetric in type, and 60% symmetric in type (Table 1). The mean area of the target dendrites of the asymmetric junctions was 0.48+0.35 btm 2, and that of the symmetric junctions was 0.34_+0.22 btm 2. The former tended to be larger than the latter, although the difference was not significant. Large terminals with areas greater than 1 btm 2 (3%, n = l l ) included terminals without recognizable junctions (n=6, Fig, 7A) and terminals showing asymmetric junctions with large dendritic shafts (mean area of the dendritic shaft: 0.96_+0.29 gm 2, n=5).

Fig. 6 Distribution of the cross-sectional areas of DA-IR terminals in the medial subdivision of the macaque NAC. The peak area is 0.1-0.2 btm 2

were regions with few DA-IR varicose fibers and a moderate amount of immunoreactive puncta (Fig. 2B).

The DA immunohistochemistry in our present materials displayed progressively more fibers in accordance with DA content. That is; the thalamus demonstrated sparse DA immunoreactivity with the same concentrations of reagents (Fig. 3).

There were many DA-IR axons and axon terminals. The DA-IR terminals (0.02-1.53 btm 2 in area) contained mitochondria and synaptic vesicles of different sizes. Most of the synaptic vesicles, 30-40 nm in diameter, were clear and round (Fig. 4A), while some of them were large in diameter (70-100 nm; Figs. 4B, 5A). Most of the small labeled terminals contained 20-30 synaptic vesicles per single ultrathin section (Fig. 4A, B), and the maximal number of vesicles was approximately 300. There was a tendency for larger DA-IR terminals to pos- sess more vesicles. The mean area of the DA-IR terminals estimated to be complete profile (n=311) was 0.33+0.27 btm 2, and the 0.1-0.2 btm 2 area was the most frequent, as shown in Fig. 6.

In the present study a total of 353 DA-IR terminals was observed in single sections, and approximately half of the terminals (52%, n=184) showed single or multiple synaptic junction. Of the total number of synaptic junctions (n=217), symmetric junctions (i.e. junctions rarely exhibiting post-synaptic precipitation; Fig. 5D) were

Dendritic spines

The mean area of target dendritic spines was 0.03 gm 2 (0.01-0.11 gm2). Of the total axo-spinous junctions, 76% were symmetric in type and 24% were asymmetric in type (Fig. 5B, Table 1). The mean area of DA-IR terminals connected with spines by symmetric junctions (0.38+0.30 btm z) was larger than that of terminals connected with spines by asymmetric junctions (0.28+ 0.22 btm:), though the difference was not significant.

Somata

As mentioned above, only a few DA-IR terminals (n=l 1) showed contact with somata, but asymmetric junctions were frequently observed (approximately half, Table 1, Fig. 5C). The target somata possessed abundant cyto- plasm. In our material, however, the morphological characteristics of the cell nucleus could not be specified.

Special profiles of DA-IR terminals

Of the total number of DA-IR terminals exhibiting recognizable junctional membranes, 18% (n=34) showed contact with more than one dendritic element. In contrast, there were some target dendrites with multiple DA- IR terminals. In many cases, they composed the synaptic complex ("synaptic triad", i.e., terminals showing a close contact with dendritic elements which are recipients of

46

Fig. 7A-D Electron micrographs of DA-IR structures in the macaque NAC. A Large DA-IR terminal showing no synaptic specialization. The terminat contains large mitochondria and large vesicles. B Two adjacent DA-IR axonal elements showing a close apposition. The lower DA-IR axonal element shows contact with the presumptive primary dendritic branch (asterisk), which contains multivesicular body (arrow) without forming synaptic specialization. C Synaptic complex ("synaptic triad") consisting of unlabeled dendritic shaft (D) which is the recipient of both unlabeled terminal (u) and DA-IR terminal. Note the asymmetric synapse (arrow). D Dopamine-IR terminal profile en passant. Dopamine- IR axonal component makes a close but non-synaptic contact with an unlabeled dendritic spine (S), which synapses with an unlabeled axonal component (u) by way of an asymmetric synapse (arrow). Bars 0.5 gm

unlabeled terminals) in the NAC (Fig. 7C). Some DA-IR axons showed terminal profiles en passant which rarely showed synaptic specialization with dendrite or spine, or did not show distinct junctional membrane (Fig. 7D). There were two adjacent DA-IR axonal elements showing a close apposition (Fig. 7B).

Discussion

The purpose of the present study was to elucidate the anatomical basis of functional aspects of DA neuronal system in the medial subdivision of the monkey NAC. The present report is, to our knowledge, the first to show DA terminal structures in the monkey NAC by using a DA antibody.

In contrast to the dorsal striatum, the NAC receives a considerable amount of noradrenergic fibers in primates (Susan and Barry 1981; Gasper et a1.1985) compared with rodents (Robert and Patric 1984). Indeed, our study using NA antibody revealed many NA-IR fibers in the dorsal parts of the medial and dorsolateral subdivisions of the monkey NAC (unpublished data). Thus, it was es- sential to use a DA antibody, but not the antibody against tyrosine hydroxylase (TH), the synthesizing enzyme of DA as well as NA, to study the morphological characteristics of DA structures in the NAC of higher mammalian species.

47

Comparison with the rat NAC

In the rat NAC, a large proportion of synapses of DA-IR terminals was shown to be of a symmetric type (Voorn et al. 1986). Similar results were obtained in studies in which TH antibodies were used (Arluison et al. 1984; Bouyer et al. 1984). Compared with the rat, more asymmetric junctions were observed (33.2%) in the monkey NAC medial subdivision, indicating a large number of specialized membranes in DA terminals. Thus, the pres- ence of a species difference in the synaptology of DA terminals is clearly demonstrated.

In the medial subdivision, the targets of DA terminals were dendritic shafts (50.7%), dendritic spines (44.2%), and somata (5.1%). The proportion of targets did not greatly differ from those given in previous reports on the rat NAC, which were obtained using TH or DA antibody (Arluison et al. 1984; Bouyer et al. 1984; Voorn et al. 1986). It is worth mentioning that asymmetric junctions were frequently observed even in the axo-somatic con- nection in the monkey NAC.

Comparison with DA terminals in other areas of the monkey central nervous system

1. Computer analysis of DA-IR terminals in the medial NAC revealed that the mean area (0.33 _ 0.27 gm 2) and the peak area (0.1-0.2 gm 2) of the DA-IR terminals were larger than those of the TH-IR terminals in the striatum of the squirrel monkey (Smith et al. 1994). The reports of findings in the rat showed that the sizes of DA-IR terminals in the striatum were also small (Freund et al. 1984; Kojima et al. 1992). The DA-IR terminals in the monkey NAC medial subdivision was particularly large. 2. The most commonly encountered type of synaptic contacts of TH-IR terminals in the monkey striatum were of a represented symmetric type (Smith et al. 1994). In the medial subdivision of the NAC, the asymmetric type ac- counted for 33.2% of all junctions formed by DA-IR terminals. The higher frequency of asymmetric junctions observed in the macaque medial subdivision than in other forebrain structures is noteworthy. In the dorsal bank of the principal sulcus (Walker's area 46), the anterior cingulate gyrus (Brodmann's area 24) and the primary motor cortex (Brodmann's area 4) in the cerebral cortex, DA-IR terminals are reported to make symmetric junctions without exception in the rhesus monkey (Goldman-Rakic et al. 1989), while 17% of junctions in the cingulate cortex (Brodmann's area 24) of Macaca fuscata are reported to be of an asymmetric type (Maeda et al. 1995). 3. In the striatum of the squirrel monkey the large terminals have been described (maximal diameter larger than 1.0 gm) as lacking synaptic specialization (Smith et al. 1994), while the results of our study demonstrated that half of the large terminals (area greater than 1 ~tm 2) in the medial subdivision showed synaptic contacts with large dendrites by asymmetric junctions. 4. Smith et al. (1994) have reported that the majority of the target structures of striatal TH terminals, presumably

DA terminals, are dendritic shafts and fewer are dendritic spines. The present study showed that in the medial subdivision of the NAC, dendritic shafts and dendritic spines were both present in the same proportions.

The present electron-microscopic observation indicates that the morphological characteristics of the DA-IR terminals in the NAC medial subdivision are different from those in the striatal region: (1) higher frequency of asymmetric junctions, (2) larger DA-IR terminals, (3) lower percentage of dendritic shafts and higher percentage of dendritic spines as targets.

Relation between other transmitters

Counterpart of synaptic contacts in the NAC

In the monkey NAC medial subdivision, most of the targets of DA terminals were dendritic shafts or dendritic spines, and few were somata. In the rat medial NAC, a proportion of TH-IR terminals (4%) is reported to synapse onto somata of GABA positive neurons, forming symmetric synapses with GABA-IR proximal dendrites (14%) (Pickel et al. 1988a). The TH-IR terminals in the rat NAC also synapse with spiny neurons lacking choline acetyltransferase immunoreactivity (Pickel and Chan 1990). This fact might provide the anatomical basis for the DA-GABA interaction in the NAC. For example, pharmacological study has shown that long-term treatment with the D 2 blocker haloperidol increases GABA content in the NAC (Mao et al. 1977; Costa et al. 1978). It is possible that the target somata of the DA-IR terminals observed in the present study are in part GABA- ergic.

Synaptic triads in the NAC

The rat NAC is reported to receive inputs from the cortex, hippocampus, thalamus and amygdala (for review see Groenewegen et al. 1991), and the transmitters re- leased from the fibers originating from the thalamus and amygdala are believed to be glutamate and/or aspartate (Christie et al. 1987). A recent study has shown that immunoreactivity of glutamate receptor (GluR1) is dense in the monkey NAC medial subdivision, and ultrastructural- ly GluRI-IR dendritic shafts and dendritic spines form asymmetric synapses (Martin et al. 1993). Furthermore, the ultrastructural evidence that some single dendrites are post-synaptic to both SP- and TH-IR terminals or dynorphin and TH-IR terminals; this suggests that SP and dynorphin axonal elements possibly contribute to the synaptic complex in the rat NAC (Pickel et al. 1988b; Van Bockstaele et al. 1994).

Some synaptic complexes containing DA terminals in the monkey NAC formed triads. In some triads DA-IR terminals showed synaptic contacts with dendritic elements which are recipients of unlabeled terminals. In others, no membrane specialization was observed be-

48

tween DA-IR and unlabeled components of the triads, which were in close apposition. Previous studies have also described the synaptic triads in the rat NAC (Totter- dell and Smith. 1989; Groenewegen et al. 1991; Sesack and Pickel 1992). Ultrastructural studies using a dual-la- beling technique demonstrated that the TH-IR terminals and the inputs from the hippocampus and prefrontal cortex converge onto single dendrites, and in some cases, make triads in the rat NAC (Totterdell and Smith. 1989; Groenewegen et al. 1991; Sesack and Pickel 1992). In the triads of synaptic complexes of the rat and monkey neocortex, the spines and dendritic shafts of pyramidal cells receive dopaminergic and non-dopaminergic inputs. In the dopaminergic contacts, no asymmetric junctions were found, while non-dopaminergic inputs were always asymmetric boutons which were very likely to be gluta- matergic (Van Eden et al. 1987; S6gu61a et al. 1988; Goldman-Rakic et al. 1989). Dopamine is thought to in- hibit the action of glutamate in the triad of the rat neocortex (Law-Tho et al. 1994). In this context, it is important to identify the unlabeled terminals constituting the triads including dopaminergic terminals which show asymmetric junctions in the monkey NAC medial subdivision.

The mode of DA transmission in the NAC medial subdivision

It is proposed that the mode of DA transmission in the striatum is mainly volume transmission or neurohumoral transmission (Fuxe and Agnati 1991; Maeda et al. 1989). On the other hand, a study of the rat locus coeruleus using DA antibody reveals that dopaminergic contacts have a very high frequency of asymmetric junctions in this area (Kojima et al. 1992; Maeda et al. 1994), and the mode of DA transmission is assumed to be mainly wiring or neural transmission (Fuxe and Agnati 1991; Maeda et al. 1994). Parnavelas and Papadopoulos (1989) assert that monoaminergic terminals in the forebrain form conven- tional synapses in certain regions in boutons, based on the observation of serial ultrathin sections. However, in the present study, asymmetric junctions were often demonstrated in the NAC even in single sections. It is possible that both the volume (or neurohumoral) DA transmission and the wiring (or neural) transmission are present in the primate NAC medial subdivision. The frequent observation of asymmetric junctions even in large terminals is in accordance with our assumption that neural DA transmission predominates in the medial subdivision in contrast to the striatum.

Acknowledgements The authors thank Dr. N. Saito for his tech- nical assistance. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture in Japan (B-02454292, C-06670959).

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electron-microscopic study of dopaminergic structures in the medial subdivision of the monkey...

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