Opioid receptors in midbrain dopaminergic regions of the rat I. MU receptor autoradiography

Summary Several lines of evidence indicate that an interaction exists between opioid peptides and midbrain dopaminergic neurons. The purpose of this study was to map and quantify the density of the mu opioid receptor subtype relative to the location of the dopaminergic (DA) neurons in the retrorubral field (nucleus A8), substantia nigra (nucleus A9), and ventral tegmental area and related nuclei (nucleus A10) in the rat. Sections through the rostral-caudal extent of the midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the mu-selective ligand,³H-Tyr-D-Ala-N-MePhe-Gyl-ol enkephalin. In the nucleus A8 region, there were low levels of mu binding. In the rostral portion of nucleus A9, there was prominent mu binding both in the ventral pars compacta, which contains numerous DA neurons, and in regions that correspond to the location of the DA dendrites which project ventrally into the underlying substantia nigra pars reticulata. In the caudal portion of nucleus A9, mu binding was greatest in the substantia nigra pars reticulata, but also in the same region that contains DA neurons. In nucleus A10, mu receptor densities differed depending upon the nucleus A10 subdivision, and the rostral-caudal position in the nucleus. Low receptor densities were observed in rostral portions of the ventral tegmental area and interfascicular nucleus, and there was negligible binding in the parabrachial pigmented nucleus and paranigral nucleus at the level of the interpeduncular nucleus; all regions where there are high densities of DA somata. Mu binding was relatively high in the central linear nucleus, and in the dorsal and medial divisions of the medial terminal nucleus of the accessory optic system, which has been shown to contain DA dendrites. These data indicate that mu opioid receptors are located in certain regions occupied by all three midbrain DA nuclei, but in a highly heterogeneous fashion.     

Efferent projections of the retrorubral nucleus to the substantia nigra and ventral tegmental area in cats as shown by anterograde tracing

The aim of the present study was to determine whether the retrorubral nucleus projects to the dopaminergic nuclei in the ventral midbrain of the cat. For this purpose, injections of biotinylated dextran-amine or Phaseolus vulgaris-leucoagglutinin were placed into the retrorubral nucleus under stereotaxic guidance. The tracers were visualized by means of (immuno) histochemical procedures. In addition, tyrosine hydroxylase immunohistochemistry was used to evaluate the location of the injection sites and the distribution of the anterogradely labeled fibers. Both tracers reveal the same topography of labeled fibers in the ventral mesencephalon. Labeled fibers with varicosities were found ipsilaterally in the substantia nigra pars compacta, the substantia nigra pars lateralis, the ventral tegmental area and, contralaterally, in the substantia nigra pars compacta, the ventral tegmental area, and the retrorubral nucleus. A considerable number of labeled axons with varicosities were observed to be wrapped around the dendrites and perikarya of tyrosine hydroxylase-positive neurons in these areas. The present results are discussed in view of the possible role of the A8 dopaminergic cell group in the coordination of A9 nigrostriatal and A10 mesolimbic systems, as well as in the progressive pathology seen in patients suffering from Parkinson's disease.     

Dopaminergic neurons in rat ventral midbrain express brain-derived neurotrophic factor and neurotrophin-3 mRNAs

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with ³⁵S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25–50% in the ventral tegmental area and 10–30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia. © 1994 Wiley-Liss, Inc.     

Midbrain dopaminergic neurons (nuclei A8, A9, and A10): Three-dimensional reconstruction in the rat

The dopaminergic neurons in the midbrain of the rat are located in three cell groups: nucleus A8 cells in the retrourbal field, nucleus A9 cells in the substantia nigra, and nucleus A10 cells in the ventral tegmental area and related nuclei. The purpose of the present study was to map and quantify the midbrain dopaminergic neurons in two and three dimensions in the rat brain, using immunohistochemical staining and computer imaging techniques. The cells were identified with an antibody against tyrosine hydroxylase, and counted in six midbrain nuclei: the retrorubral field, substantia nigra pars compacta, substantia nigra pars reticulata, central linear nucleus, ventral tegmental area, and interfascicular nucleus. Outlines were traced around the perimeters of the coronal tissue sections, and the locations of all immunoreactive ventral midbrain cells were mapped. On one side of the brain there are approximately 1,300 nucleus A8 cells, 10,500 nucleus A9 cells, and 10,200 nucleus A10 cells. The 2- and 3-dimensional reconstructions illustrate the region-specific density of dopaminergic neurons throughout the midbrain cell complex, and provide a visual appreciation of the location and distribution of the three dopaminergic cell groups in relation to their position in the midbrain. Information about the number and location of midbrain dopaminergic neurons will be useful in conjunction with future studies that characterize these cells more specifically, for example, in terms of their co-transmitters, and afferent and efferent projections. © 1993 Wiley-Liss, Inc.     

Midbrain dopaminergic cell loss in Parkinson's disease: computer visualization

Computer visualization techniques were used to map the distribution of dopaminergic neurons within midbrain tissue sections from 5 parkinsonian patients and 3 age-matched control subjects. The Parkinsonian brains had over 50% fewer dopaminergic neurons within the midbrain than age-matched normal brains. The cell loss occurred within the combined substantia nigra (dopaminergic nucleus A9) and retrorubral (dopaminergic nucleus A8) areas (greater than 61%) and the ventral tegmental area (dopaminergic nucleus A10) (greater than 42%). The cell loss was greatest within the ventral portion of the substantia nigra zona compacta. The specific pattern of cell loss is very similar to the pattern of cells that project to the striatum (as opposed to cortical and limbic sites) in animal neuroanatomical tracing experiments. These data suggest that Parkinson's disease preferentially destroys midbrain dopaminergic neurons in nuclei A8, A9, and A10, which project to the striatum.     

Characterization and autoradiographic distribution of neurotensin binding sites in the human brain

The characteristics and topographical distribution of monoiodo¹²⁵I-Tyr₃-neurotensin (NT) binding sites in normal human brain tissue were studied on brain sections and by quantitative autoradiography. Sections at the level of the substantia nigra show a dissociation constant and maximal binding capacity of4.8 ± 0.8nM and 70 ± 7fmol/mg protein, respectively. High density of¹²⁵I-NT binding sites were mainly found in dopaminergic (DA)-rich areas such as the substantia nigra, the ventral tegmental area, the striatum and the nucleus accumbens, further supporting an interaction between NT and DA neurons in human brain.     

Autoradiographic localization of delta opioid receptors within the mesocorticolimbic dopamine system using radioiodinated [2-D-penicillamine, 5-D-penicillamine]enkephalin (125I-DPDPE)

The enkephalin analog [2-D-penicillamine, 5-D-penicillamine]enkephalin was radioiodinated (125I-DPDPE) and shown to retain a pharmacological selectivity characteristic of the delta opioid receptor in in vitro binding studies. The distributions of 125I-DPDPE binding, using in vitro autoradiographic techniques, were similar to those previously reported for the delta opioid receptor. The nucleus accumbens, striatum, and medial prefrontal cortex contain dense gradients of 125I-DPDPE binding in regions known to receive dopaminergic afferents emanating from the mesencephalic tegmentum. Selective chemical lesions of the ventral tegmental area and substantia nigra were employed to deduce the location of the 125I-DPDPE binding within particular regions of the mesocorticolimbic dopamine system. Unilateral lesions of dopamine perikarya (A9 and A10) within the ventral tegmental area and substantia nigra produced by mesencephalic injection of 6-hydroxydopamine resulted in significant (20-30%) increases in 125I-DPDPE binding contralateral to the lesion within the striatum and nucleus accumbens. Lesions of the perikarya (dopaminergic and nondopaminergic) of the ventral tegmental area, induced by quinolinic acid injections, caused increases of less magnitude within these same nuclei. No significant alterations in 125I-DPDPE binding were observed within the mesencephalon as a result of either treatment. The specificity of the lesions was confirmed by immunocytochemistry for tyrosine hydroxylase. These results suggest that the enkephalins and opioid agonists acting through delta opioid receptors do not directly modulate dopaminergic afferents but do regulate postsynaptic targets of the mesocorticolimbic dopamine system.     

Midbrain dopaminergic neurons in the mouse: Computer-assisted mapping

The dopaminergic (DA) neurons in the midbrain play a role in cognition, affect and movement. The purpose of the present study was to map and quantify the number of DA neurons in the midbrain, within the nuclei that constitute cell groups A8, A9 and A10, in the mouse. Two strains of mice were used; the C57BL/6 strain was chosen because it is commonly used in neurobiological studies, and the FVB/N strain was chosen because it is used frequently in transgenic studies. DA neurons were identified, in every fifth 20-μm-thick coronal section, using an antibody against tyrosine hydroxylase. Cell locations were entered into a computer imaging system. The FVB/N strain has 42% more midbrain DA neurons than the C57BL/6 strain; on one side of the brain there were 15,135 ± 356 neurons (mean ± S.E.M.) in the FVB/N strain, and 10,645 ± 315 neurons in the C57BL/6 strain. In both strains, approximately 11% of the neurons were located in nucleus A8 (the DA neurons in the retrorubral field), 38% in nucleus A9 (the DA neurons in the substantia nigra pars compacta, pars reticulata, and pars lateralis), and 51% in nucleus A10 (the DA neurons in midline regions such as the ventral tegmental area, central linear nucleus, and interfascicular nucleus). The number of midbrain DA cells, and their distribution within the three nuclear groups, is discussed with respect to findings in other species. © 1996 Wiley-Liss, Inc.     

Immunohistochemical localization of monoamine oxidase-B in the cat brain: Clues to understanding N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity

The immunohistochemical localization of monoamine oxidase-B in normal cat brain was examined. The enzyme was localized in both neural and nonneural elements of the cat brain. Neurons in the hypothalamus (lateral, dorsal, ventromedial, dorsomedial, and supraoptic nuclei), raphe system, dorsal tegmental nucleus, locus ceruleus, K?lliker-Fuse nucleus, dorsal parabrachial region, and central tegmental field were positive. No substantia nigra pars compacta, retrorubral, or ventral tegmental neurons stained positively. Glial cells (astrocytes) stained positively for monoamine oxidase-B in many regions of the central nervous system, however, there was a significantly greater number of monoamine oxidase-B-positive glial cells in the substantia nigra pars compacta than in other adjacent dopaminergic regions. Because nigra compacta neurons are specifically damaged by the neurotoxin MPTP and because the toxicity of the drug is expressed only in the presence of monoamine oxidase-B, it is possible that the preferential loss of substantia nigra pars compacta neurons in the cat brain may be related to the regional and cellular localization of monoamine oxidase-B.     

Estrogen receptor-beta immunoreactivity in the midbrain of adult rats: regional, subregional, and cellular localization in the A10, A9, and A8 dopamine cell groups

Estrogen modulates dopamine synthesis, release, and metabolism in corticolimbic and striatal targets of midbrain dopamine neurons. The relevant sites of receptor-mediated action, however, had been elusive, because all available evidence suggested a paucity of intracellular estrogen receptors in the A8, A9, and A10 dopamine regions and their afferent targets. More recent evidence of a relative abundance of the beta isoform of the estrogen receptor (ER) in the substantia nigra and ventral tegmental area (VTA), however, suggests that this newly described receptor may be important in estrogen's stimulation of midbrain DA systems. It is unknown, however, precisely how ERbeta is distributed with respect to the functionally and neurochemically diverse cell populations of the ventral midbrain. To address these issues, this study used single- and double-label immunocytochemistry to detail the regional, subregional, and cellular distributions of ERbeta immunoreactivity in and around midbrain dopamine-containing cell groups in hormonally intact adult male and female rats. These analyses revealed that ERbeta-immunoreactive nuclei were found only in neurons, more specifically, within subsets of both dopaminergic and nondopaminergic neurons in the dorsal VTA, the parabrachial pigmented nucleus, the substantia nigra pars lateralis, the retrorubral fields, and to a lesser extent the linear midline nuclei. These regional and cellular receptor distributions thus place the ERbeta isoform in anatomical register with midbrain dopamine systems known to participate in a spectrum of motor, cognitive, and affective functions.     

The weaver gene continues to target late-generated dopaminergic neurons in midbrain areas at P90

To determine if lethal action of the weaver gene is more intense in late-generated dopaminergic neurons in midbrain areas on postnatal day (P) 90 [3H] thymidine autoradiography and tyrosine hydroxylase immunohistochemistry were combined in the same tissue section in homozygous weaver mice and normal controls. The experimental animals were the offspring of pregnant dams injected with [3H] thymidine on embryonic days (E) 11-12, E12-13, E13-14 and E14-15. Neurogenetic timetables of dopaminergic neurons were different between wild type and homozygous weavers in all midbrain areas analyzed. A substantial number of late-generated neurons in the substantia nigra pars compacta and in the ventral tegmental area are missing at P90, in these dopaminergic areas the loss is greater than at P20 indicating that neuronal loss is progressive. The greatest loss is in the substantia nigra pars compacta, confirming the report of Bayer et al. [Exp. Brain Res. 105 (1995) 200] at P20, while in the retrorubral field and the interfascicular nucleus late-generated neuron loss was less severe. These results furnish more evidence that dopaminergic neuron loss in homozygous weaver midbrain is a phenomenon linked to development.     

Neurotensin in projection neurons of the striatum and nucleus accumbens, with reference to coexistence with enkephalin and GABA: an immunohistochemical study in the cat

Neurotensin-like immunoreactivity (NT-LI) was demonstrated in projection neurons of the striatum and nucleus accumbens in the cat by combining immunohistochemistry and the fluorescent retrograde neuronal labeling method. In colchicine-treated cats, many neurons with NT-LI were found in the caudate nucleus, nucleus accumbens, and putamen. Most of these neurons were medium-sized neurons with spiny dendrites. NT-LI of neuronal elements in the caudate nucleus and nucleus accumbens formed dense aggregates with irregular figures, which appeared to correspond to the striosomes of Graybiel et al. (Proc. Natl. Acad. Sci. USA 75:5723-5726, '78; Exp. Brain Res. 34:189-195, '79; Neuroscience 6:377-397, '81). Fibers with NT-LI were distributed massively to the globus pallidus and ventral midbrain regions, but not to the entopeduncular nucleus. In the ventral midbrain regions, many fine varicose fibers with NT-LI were distributed to the pars compacta and pars lateralis of the substantia nigra, ventral tegmental area, and retrorubral area. In the pars reticulata of the substantia nigra, however, fibers with NT-LI were rather sparse. Examination of consecutive sections immunostained for NT, enkephalin (Enk), GABA, and substance P (SP) revealed that 50% of neurons with NT-LI in the caudate nucleus and nucleus accumbens exhibited Enk-LI, 15% showed GABA-LI, and 5% manifested both Enk-LI and GABA-LI; no NT-positive neurons in the striatum and nucleus accumbens showed SP-LI. No morphological differences were found between NT-positive neurons with Enk-LI and/or GABA-LI and those without Enk-LI and GABA-LI. Most neurons with NT-LI in the striatum and nucleus accumbens were retrogradely labeled with True Blue injected into the globus pallidus, pars compacta and pars lateralis of the substantia nigra, and ventral tegmental area. After hemitransection severing neuronal connections between the ventral midbrain regions and the forebrain structures, fibers with NT-LI and those with Enk-LI in the ventral midbrain regions were markedly reduced in number.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamatergic inputs from the pedunculopontine nucleus to midbrain dopaminergic neurons in primates: Phaseolus vulgaris-leucoagglutinin anterograde labeling combined with postembedding glutamate and GABA immunohistochemistry

To verify the possibility that the pedunculopontine nucleus is a source of glutamatergic terminals in contact with midbrain dopaminergic neurons in the squirrel monkey, we used the anterograde transport of Phaseolus vulgaris-leucoagglutinin in combination with preembedding immunohistochemistry for tyrosine hydroxylase and for calbindin D-28k and postembedding immunocytochemistry for glutamate and for γ-aminobutyric acid. Following tracer injections in the pedunculopontine nucleus, numerous anterogradely labeled fibers emerged from the injection sites to innervate densely the pars compacta of the substantia nigra and ventral tegmental area. The major type of labeled fibers were thin with multiple collaterals and varicosities that established intimate contacts with midbrain dopaminergic neurons. At the electron microscopic level, the anterogradely labeled boutons were medium sized (maximum diameter between 0.9 μm and 2.5 μm) and contained numerous round vesicles and mitochondria. Postembedding immunocytochemistry revealed that 40–60% of anterogradely labeled terminals were enriched in glutamate and formed asymmetric synapses with dendritic shafts of substantia nigra and ventral tegmental area neurons. In triple-immunostained sections, some of the postsynaptic targets to these terminals were found to be dopaminergic. In addition, 30–40% of the anterogradely labeled terminals in both regions displayed immunoreactivity for γ-aminobutyric acid and, in some cases, formed symmetric synapses with dendritic shafts. In conclusion, our results provide the first ultrastructural evidence for the existence of synaptic contacts between glutamate-enriched terminals from the pedunculopontine nucleus and midbrain dopaminergic neurons in primates. Our results also show that the pedunculopontine nucleus is a potential source of γ-aminobutyric acid input to this region. These findings suggest that the pedunculopontine nucleus may play an important role in the modulation of the activity of midbrain dopaminergic cells by releasing glutamate or γ-aminobutyric acid as neurotransmitter. © 1996 Wiley-Liss, Inc.     

Subtypes of substantia nigra dopaminergic neurons revealed by apamin: autoradiographic and electrophysiological studies.

In the intact animal, some substantia nigra dopaminergic neurons exhibit regular, and some exhibit burst firing patterns. In the in vitro slice preparation, however, all dopaminergic neurons exhibit a nonburst firing pattern. Burst firing patterns are thought to be regulated, in part, by a small conductance calcium-activated potassium channel (SK channels). To test whether SK channels reside within the midbrain dopaminergic cell regions of the mouse, receptor autoradiographic experiments were conducted with the SK channel antagonist, 125I-apamin. To determine whether SK channels play a role in burst firing pattern generation in substantia nigra dopaminergic neurons, changes in firing patterns of these cells were examined in the in vitro slice preparation following apamin superfusion (1-1000 nM). It was demonstrated that a) specific binding of radiolabeled apamin was found within the dopaminergic cell regions of the substantia nigra pars compacta, and ventral tegmental area (2.7-4.7 fmol/mg tissue); b) the firing patterns of less than half of the dopaminergic neurons were changed from a regular pattern to that of a burster with concentrations as low as 1 nM, but the firing patterns of many neurons were not changed by the drug; and c) blockade of the SK channel did not interfere with the inhibitory effects of dopamine on dopaminergic neuronal impulse flow, indicating that the known hyperpolarizing effects mediated by this dopamine receptor are not importantly mediated via the SK channel.     

Mesostriatal projections in BALB/c and CBA mice: a quantitative retrograde neuroanatomical tracing study

A major portion of the midbrain dopamine (DA)-containing neurons project to the striatum and make up the mesostriatal DA system. The purpose of the present experiment was to map the location and quantify the density of mesostriatal neurons within two inbred mouse strains (BALB/c and CBA) known to possess different numbers of midbrain DA neurons. Computer-assisted reconstructions were made of both the wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) striatal injection site and the retrogradely labeled midbrain cells. There was no strain differences in the major source or topographical pattern of innervation of the striatum from the midbrain cellular regions. Even following small striatal injections, the labeled midbrain cells were found throughout most of the rostrocaudal extent of the midbrain DA nuclei; some labeled cells were found within the substantia nigra, the ventral tegmental area and the adjacent retrorubral field. Although the BALB/c strain has 20-25% more midbrain DA neurons than the CBA, given comparable striatal injection volumes, there was no significant difference in the number of HRP-filled mesostriatal neurons between the two mouse strains. These data suggest that the mesostriatal neurons give rise to comparable axonal branching within the striatum in the two mouse strains.     

Forebrain projections from cholecystokininlike-immunoreactive neurons in the rat midbrain

The purpose of the present study was to analyze the distribution of cholecystokininlike-immunoreactive (CCK-I) neurons within the rat ventral mesencephalon which project to several forebrain areas. The peroxidase-antiperoxidase immunocytochemical technique was used to examine the anatomical localization of CCK-I within the ventral midbrain and in the following forebrain regions: caudate-putamen, nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, septum, amygdala, and prefrontal, anterior cingulate, and piriform cortices. CCK-I perikarya were distributed throughout the substantia nigra, ventral tegmental area, and several midline raphe nuclei to a greater extent than previously reported, particularly in the substantia nigra pars compacta. Terminallike immunoreactivity for CCK was observed in all of the above forebrain sites. In addition, infrequent CCK-I cell bodies were localized in the caudate-putamen, nucleus accumbens, olfactory tubercle, septum, and bed nucleus of the stria terminalis. To analyze forebrain projections of the ventral midbrain CCK-I neurons, indirect immunofluorescence was combined with fluorescence retrograde tracing. CCK-I neurons of the substantia nigra and/or ventral tegmental area were found to project, to varying extents, to all of the above CCK-I forebrain terminal fields. The nucleus accumbens, olfactory tubercle, and septal and prefrontal cortical projections arose primarily from CCK-I perikarya in the ventral tegmental area whereas the projections to the caudate-putamen and anterior cingulate cortex arose predominantly from immunoreactive neurons in the substantia nigra pars compacta. The amygdala received innervation mainly from CCK-I cell bodies located in the substantia nigra pars lateralis. CCK-I afferents to the bed nucleus of the stria terminalis and piriform cortex originated from perikarya distributed approximately equally across the ventral tegmental area and substantia nigra pars compacta. The general topography of CCK-I forebrain innervation observed in this study is similar to that previously reported for the ascending dopaminergic projections from ventral mesencephalic neurons. CCK-I neurons of the midline raphe nuclei were found to provide relatively minor afferents to the caudate-putamen, bed nucleus of the stria terminalis, septum, and prefrontal cortex and more substantial projections to the amygdala. The results of this study demonstrate that CCK-I neurons of the ventral midbrain supply a much broader innervation of forebrain regions than previously appreciated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leucine5-enkephalin afferents to midbrain dopaminergic neurons: Light and electron microscopic examination

The relationship between leucine⁵-enkephalin-containing nerve terminals and midbrain dopaminergic neurons was studied in the adult rat by light and electron microscopy. For light microscopy, alternate midbrain sections were immunostained with rabbit polyclonal antibodies against leucine⁵-enkephalin and tyrosine hydroxylase, by means of the peroxidase antiperoxidase technique. Leucine⁵-enkephalin stained fibers and terminals were observed with varying density in the retrorubral field (dopaminergic nucleus A8 region), substantia nigra pars compacta (dopaminergic nucleus A9 region), and ventral tegmental area and related nuclei (dopaminergic nucleus A10 region). For electron microscopy, midbrain sections were immunostained with a mouse monoclonal antibody against leucine⁵-enkephalin and a rabbit polyclonal antibody against tyrosine hydroxylase, by means of the peroxidase antiperoxidase technique and silver-intensified colloidal gold reactions, respectively. The nucleus A10 area was examined at the electron microscopic level, and there were (a) both symmetric (75%) and asymmetric (25%) synapses made between leucine⁵-enkephalin axon terminals and dopaminergic dendrites, and also synaptic contacts with unlabeled dendrites; (b) leucine⁵-enkephalin synaptic contacts with dopaminergic dendrites that were covered with astrocytic membranes; and (c) leucine⁵-enkephalin appositions with unlabeled nerve terminals that made synaptic contacts with dopaminergic dendrites, suggestive of axo-axonic connections. These findings provide the structural basis for both direct and indirect control of A10 dopaminergic neurons by enkephalin-containing nerve terminals. © 1993 Wiley-Liss, Inc.