Self-stimulation of the nucleus accumbens and ventral tegmental area of tsai attenuated by microinjections of spiroperidol into the nucleus accumbens

The contribution of dopaminergic neurons to self-stimulation of the ventral tegmental area, nucleus accumbens and prefrontal cortex was investigated. The ventral tegmental area is the site of non-striatal dopaminergic neurons and their axons project to the nucleus accumbens and prefrontal cortex. Injections of spiroperidol, a dopamine antagonist, into the nucleus accumbens significantly reduced self-stimulation of the ipsilateral ventral tegmental area but did not influence self-stimulation of the contralateral ventral tegmental area. Injections of spiroperidol into the prefrontal cortex did not reduce self-stimulation of the ipsilateral or contralateral ventral tegmental area. Electrical stimulation of sites in the nucleus accumbens positive for self-stimulation antidromically activated neurons of the ventral tegmental area, and a reduction of discharge of these neurons following administration of apomorphine suggested that they were dopaminergic neurons. These observations provide additional evidence implicating dopaminergic neurons in brain-stimulation reward and suggest that dopaminergic neurons contribute to self-stimulation of the nucleus accumbens but not the prefrontal cortex.     

Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate-putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde-fixed rat brain were processed 1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine-synthesizing enzyme choline acetyltransferase (CAT) and 2) for the immunoautoradiographic localization of a rabbit polyclonal antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate-putamen versus NAS. Immunoperoxidase reaction for CAT versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2-3% of the CAT-labeled terminals (n = 136) and less than 1% of the TH-labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH-labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH-labeled terminals. Thirteen percent of the CAT-labeled and 14% of TH-labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudate-putamen and NAS cholinergic and catecholaminergic terminals 1) form symmetric, most likely inhibitory, synapses primarily with non-cholinergic neurons, 2) differentially synapse on shafts or spines of separate dendrites, and 3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic-dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.     

Synaptic innervation of midbrain dopaminergic neurons by glutamate-enriched terminals in the squirrel monkey

The excitatory amino acid, glutamate, has long been thought to be a transmitter that plays a major role in the control of the firing pattern of midbrain dopaminergic neurons. The present study was aimed at elucidating the anatomical substrate that underlies the functional interaction between glutamatergic afferents and midbrain dopaminergic neurons in the squirrel monkey. To do this, we combined preembedding immunocytochemistry for tyrosine hydroxylase and calbindin D-28k with postembedding immunostaining for glutamate. On the basis of their ultrastructural features, three types (so-called types I, II, and III) of glutamate-enriched terminals were found to form asymmetric synapses with dendrites and perikarya of midbrain dopaminergic neurons. The type I terminals accounted for more than 70% of the total population of glutamate-enriched boutons in contact with dopaminergic cells in the dorsal and ventral tiers of the substantia nigra pars compacta as well as in the ventral tegmental area, whereas 5–20% of the glutamatergic synapses with dopaminergic neurons involved the two other types of terminals. The major finding of our study is that the glutamate-enriched boutons were involved in 70% of the axodendritic synapses in the ventral tegmental area. In contrast, less than 40% of the boutons in contact with dopaminergic dendrites were immunoreactive for glutamate in the dorsal and ventral tiers of the substantia nigra pars compacta. Approximately 50% of the terminals in contact with the perikarya of the different populations of midbrain dopaminergic neurons displayed glutamate immunoreactivity. In conclusion, our findings provide the first evidence that glutamate-enriched terminals form synapses with midbrain dopaminergic neurons in primates. The fact that the proportion of glutamatergic boutons in contact with dopaminergic cells is higher in the ventral tegmental area than in the substantia nigra pars compacta suggests that the different groups of midbrain dopaminergic neurons are modulated differently by extrinsic glutamatergic afferents in primates. © 1996 Wiley-Liss, Inc.     

Increase of tyrosine hydroxylase-like immunoreactive neurons in the nucleus accumbens and the olfactory bulb in the rat with the lesion in the ventral tegmental area of the midbrain

A small number of neurons in the nucleus accumbens of the rat showed tyrosine hydroxylase-like immunoreactivity (TH-LI). These TH-LI neurons had medium-sized cell bodies with several spiny dendrites. When a lesion was produced in the ventral tegmental area of the midbrain by injecting 6-hydroxydopamine, TH-LI neurons in the nucleus accumbens, as well as those in the olfactory bulb, were increased in number on the side ipsilateral to the lesion. The results indicated that expression of TH might be enhanced in some neurons deprived of dopaminergic afferent fibers.     

Dopamine terminals synapse on callosal projection neurons in the rat prefrontal cortex

Dopamine (DA) afferents to the prefrontal cortex (PFC) play an important role in the cognitive functions subserved by this cortical area. Within the PFC, DA terminals synapse onto the distal dendrites of both local circuit neurons and pyramidal projection cells. We have previously demonstrated in the rat PFC that some of the dendrites and spines postsynaptic to DA terminals arise from pyramidal neurons that project to the nucleus accumbens. However, it is not known whether the pyramidal cells that give rise to callosal intercortical connections of the PFC also receive DA synaptic input. To address this question, retrograde tract tracing using an attenuated strain of pseudorabies virus (PRV-Bartha) was combined with immunocytochemistry for tyrosine hydroxylase (TH) to identify DA terminals in the PFC. Thirty-six to 40 hours following injection of PRV into the contralateral PFC, numerous callosal projection neurons were extensively labeled throughout their dendritic trees, with no evidence of PRV trans-synaptic passage. In tissue prepared for electron microscopy, labeling for PRV was distributed throughout pyramidal cell somata and extended into distal dendrites and dendritic spines. Some PRV-labeled dendrites and spines received symmetric synaptic input from terminals containing peroxidase labeling for TH. These results demonstrate that DA terminals synapse onto the distal dendrites of callosally projecting PFC neurons and suggest substrates through which DA may modulate interhemispheric cortical communication.     

Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents

Neurons containing gamma-aminobutyric acid (GABA) in the medial portion of the adult rat nucleus accumbens were characterized with respect to their ultrastructure, sites of termination, and catecholaminergic input. Antisera against GABA-conjugates and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), were localized within single sections by means of peroxidase-antiperoxidase (PAP) and immunoautoradiographic labeling methods. Peroxidase reaction product indicating GABA-like immunoreactivity (GABA-LI) was seen in medium-size (15-20 microns) perikarya containing either round and unindented or invaginated nuclear membranes. The cells with invaginated nuclei were few in number and usually exhibited more intense peroxidase reaction product in sections collected at the same distance from the surface of the tissue. Reaction product for GABA was also detected in proximal (1.5-3.0 microns) dendrites, axons, and terminals. Terminals with GABA-LI formed symmetric junctions on perikarya, proximal dendrites, and dendritic spines of neurons that usually lacked detectable immunoreactivity. Many of the GABAergic terminals also were apposed directly to other unlabeled terminals and to terminals exhibiting either peroxidase labeling for GABA or immunoautoradiographic labeling for TH. Many of the unlabeled terminals associated with the GABAergic axons formed asymmetric junctions on dendritic spines. From 138 TH-labeled, principally dopaminergic terminals that were examined in the medial nucleus accumbens, 4% were associated with the somata of GABAergic neurons and another 14% formed symmetric junctions with proximal dendrite showing GABA-LI. The remaining TH-immuno-reactive terminals either lacked recognizable densities or formed symmetric synapses on unlabeled dendrites and spines. A few of the unlabeled dendrites, as well as those containing GABA-LI, received symmetric synapses from both catecholaminergic and GABAergic terminals. We conclude that in the medial portion of the rat nucleus accumbens, GABA is localized to two morphologically distinct types of neurons, one or both of which receive monosynaptic input from catecholaminergic afferents, and that GABAergic terminals form symmetric synapses on other principally non-GABAergic neurons. The results also support earlier physiological evidence showing that GABA may modulate the output of other GABAergic and non-GABAergic neurons through presynaptic associations.     

Neurotensin terminals form synapses primarily with neurons lacking detectable tyrosine hydroxylase immunoreactivity in the rat substantia nigra and ventral tegmental area.

A light and electron microscopic double antigen localization technique was employed to examine the fine structural relationship between neurotensin-containing axon terminals and dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat. At the light microscopic level, neurotensin-immunoreactive terminals were densely distributed throughout the substantia nigra pars compacta and ventral tegmental area in close proximity to tyrosine hydroxylase-immunoreactive somata and dendrites. On electron microscopic examination, direct synaptic connections were identified between neurotensin-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive perikarya and dendrites. However, only 8.2% and 8.8% of the neurotensin-immunoreactive axonal profiles detected in the substantia nigra and ventral tegmental area, respectively, were found in direct apposition with tyrosine hydroxylase-immunostained elements. In turn, only 9.3% and 10.0% of tyrosine hydroxylase immunoreactive dendrites sampled from the substantia nigra and ventral tegmental area, respectively, were seen in contact with neurotensin immunopositive axon terminals. However, neurotensin-immunoreactive and tyrosine hydroxylase-immunolabelled elements were frequently identified in close anatomical proximity (less than 5 microns) to one another. These results are interpreted in light of the selective association of neurotensin receptors with dopaminergic neurons in the substantia nigra and ventral tegmental area to suggest a predominantly parasynaptic mechanism of action for neurotensin in the ventral midbrain.     

Ultrastructural features of the nitric oxide synthase-containing interneurons in the nucleus accumbens and their relationship with tyrosine hydroxylase-containing terminals

The ultrastructural features of neuronal nitric oxide synthase (NOS) -immunoreactive interneurons of rat nucleus accumbens shell and core were studied and compared. The NOS-containing subpopulation displayed characteristics similar to those previously described for nicotinamide adenine dinucleotide phosphate diaphorase-, neuropeptide Y, or somatostatin-containing striatal neurons, but also showed properties not previously associated with them, particularly the formation of both asymmetric and symmetric synaptic junctions. Inputs derived mainly from unlabeled terminals, but some contacts were made by NOS-immunolabeled terminals, by means of asymmetric synapses. Immunopositive endings that formed symmetric synapses were mainly onto dendritic shafts, whereas those that formed asymmetric synapses targeted spine heads. Morphometric analysis revealed that the core and shell NOS-stained neurons had subtly different innervation patterns and that immunostained terminals were significantly larger in the shell. A parallel investigation explored synaptic associations with dopaminergic innervation identified by labeling with an antibody against tyrosine hydroxylase (TH). In both shell and core, TH-positive boutons formed symmetric synapses onto NOS-containing dendrites, and in the core, TH- and NOS-immunolabeled terminals converged on both a single spiny dendrite and a spine. These results suggest that, in the rat nucleus accumbens, NOS-containing neurons may be further partitioned into subtypes, with differing connectivities in shell and core regions. These NOS-containing neurons may be influenced by a dopaminergic input. Recent studies suggest that nitric oxide potentiates dopamine release and the current study identifies the medium-sized, densely spiny neurons as a possible site of such an interaction.     

Nucleus accumbens in the lizard Psammodromus algirus: chemoarchitecture and cortical afferent connections

To better understand the organization and evolution of the basal ganglia of vertebrates, in the present study we have analyzed the chemoarchitecture and the cortical input to the nucleus accumbens in the lacertid lizard Psammodromus algirus. The nucleus accumbens contains many gamma-aminobutyric acid (GABA)-positive neurons and calbindin-positive neurons, the majority of which may be spiny projection neurons, and a few dispersed neuropeptide Y-positive neurons that likely represent aspiny interneurons. The nucleus accumbens contains two chemoarchitectonically different fields: a rostromedial field that stains heavily for substance P, dopamine, GABA(A) receptor, and a caudolateral field that stains only lightly to moderately for them, appearing more similar to the adjacent striatum. Injections of biotinylated dextran amine were placed in either the medial, dorsomedial, or dorsal cortices of Psammodromus. The medial and the dorsal cortices project heavily to the rostromedial field of the accumbens, whereas they project lightly to moderately to the caudolateral field. Cortical terminals make asymmetric, presumably excitatory, synaptic contacts with distal dendrites and the head of spines. Our results indicate that the hippocampal-like projection to the nucleus accumbens is similar between mammals and reptiles in that cortical terminals make mainly excitatory synapses on spiny, putatively projection neurons. However, our results and results from previous investigations indicate that important differences exist between the nucleus accumbens of mammals and reptiles regarding local modulatory interactions between cortical, dopaminergic, and cholinergic elements, which suggest that the reptilian nucleus accumbens may be as a whole comparable to the shell of the mammalian nucleus accumbens.     

A catecholaminergic projection from the ventral tegmental area to the diagonal band of broca: Modulation by neurotensin

The ventral tegmental area contains a high density of dopaminergic perikarya having ascending projections to a number of limbic forebrain regions. In this study, we use combined retrograde labeling with horseradish peroxidase (HRP) and immunohistochemical staining for tyrosine hydroxylase to examine the catecholaminergic projection from the ventral tegmental area to the diagonal band of Broca. When injection of HRP was restricted to the diagonal band, only neurons in the nucleus linearis, nucleus interfascicularis and ventromedial portion of the nucleus paranigralis were labeled. In contrast, HRP injection into the adjacent nucleus accumbens labeled neurons throughout these nuclei, plus the nucleus parabrachialis pigmentosus, nucleus retroruber and substantia nigra, pars compacta. Approximately 60% of neurons in the ventral tegmental area labeled from the diagonal band contained tyrosine hydroxylase, compared with 79% of the neurons labeled from the nucleus accumbens. Neurotensin is a tridecapeptide found in the ventral tegmental area which has been shown to activate dopamine neurons projecting to the nucleus accumbens. In this study, microinjection of neurotensin into ventral tegmental nuclei which contained neurons retrogradely labeled from the diagonal band significantly elevated the levels of dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the diagonal band. The results of this study demonstrate that a catecholaminergic projection exists from the ventral tegmental area to the diagonal band of Broca, and that this pathway can be stimulated by intra-ventral tegmental injection with neurotensin.     

Ultrastructural double-labeling demonstrates synaptic contacts between dopaminergic terminals and substance P-containing striatal neurons in pigeons.

Immunohistochemical studies in rats have demonstrated dopaminergic input onto medium spiny neurons of the striatum. Medium spiny neurons, however, are known to consist of two major neuropeptide-specific types, those containing substance P (SP) and those containing enkephalin. Although both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites, the extent to which both types also receive direct dopaminergic input onto distal dendritic shafts or onto dendritic spines is uncertain. In the present study, we used EM immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto SP+ striatal neurons. We examined the striatum of pigeons, in whom SP+ striatal neurons, including their dendritic shafts and spines, can be readily labeled. Antibodies against tyrosine hydroxylase (TH) were used to identify dopaminergic terminals, which were labeled using silver-intensified immunogold. The SP+ neurons were labeled immunohistochemically using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts and dendritic spines of SP+ neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto SP+ striatal neurons in a manner similar to that described for dopaminergic input onto striatal medium spiny neurons in general.     

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.     

The Convergence of Axon Terminals from the Mediodorsal Thalamic Nucleus and Ventral Tegmental Area on Pyramidal Cells in Layer V of the Rat Prelimbic Cortex

We investigated the ultrastructural basis of the synaptic convergence of afferent fibres from the mediodorsal thalamic nucleus (MD) and the ventral tegmental area (VTA) on the prefrontal cortical neurons of the rat by examining the synaptic relationships between thalamocortical or tegmentocortical terminals labelled with anterograde markers [lesion-induced degeneration or transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA—HRP)] and randomly selected unlabelled apical dendrites of layer V pyramidal cells in the prelimbic cortex. WGA—HRP-labelled terminals from the VTA ranged in diameter from 0.7 to 2.8 μm and established synaptic contacts with large dendritic profiles, i.e. proximal segments of apical dendritic shafts and spines from layer V pyramidal cells. Symmetrical synapses, i.e. inhibitory synapses, were more often seen than asymmetrical ones. Degenerating terminals from the MD formed asymmetrical synapses on dendritic spines or occasionally on small dendritic shafts of apical dendrites from layer V pyramidal cells, which received tegmentocortical synapses, mostly within layer III. Thalamocortical synapses were more distally distributed over common apical dendrites than tegmentocortical synapses, although some of them overlapped. The numerical density of direct synaptic inputs from the MD and VTA was low. These results suggest that fibres from the VTA exert their inhibitory effects directly on pyramidal cells in layer V via synaptic junctions with apical dendrites of these pyramidal cells, and that the tegmentocortical fibres are in an ideal anatomical position to modulate the reverberatory circuits between the MD and the prelimbic cortex.     

Synaptic relationships between axon terminals from the mediodorsal thalamic nucleus and gamma-aminobutyric acidergic cortical cells in the prelimbic cortex of the rat

Although the reciprocal interconnections between the prefrontal cortex and the mediodorsal nucleus of the thalamus (MD) are well known, the involvement of inhibitory cortical interneurons in the neural circuit has not been fully defined. To address this issue, we conducted three combined neuroanatomical studies on the rat brain. First, the frequency and the spatial distribution of synapses made by reconstructed dendrites of nonpyramidal neurons were identified by impregnation of cortical cells with the Golgi method and identification of thalamocortical terminals by degeneration following thalamic lesions. Terminals from MD were found to make synaptic contacts with small dendritic shafts or spines of Golgi-impregnated nonpyramidal cells with very sparse dendritic spines. Second, a combined study that used anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and postembedding gamma-aminobutyric acid (GABA) immunocytochemistry indicated that PHA-L-labeled terminals from MD made synaptic junctions with GABA-immunoreactive dendritic shafts and spines. Nonlabeled dendritic spines were found to receive both axonal inputs from MD with PHA-L labelings and from GABAergic cells. In addition, synapses were found between dendritic shafts and axon terminals that were both immunoreactive for GABA. Third, synaptic connections between corticothalamic neurons that project to MD and GABAergic terminals were investigated by using wheat germ agglutinin conjugated to horseradish peroxidase and postembedding GABA immunocytochemistry. GABAergic terminals in the prelimbic cortex made symmetrical synaptic contacts with retrogradely labeled corticothalamic neurons to MD. All of the synapses were found on cell somata and thick dendritic trunks. These results provide the first demonstration of synaptic contacts in the prelimbic cortex not only between thalamocortical terminals from MD and GABAergic interneurons but also between GABAergic terminals and corticothalamic neurons that project to MD. The anatomical findings indicate that GABAergic interneurons have a modulatory influence on excitatory reverberation between MD and the prefrontal cortex.     

Histological and ultrastructural evidence that D-amphetamine causes degeneration in neostriatum and frontal cortex of rats

D-Amphetamine sulfate, continuously administered for 3 days subcutaneously via an implanted minipump, induced neural degeneration in Long-Evans and Sprague-Dawley rats at doses between 20 and 60 mg/kg/day. Using Fink-Heimer silver staining, axonal degeneration was detected in the neostriatum and the dorsal agranular insular cortex and degenerating pyramidal cells were observed in portions of the somatosensory neocortex in both strains. In contrast, dense axonal degeneration largely confined to layers 2 and 3 of frontal motor areas (Fr1, Fr2 and Fr3 of Zilles36) with occasional degenerating cells was seen reliably in Long-Evans rats but rarely in Sprague-Dawley rats. In the electron microscope, cortical degeneration consisted mainly of disrupted cell bodies and dark processes, including axons making asymmetric synapses. Damage in all cortical areas represents damage to non-monoamine neurons and processes since tyrosine hydroxylase and serotonin immunolabeling were normal. In contrast, the damage in neostriatum probably includes damage to dopamine axonal terminals since tyrosine hydroxylase immunolabeling was patchy with many swollen and distorted labeled axons. Serotonin and Leu-enkephalin labeling were normal. Electron microscopy confirmed that the neostriatum contained many tyrosine hydroxylase-labeled axons that were swollen and disrupted, although other labeled processes made normal symmetric synapses onto spines and dendrites. Additional degeneration found only in amphetamine-treated rats included many dark, shrunken profiles. Some of these appeared to be astrocytic processes and a few were myelinated axons, suggesting that some non-monoamine, possibly cortical afferents, are also degenerating in the neostriatum. Since similar degrees of behavioral activation, weight loss and lethality were seen in both strains, a genetic predisposition constrain amphetamine-induced motor cortex damage but not neostriatal damage.     

Dopaminergic terminals form synaptic contacts with enkephalinergic striatal neurons in pigeons: an electron microscopic study

Medium spiny projection neurons of the striatum consist of two major neuropeptide-specific types, one type containing substance P and another type containing enkephalin. Both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites. However, whether each of these types receives direct dopaminergic input onto distal dendritic shafts and onto dendritic spines has not been explored in depth. In the present study, we used electron microscopic immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto enkephalin-positive (ENK +) striatal neurons in pigeons, in whom ENK + striatal perikarya, dendritic shafts and spines can be readily labeled. Antibodies against tyrosine hydroxylase were used to label dopaminergic terminals using a silver-intensified immunogold method. ENK + neurons were labeled using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts, and dendritic spine necks of ENK + striatal neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto ENK + striatal neurons in a manner similar to that described previously by us for substance P-positive striatal medium spiny neurons.     

Tyrosine hydroxylase immunochemistry and quantitative light microscopic studies of the mesolimbic dopamine system in rat brain: effects of chronic methamphetamine administration

Long-term treatment of rats with methamphetamine (20 mg/kg, IP, every 12 hours for 10 days) resulted in a large decrease in tyrosine hydroxylase staining axons and terminal boutons in the nucleus accumbens and frontal cortex, as well as the ventral tegmental area of the midbrain, when examined 60 days following termination of the drug treatment regimen. Quantitative analysis showed a 71 and 78% decrease in tyrosine hydroxylase staining processes in the nucleus accumbens and frontal cortex, respectively, and a 90% decrease in tyrosine hydroxylase positive material in the ventral tegmental area. Thus, tyrosine hydroxylase enzyme in both the cell bodies of the midbrain ventral tegmental area as well as in the nerve terminals in post-synaptic target regions of the forebrain is depleted by chronic methamphetamine administration.     

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.     

Ultrastructural localization of tyrosine hydroxylase in rat nucleus accumbens

Immunocytochemical localization of tyrosine hydroxylase (TH) was used to determine the ultrastructural morphology and synaptic associations of catecholaminergic terminals in the nucleus accumbens of the rat. The brains were fixed by vascular perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde. Coronal sections cut with a vibrating microtome were incubated with rabbit antiserum to TH then immunocytochemically labeled by the peroxidase-antiperoxidase method. Immunoreactivity for the enzyme was found within unmyelinated axons and axon terminals. These terminals contained either all small clear or combined small clear and large dense core vesicles. Approximately 40% of the labeled terminals formed symmetric synapses with unlabeled proximal or distal dendritic shafts. The dendrites showed a spare distribution of spines. Axosomatic synapses and axonal associations of the TH-containing terminals also were detected. The recipient perikarya were usually 10-20 micrometers in diameter and contained an indented nucleus and abundant cytoplasm. The content of large dense vesicles and synaptic associations with somata and proximal dendrites suggest that a certain proportion of the TH-containing terminals within the nucleus accumbens are morphologically distinct from catecholaminergic terminals within the dorsal striatum. These differences are discussed in relation to neuropeptides and functions of the dopaminergic mesolimbic and nigrostriatal pathways.