Ultrastructural single- and double-label immunohistochemical studies of substance P-containing terminals and dopaminergic neurons in the substantia nigra in pigeons.

The vast majority of striatonigral projection neurons in pigeons contain substance P (SP), and the vast majority of SP-containing fibers terminating in the substantia nigra arise from neurons in the striatum. To help clarify the role of striatonigral projection neurons, we conducted electron microscopic single- and double-label immunohistochemical studies of SP+ terminals and/or dopaminergic neurons (labeled with either anti-dopamine, DA, or anti-tyrosine hydroxylase, TH) in pigeons to determine: (1) the synaptic organization of SP+ terminals, (2) the synaptic organization of TH+ perikarya and/or dendrites, and (3) the synaptic relationship between SP+ terminals and TH+ neurons in the substantia nigra. Tissue single-labeled for SP revealed numerous SP+ terminals contacting thin unlabeled dendrites in the substantia nigra, but few SP+ terminals were observed contacting perikarya or large-diameter dendrites. SP+ terminals contained round, densely packed, clear vesicles, and often contained one or more dense-core vesicles. Synaptic junctions between SP+ terminals and their targets were more often symmetric (86%) than asymmetric. In tissue single-labeled for DA, we observed few terminals contacting DA+ perikarya, whereas terminals contacting DA+ dendrites were more abundant. Terminals contacting DA+ structures comprised at least four different morphologically distinct types based on the morphology of the clear synaptic vesicles and the type of synaptic junction. One type of terminal contained round clear vesicles and made symmetric synapses, and thus resembled the predominant type of SP+ terminal. The second type contained round clear vesicles and made asymmetric synapses, the third type contained medium-size pleomorphic clear vesicles and made symmetric synapses, and the fourth type contained small pleomorphic clear vesicles and made symmetric synapses. The presence of contacts between SP+ terminals and dopaminergic dendrites in the substantia nigra was directly demonstrated in tissue double-labeled for SP (by the peroxidase-antiperoxidase procedure, or PAP, with diaminobenzidine) and TH (by either the silver-intensified immunogold procedure or the PAP procedure with benzidine dihydrochloride). SP+ terminals commonly contacted thin TH+ dendrites in the substantia nigra, but few SP+ terminals contacted large-diameter TH+ dendrites or perikarya. Synapses between SP+ terminals and TH+ neurons were always symmetric. TH+ dendrites also were contacted by terminals not labeled for SP, which were more abundant than were SP+ terminals. Non-TH+ neurons were also contacted by both SP+ terminals and non-SP+ terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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-positive fibers and neurons in transplanted striatal tissue in rats with quinolinic acid lesions of the striatum

Using the quinolinic acid (QA) animal model of Huntington's disease (HD) the dopaminergic afferent input to intrastriatal striatal grafts was examined. After bilateral striatal lesions with QA (15 nmol), 4 microliters of fetal (E17) striatal tissue were delivered into the lesioned striata. Twenty-eight weeks posttransplantation the tissue was processed for TH immunocytochemistry and cresyl violet staining. In addition fetal intact brains (E17) were also processed for TH immunocytochemistry and cresyl violet staining. Viable striatal grafts were located within the host striatum and in some cases within the lateral ventricles. TH-positive fibers were present within the graft and also groups of TH-positive cell bodies were seen in some of the grafts. TH immunocytochemistry on E17 fetuses revealed several groups of TH-positive neurons one of which was placed immediately ventral to the developing striatal ridge. The origin of TH-positive innervation within the graft is discussed.     

Human ventral mesencephalic xenografts to the catecholamine-depleted striata of athymic rats: ultrastructure and immunocytochemistry

On the basis of animal studies, grafts of fetal human dopaminergic cells have been suggested as a therapy for Parkinson's disease. The purpose of this study was to characterize the ultrastructure and immunocytochemistry of human ventral mesencephalic xenografts placed into the catecholamine-depleted striata of athymic "nude" rats. Human fetal tissue was obtained from tissue fragments derived from elective abortions during the first trimester of pregnancy. Small pieces of the basal mesencephalon were grafted into the catecholamine-depleted striata of four athymic nude rats. The rats were allowed to survive from 3 to 6 months after grafting; following fixation, the striatal tissue containing the grafts was labeled with antibodies against tyrosine hydroxylase and serotonin. Immunocytochemistry revealed tyrosine-hydroxylase-like-immunoreactive (THLI) and serotoninlike-immunoreactive (5HTLI) cell bodies within the human grafts. Both 5HTLI and THLI fibers crossed the graft-host interface and innervated the previously lesioned striatum. Both types of fibers also entered the host cortex from the adjacent human graft. At the ultrastructural level, THLI and 5HTLI fibers and synaptic terminals were observed in the host neuropil. THLI and 5HTLI dendrites and axon terminals were also observed in the neuropil of the grafts themselves. THLI axon terminals are not normally present in the substantia nigra. The results of our study indicate that human xenografts can survive in the neuropil of the host striatum and form morphologically appropriate synapses within the host brain.     

Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat

Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional‐motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits. It has previously been proposed that dopaminergic neurons in the ventral mesencephalon may play a role in this circuit interaction. Therefore, we studied in rats convergence of basal ganglia circuits by depositing an anterograde neuroanatomical tracer into the ventral striatum together with a retrograde fluorescent tracer ipsilaterally in the dorsolateral striatum. In the mesencephalon, using confocal microscopy, we looked for possible appositions of anterogradely labeled fibers and retrogradely labeled neurons, “enhancing” the latter via intracellular injection of Lucifer Yellow. Tyrosine hydroxylase (TH) immunofluorescence served to identify dopaminergic neurons. In neurophysiological experiments, we combined orthodromic stimulation in the medial ventral striatum with recording from ventral mesencephalic neurons characterized by antidromic stimulation from the dorsal striatum. We observed terminal fields of anterogradely labeled fibers that overlap populations of retrogradely labeled nigrostriatal cell bodies in the substantia nigra pars compacta and lateral ventral tegmental area (VTA), with numerous close appositions between boutons of anterogradely labeled fibers and nigrostriatal, TH‐immunopositive neurons. Neurophysiological stimulation in the medial ventral striatum caused inhibition of dopaminergic nigrostriatal neurons projecting to the ventrolateral striatal territory. Responding nigrostriatal neurons were located in the medial substantia nigra and adjacent VTA. Our results strongly suggest a functional link between ventromedial, emotional‐motivational striatum, and the sensorimotor dorsal striatum via dopaminergic nigrostriatal neurons.     

Glial responses associated with dopaminergic striatal reinnervation following lesions of the rat substantia nigra

Lesioning of dopaminergic substantia nigra pars compacta (SNpc) neurons leads to depletion of dopamine (DA) and dopaminergic axons in the dorsal striatum, followed by subsequent compensatory sprouting of dopaminergic fibers and striatal reinnervation. In this study, the response of striatal glia (microglia and astroglia) was compared with the degeneration and regeneration of dopaminergic axons following SNpc lesions. Following partial SNpc lesions, density of dopamine transporter (DAT) immunoreactive (-ir) terminals in the dorsal striatum returned to normal within 16 weeks of injury, suggesting that dopaminergic reinnervation of the striatum was complete. In conjunction, the glial responses in the dorsal striatum consisted of two peaks. The first peak in glial density occurred immediately after lesioning, peaking at 7 days, implying that it was likely to be associated with removal of debris from degenerating terminals. The second glial response commenced 8 weeks after lesioning and peaked some time after 16 weeks. The time of onset of the second peak suggests that it may be associated with the establishment of synapses rather than with axonal guidance.     

Host brain regulation of dopaminergic grafts function: role of the serotonergic and noradrenergic systems in amphetamine-induced responses

The indirect dopaminergic (DA) agonist amphetamine has frequently been used to study functional responses of DA grafted neurons. However, it is not known if striatal responses, primarily related to DA release by the grafted neurons, are modulated by the host striatal afferents. We investigated the changes in amphetamine-induced rotational behavior and striatal expression of Fos in DA-denervated and grafted rats subjected to serotonergic denervation and/or treatment with the alpha(1)-adrenergic receptor antagonist Prazosin. Acute serotonergic lesions with p-chlorophenylalanine suppressed the expression of Fos induced by 1 mg/kg of amphetamine in both the grafted and the contralateral striatum. Chronic serotonergic denervation with 5,7-dihydroxytryptamine induced a significant reduction in Fos expression in both the grafted and nongrafted striata and a nonsignificant reduction in the contraversive rotation. In DA-innervated striata, Prazosin significantly reduced the expression of Fos but only in the presence of serotonergic innervation. However, Prazosin did not decrease the expression of Fos induced by grafts located in striata not subjected to serotonergic denervation. The present results suggest functional integration of transplanted DA neurons and major host striatal afferent systems, particularly the serotonergic system, in modulating responses of the host striatal neurons. However, indirect effects exerted by the noradrenergic system on the normal striatum were not observed in the DA-denervated and grafted striata.     

Disparity in the temporal appearance of methamphetamine-induced apoptosis and depletion of dopamine terminal markers in the striatum of mice

Methamphetamine (METH) causes damage in the striatum at pre- and post-synaptic sites. Exposure to METH induces long-term depletions of dopamine (DA) terminal markers such as tyrosine hydroxylase (TH) and DA transporters (DAT). METH also induces neuronal apoptosis in some striatal neurons. The purpose of this study is to demonstrate which occurs first, apoptosis of some striatal neurons or DA terminal toxicity in mice. This is important because the death of striatal neurons leaves the terminals in a state of deafferentation. A bolus injection (i.p.) of METH (30 mg/kg) induces apoptosis (TUNEL staining) in approximately 25% of neurons in the striatum at 24 h after METH. However, in contrast to apoptosis, depletion of TH (Western blotting) begins to appear at 24 h after METH in dorsal striatum while the ventral striatum is unaffected. The peak of TH depletion (approximately 80% decrease relative to control) occurs at 48 h after METH. Autoradiographic analysis of DAT sites showed that depletion begins to appear 24 h after METH and peaks at 2 days (approximately 60% depletion relative to control). Histological analysis of the induction of glial fibrillary acidic protein (GFAP) by METH in striatal astrocytes revealed an increase at 48 h after METH that peaked at 3 days. These data demonstrate that striatal apoptosis precedes the depletion (toxicity) of DA terminal markers in the striatum of mice, suggesting that the ensuing state of deafferentation of the DA terminals may contribute to their degeneration.     

Anatomical and functional reconstruction of the nigrostriatal system in vitro: selective innervation of the striatum by dopaminergic neurons.

To study development of the nigrostriatal pathway in an in vitro model system, organotypic slices obtained from rat pups (P4) and containing the striatum and the cortex were grown together with apposed embryonic (E13.5) mesencephalic blocks according to the static slice culture method of Stoppini et al. (1991; J. Neurosci. Methods 37:173-182). Under these conditions, mesencephalic dopaminergic (DA) fibers rapidly grow through the slice, preferentially its striatal portion. This innervation provides a true synaptic innervation to the striatum, as shown by the presence of DA terminals on striatal neurons. DA fibers are able to exert a functional influence, as seen by their ability to modulate c-Fos expression in striatal neurons in the same way as in vivo. Thus, blockade, under basal conditions, of the effect of spontaneously released dopamine by the D2 receptor antagonist haloperidol leads to the activation of c-Fos expression in the striatum. Furthermore, stimulation of DA release by amphetamine induces striatal c-Fos expression in a D1 receptor-dependent manner. Next, the mechanisms of the selective striatal innervation were examined. Indeed, DA fibers innervated specifically the striatum, avoiding the cortical portion of the slice. This selectivity seems to be specific for DA neurons; no selectivity could be observed when noradrenergic neurons were substituted for DA neurons. Short-term cocultures in a collagen gel of mesencephalic blocks with striatal blocks failed to reveal any oriented outgrowth of DA fibers from the mesencephalon, suggesting that the selective innervation observed in the organotypic slices results from some contact-dependent, presumably adhesive interactions rather than from the presence of some diffusible substance orienting the growth of DA fibers towards the striatum. On the other hand, DA neurons seeded onto striatal slices did not attach selectively onto the striatal portion of the slice, indicating that the putative specific adhesive interactions governing the selective striatal innervation are not the same as those determining the adhesion of the DA neurons. These results show that cocultures of cortex-striatum and mesencephalic slices result in a system that displays a number of the morphological and functional traits of the normal nigrostriatal system and that can be relied on as a good in vitro model of in vivo development.     

Gamma-aminobutyric acid, glutamic acid decarboxylase and tyrosine hydroxylase in rat striatum demonstrated by single and dual immunocytochemistry.

By means of dual ultrastructural immunostaining the followings patterns are visualized: gamma-aminobutyric acid (GABA) immunoreactive neurons, dendrites, axons and axon terminals and tyrosine hydroxylase (TH) immunopositive fibers, varicosities and boutons in rat striatum. Additionally single glutamic acid decarboxylase (GAD) immunolabeling is carried out. Four subgroups of GABA and GAD immunoreactive striatal neurons are revealed. These neuronal types are identified on the basis of sectional diameters, nuclear form and nuclear envelope invaginations, quantity of cytoplasm and cell organelles. Plasmalemmal appositions between GABAergic and between GABAergic and immunonegative neurons are observed. All subgroups of striatal GABAergic neurons contact with GABA and GAD immunoreactive, TH immunoreactive and immunonegative boutons. In the striatal neuropil numerous GABAergic, dopaminergic and immunonegative axonal endings synapsed with dendrites and spines are found out. Massive dopaminergic striatal structures using dual immunostaining is evident. Some GABA and GAD immunoreactive dendrites are revealed in direct contact with capillary walls.     

Dopamine-acetylcholine interaction in the rat striatum: A dual-labeling immunocytochemical study

The relationship between cholinergic neurons and dopaminergic axons in the rat striatum was examined by a dual-labeling immunocytochemical method. Cholinergic neurons were identified by their immunoreactivity for choline acetyltransferase (ChAT), and dopaminergic axon terminals were identified by their positive immunoreactivity for tyrosine hydroxylase (TH). Electron microscopic analysis of dual-labeled sections revealed that while most TH-positive terminals formed synapses with unlabeled striatal neurons and dendrites, a number of TH-positive terminals formed close appositions, highly suggestive of synapses, with both large and small dendrites as well as somata of ChAT-positive neurons. Tight appositions were also found between TH-positive terminals and ChAT-positive terminals. Moreover, TH-positive terminals and ChAT-positive terminals were found to form synapses with common dendrites of unlabeled striatal neurons. These results indicated that 1) dopaminergic axon terminals could interact directly with striatal cholinergic interneurons via tight appositions with distances comparable to conventional synapses; and 2) there is a convergence of dopaminergic and cholinergic axon terminals on noncholinergic striatal neurons.     

The distribution of GABA-containing perikarya,fibers, and terminals in the forebrain and midbrain of pigeons,with particular reference to the basal ganglia and its projection targets

Immunohistochemical techniques were used to study the distributions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid (GABA) in pigeon forebrain and midbrain to determine the organization of GABAergic systems in these brain areas in birds. In the basal ganglia, numerous medium-sized neurons throughout the striatum were labeled for GABA, while pallidal neurons, as well as a small population of large, aspiny striatal neurons, labeled for GAD and GABA. GAD+ and GABA+ fibers and terminals were abundant throughout the basal ganglia, and GABAergic fibers were found in all extratelencephalic targets of the basal ganglia. Most of these targets also contained numerous GABAergic neurons. In pallial regions, approximately 10-12% of the neurons were GABAergic. The outer rind of the pallium was more intensely labeled for GABAergic fibers than the core. The olfactory tubercle region, the ventral pallidum, and the hypothalamus were extremely densely labeled for GABAergic fibers, while GABAergic neurons were unevenly distributed in the hypothalamus. GABAergic neurons and fibers were abundant in the dorsalmost part of thalamus and the dorsal geniculate region, while GABAergic neurons and fibers were sparse (or lightly labeled) in the thalamic nuclei rotundus, triangularis, and ovoidalis. Further, GABAergic neurons were abundant in the superficial tectal layers, the magnocellular isthmic nucleus, the inferior colliculus, the intercollicular region, the central gray, and the reticular formation. GABAergic fibers were particularly abundant in the superficial tectal layers, the parvocellular isthmic nucleus, the inferior colliculus, the intercol-licular region, the central gray, and the interpeduncular nucleus. These results suggest that GABA plays a role as a neurotransmitter in nearly all fore- and midbrain regions of birds, and in many instances the observed distributions of GABAergic neurons and fibers closely resemble the patterns seen in mammals, as well as in other vertebrates. © 1994 Wiley-Liss, Inc.     

Histological evidence supporting a role for the striatal neurokinin-1 receptor in methamphetamine-induced neurotoxicity in the mouse brain

Several studies have documented the effect of methamphetamine (METH) on the toxicity of the dopamine (DA) terminals of the striatum but only a few studies have assessed the damaging effects of METH on striatal neurons postsynaptic to the nigrostriatal DA terminals. In the present study, we employed histological methods to study the effect of METH on DA terminals and striatal neurons. We also assessed the role of the striatal neurokinin-1 (NK-1) receptor on pre- and post-synaptic METH-induced damage. Male mice were treated with METH (10 mg/kg) four times at 2-h intervals and were sacrificed 3 days after the treatment. A number of animals received the non-peptide NK-1 receptor antagonist WIN-51,708 (10 mg/kg) 30 min before the first and fourth injections of METH. Immunocytochemical staining for tyrosine hydroxylase (TH) showed significant deficits throughout all aspects of the caudate-putamen in animals exposed to METH. Pretreatment with WIN-51,708 prevented the METH-induced loss of TH immunostaining. Sections from a separate set of mice were stained with Fluoro-Jade B (FJB), a fluorochrome that binds specifically to degenerating fibers and cell bodies of neurons. Treatment with METH shows Fluoro-Jade B positive cell bodies in the striatum and pretreatment with WIN-51,708 abolished Fluoro-Jade B staining. Moreover, double labeling with Fluoro-Jade B and glial fibrillary acidic protein (GFAP) shows reactive astrocytosis in the area adjacent to the Fluoro-Jade B-positive cells but no Fluoro-Jade B staining of the astrocytes. This observation suggests that the degenerating cells must be striatal neurons and not astrocytes. The data demonstrate that METH induces pre- and post-synaptic damage in the striatum and the damage can be prevented with pharmacological blockade of the NK-1 receptor. These findings represent a new direction in the study of the mechanism of toxicity to METH and could be useful in the treatment of some neurological disorders.     

Differentiation of the midbrain dopaminergic pathways during mouse development

Dopaminergic (DA) neurons in the substantia nigra (SN) and ventral tegmental area (VTA) of the midbrain project to the dorsolateral caudate/putamen and to the ventromedially located nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. Disruptions in this system have been implicated in Parkinson's disease, drug addiction, schizophrenia, and attention deficit hyperactivity disorder. However, progress in our understanding has been hindered by a lack of knowledge of how these pathways develop. In this study, different retrograde tracers, placed into the dorsolateral caudate/putamen and the nucleus accumbens, were used to analyze the development of the dopaminergic pathways. In embryonic day 15 mouse embryos, both SN and VTA neurons, as well as their fibers, were doubly labeled by striatal injections into the dorsolateral and ventromedial striatum. However, by birth, the SN DA neurons were labeled exclusively by DiA placed in the dorsolateral striatum, and the VTA DA neurons were labeled only by DiI injected into the ventromedial striatum. These data suggest that initial projections from midbrain DA neurons target nonspecifically to both the dorsolateral striatum and the nucleus accumbens. Later during development, the separate mesostriatal and mesolimbic pathways differentiate through the selective elimination of mistargeted collaterals.     

Striatal dopaminergic afferents concentrate in GDNF-positive patches during development and in developing intrastriatal striatal grafts

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic action on fetal dopaminergic neurons. We have used a double immunocytochemical approach with antibodies that recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP-32, to study the developmental pattern of their interactions in the rat striatum and in intrastriatal striatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchy distribution in the striatum, together with a high level of expression in the lateral striatal border, similar to that observed for the striatal marker DARPP-32 and also for TH. In the adult striatum, there was diffuse, weak immunopositivity for GDNF, together with widespread expression of DARPP-32-positive neurons and TH-immunoreactive (TH-ir) fibers. In 1-week-old intrastriatal striatal transplants, there were some GDNF immunopositive patches within the grafts and although there was not an abundance of TH-positive fibers, the ones that were seen were located in GDNF-positive areas. This was clearly evident in 2-week-old transplants, where TH-ir fibers appeared selectively concentrated in GDNF-positive patches. This pattern was repeated in 3-week-old grafts. In co-transplants of mesencephalic and striatal fetal tissue (in a proportion of 1:4), TH-ir somata were located mainly at the borders of areas that were more strongly immunostained for GDNF, and TH-ir fibers were also abundant in these areas and were found in smaller numbers in regions that were weakly positive for GDNF. These results demonstrate that GDNF-ir is coincident with that for TH and DARPP-32, and suggest that GDNF release by fetal striatal neurons both in normal development and in developing striatal grafts may have not only a trophic but also a tropic influence on TH-ir fibers and may be one of the factors that regulate dopaminergic innervation of the striatum.     

(+)MK-801 prevents the DDC-induced enhancement of MPTP toxicity in mice

In order to reach deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, MK-801, a non-competitive antagonist of NMDA receptors, has been used as a tool to study the role of excitatory amino acids. In agreement with previous reports, (+)MK-801 did not significantly affect either striatal dopamine (DA) or tyrosine-hydroxylase (TH) activity in MPTP-treated animals. On the contrary (+)MK-801, but not (−)MK-801 significantly reduced the DDC + MPTP-induced fall in striatal DA and TH activity. A similar preventing effect on DA metabolites (DOPAC and HVA) and HVA/DA ratio was observed. The number of TH⁺Mneurons in the substantia nigra (SN) of (+)MK-801-pretreated mice was not significantly different from that of control animals, indicating that this treatment specifically antagonized the extensive DDC-induced lesion of dopaminergic cell bodies in this brain area. (+)MK-801 treatment did not affect the DDC-induced changes of striatal MPP⁺ levels, suggesting that the observed antagonism of MK-801 against DDC is not due to MPP⁺ kinetic modifications. Pretreatment with the MAO-B inhibitor,l-deprenyl, or with the DA uptake blocker, GBR 12909, completely prevented the marked DA depletion elicited by DDC + MPTP within the striatum. Both treatments also protected from the fall in DA metabolites and TH activity as well. This indicates that DDC-induced potentiation is dependent upon MPP⁺ production and its uptake by the dopaminergic nerve terminals. All these findings suggest that NMDA receptors play a crucial role in the DDC-induced enhancement of MPTP toxicity.     

Connectivities of the striatal grafts in adult rat brain: a rich afference and scant striatonigral efference

Previous reports from this laboratory have indicated that fetal rat striatal grafts have the major types of neuronal and glial components known to be involved in Huntington's chorea. In this study a number of major afferent and efferent innervations seen in normal striatum were examined in the striatal grafts and were compared with embryonic striatal afferents. First, using immunocytochemistry and histochemistry, the host serotonergic (5-HT), dopaminergic (DA, stained with anti-tyrosine hydroxylase (TH) antiserum), and acetylcholinesterase (AChE) fibers exhibited vigorous growth into the grafts implanted in neostriatum, lateral ventricle, globus pallidus or substantia nigra within a period of 6 and 10 weeks. Individual characteristic terminal patterns formed in striatal grafts: 5-HT fibers were diffused; TH fibers became heavily packed, and AChE fibers were patchy. This peculiar patternization of 5-HT and TH growth into striatal graft appeared to be a recapitulation of the normal 5-HT and TH ingrowth into striatum in the embryonic stage. However, a significantly slow (6 week) onset of adult 5-HT and TH ingrowth into the fetal graft was noted, as compared with that of normal embryonic development (5-6 days from the appearance of 5-HT and TH neurons). With the anterograde-transport marker Phaseolus vulgaris agglutinin leuca method, host cortical neurons also projected to the graft, but in limited numbers. Finally, with the retrograde-transport marker (horseradish peroxidase method, the grafts implanted in neostriatum were found incapable of sending fibers to a major, distal target, substantia nigra. In a current evaluation of striatal transplants, it is shown that major input to the graft can be achieved over time, but output to the distal nigra seems an unrealistic expectation. These data suggest that: (1) the fetal brain tissue was found to be a strong stimulant for sprouting or regeneration of adult nerve fibers; (2) a number of functional recoveries reported on the tested behavior paradigm in this grafting model could be due to the survival of striatal graft and the establishment of input circuitries; further, (3) the data illustrate the necessity of seeking a bridge from the striatal transplant to the host nigra. If a proper functional recovery in Huntington's chorea requires complete striatonigral circuitry, then such a bridge is worthy of a major investigation.     

Increased dopamine release from striata of rats after unilateral nigrostriatal bundle damage

Dopaminergic control of striatal neurons is retained in rats sustaining lesions of the nigrostriatal bundle (NSB) as long as 10% of the projection remains, suggesting that enhanced efficiency of dopamine (DA) transmission may compensate for the denervation of the striatum. To examine this hypothesis we have studied the extracellular concentration of striatal DA using brain dialysis. In control rats, haloperidol (1 mg/kg, i.p.) or depolarization of striatal tissue with 25 mM KCl increased, and gamma-butyrolactone (500 mg/kg, i.p.) decreased DA and homovanillic acid (HVA) levels in striatal dialysates. Three weeks after unilateral injection of 6-hydroxydopamine (6-OHDA) to substantia nigra, DA content in the ipsilateral striatum was decreased by 60-98%. Nevertheless, extracellular DA concentration in the lesioned striata remained unchanged in rats with 60-90% DA depletions. More extensive lesions (96% DA depletion) were accompanied by 60% reduction in DA release. In contrast, extracellular HVA levels in the lesioned striata decreased proportionally to the depletion of tissue DA, indicating decreased inactivation of extracellular DA. We propose that the capacity of the residual DA terminals to maintain normal levels of extracellular DA after 60-90% NSB lesions may serve to compensate for the partial denervation of the striatal tissue. Disruption of striatal DA functions and postsynaptic supersensitivity after more extensive lesions may be associated with the failure of the NSB to fully compensate for loss of DA terminals. In striata contralateral to the 6-OHDA lesions, increased DA release was also observed. In addition, 60-90% ipsilateral DA depletions were accompanied by 32% and 42% increases in DA and HVA content in contralateral tissue, respectively. The possibility of the contralateral sprouting of DA terminals is discussed.