Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: A double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms. J. Comp. Neurol. 403:295–308, 1999. © 1999 Wiley-Liss, Inc.     

Subpopulations of mesencephalic dopaminergic neurons express different levels of tyrosine hydroxylase messenger RNA.

Subpopulations of mesencephalic dopamine containing neurons possess different electrophysiological, pharmacological, biochemical, and anatomical properties. In order to determine whether such differences are related to the regulation of tyrosine hydroxylase, the rate limiting enzyme in the synthesis of catecholamines, the regional distribution of tyrosine hydroxylase messenger RNA in these neurons was examined using in situ hybridization histochemistry. In the mouse, labelling for tyrosine hydroxylase messenger RNA associated with individual neurons was significantly less in the lateral substantia nigra pars compacta than in the medial substantia nigra pars compacta and the ventral tegmental area. A similar pattern of labelling was observed in the rat. Labelling for tyrosine hydroxylase messenger RNA was significantly less in the lateral substantia nigra pars compacta than in medial pars compacta (a densely cellular region), the area dorsal to the medial substantia nigra pars compacta (a less cell dense region), and the ventral tegmental area. Differences in levels of labelling for messenger RNA in mesencephalic dopamine neurons were not related to differences in cell size as measured in sections processed for tyrosine hydroxylase immunohistochemistry. The results suggest that tyrosine hydroxylase messenger RNA is differentially regulated in subpopulations of mesencephalic dopamine neurons, supporting the view that these neurons are physiologically distinct.     

Ventral mesencephalic neurons containing both cholecystokinin- and tyrosine hydroxylase-like immunoreactivities project to forebrain regions

The coexistence of cholecystokinin- and tyrosine hydroxylase-like immunoreactivities within neurons of the rat ventral mesencephalon was analyzed by using an indirect immunofluorescence technique for the simultaneous demonstration of two antigens in the same tissue section. A high degree of colocalization was observed in the substantia nigra pars compacta, in which 80-90% of all labeled neurons at rostral and up to 70% at intermediate levels contained both cholecystokinin and tyrosine hydroxylase. At caudal levels, the incidence of colocalization declined to approximately 30-50%. All of the immunoreactive perikarya in the substantia nigra pars lateralis were labeled with both substances. Other areas of the ventral midbrain that exhibited a moderate proportion of neurons immunoreactive for both cholecystokinin and tyrosine hydroxylase included the ventral tegmental area, interfascicular nucleus, and rostral and caudal linear nuclei. In addition, coexistence was occasionally observed within neurons of the central and ventral periaqueductal gray matter, supramammillary region, peripeduncular region, retrorubral field, and extremely rarely, within the substantia nigra pars reticulata. Cell bodies containing tyrosine hydroxylase-like immunoreactivity (indicative of dopamine) usually outnumbered those containing the peptide except in the supramammillary region and in the ventral periaqueductal gray matter, where the cholecystokinin perikarya were present in higher numbers. The double-labeling colocalization technique was combined with fluorescence retrograde tracing to determine some of the forebrain projections of these neurons. Ventral midbrain neurons containing both cholecystokinin and tyrosine hydroxylase were found to project to the caudate-putamen, nucleus-accumbens, prefrontal cortex, and amygdala. These projections originated from neurons located predominantly in the substantia nigra pars compacta and the ventral tegmental area. Thus, cholecystokinin occurs within the well-known dopaminergic nigrostriatal pathway in the rat. Overall, these results demonstrate that a significant proportion of the dopamine neurons giving rise to the ascending mesotelencephalic projections also contain the peptide cholecystokinin.     

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.     

Estrogen effects on tyrosine hydroxylase-immunoreactive cells in the ventral mesencephalon of the female rat: further evidence for the two cell hypothesis of dopamine function

The present study was undertaken to examine the differential effect of estrogen (E) on the expression of tyrosine hydroxylase (TH) in the substantia nigra compacta (SNc) and in two subdivisions of the ventral tegmental area in ovariectomized (ovx) and ovx plus estradiol benzoate (ovx+E)-treated female rats. Cell counting of TH-immunoreactive perikarya of the SNc, paranigral (PN) and interfascicular (IF) nucleus was performed and compared. Our findings demonstrate that E eliminated TH immunoreactivity from a number of midbrain neurons, while it seemingly did not affect it in others. This signifies a differential effect of E on ventral mesencephalic dopaminergic neurons.     

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.     

Calbindin D-28K in the dopaminergic mesocortical projection of a monkey (Aotus trivirgatus)

Injections of fluorescent dyes were made in the prefrontal and motor cortex of owl monkeys and retrogradely labeled neurons in the mesencephalon were analyzed for tyrosine hydroxylase and calbindin-D28K immunostaining. Numbers of mesocortical dopaminergic neurons in the dorsal substantia nigra compacta and in the ventral tegmental area also contain calbindin-D28K. This cortically projecting calbindin-D28K containing subpopulation of the dopaminergic mesencephalic cells may be characterized by different electrophysiological properties and a lesser vulnerability to cell death.     

Long-term high-frequency electro-acupuncture stimulation prevents neuronal degeneration and up-regulates BDNF mRNA in the substantia nigra and ventral tegmental area following medial forebrain bundle axotomy

Electroacupuncture (EA) has been used in China for many years to treat Parkinson's disease (PD) with reportedly effective results. However, the physiological and biological mechanism behind its effectiveness is still unknown. In the present study, different frequencies of chronic EA stimulation (0, 2, 100 Hz) were tested in a partially lesioned rat model of PD which was induced by transection of the medial forebrain bundle (MFB). After 24 sessions of EA stimulation (28 days after MFB transection), dopaminergic neurons in the ventral midbrain were examined by immunohistochemical staining, and brain-derived neurotrophic factor (BDNF) mRNA levels in ventral midbrain were measured by in situ hybridization. The results show a marked decrease of dopaminergic neurons on the lesioned side of the substantia nigra (SN) comparing with the unlesioned side. Zero Hz and 2 Hz EA stimulation had no effect on the disappearance of dopaminergic neurons. However, after 100 Hz EA, about 60% of the tyrosine hydroxylase (TH)-positive neurons remained on the lesioned side of the SN. In addition, levels of BDNF mRNA in the SN and ventral tegmental area (VTA) of the lesioned side were significantly increased in the 100 Hz EA group, but unchanged in the 0 and 2 Hz groups. Our results suggest that long-term high-frequency EA is effective in halting the degeneration of dopaminergic neurons in the SN and up-regulating the levels of BDNF mRNA in the subfields of the ventral midbrain. Activation of endogenous neurotrophins by EA may be involved in the regeneration of the injured dopaminergic neurons, which may underlie the effectiveness of EA in the treatment of PD.     

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)     

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.     

Microtopography of methionine-enkephalin, dopamine and noradrenaline in the ventral mesencephalon of human control and Parkinsonian brains

The topographical distributions of Met-enkephalin, dopamine and noradrenaline were determined in serial frontal sections of human substantia nigra (pars compacta and pars reticulata) and ventral tegmental area. Met-enkephalin was identified by Biogel and thin layer chromatography and assayed by a specific radioimmunoassay. In the substantia nigra (pars compacta and pars reticulata), the levels of Met-enkephalin increased progressively from the rostal to the caudal part of the structure. This pattern closely resembled that of dopamine levels, particularly in the pars compacta. Noradrenaline levels in the substantia nigra and those of Met-enkephalin, dopamine, and noradrenaline in the ventral tegmental area, exhibited only limited fluctuations from the anterior to the posterior part of each structure.Highly significant decreases in Met-enkephalin, dopamine and noradrenaline levels were observed in the substantia nigra and ventral tegmental area of Parkinsonian brains. This observation, together with the close topographical association of dopamine and Met-enkephalin in the substantia nigra, further supports the likely existence of important functional relationships between dopaminergic and enkephalinergic neurons in the human brain.     

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.     

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.     

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.     

Trophic factor distribution predicts functional recovery in parkinsonian monkeys

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival, growth, and regeneration of dopamine neurons in the midbrain that degenerate in Parkinson's disease. However, translating successful animal studies into effective clinical therapy for Parkinson's disease has proved difficult. In this article, using pulsed infusion for convection-enhanced delivery of GDNF, we have analyzed two variables hypothesized to be important for achieving efficacy: dose and GDNF distribution in the target tissue. Motor functions were significantly improved in rhesus monkeys with unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism that received midbrain infusion of GDNF for 10 weeks. The volume of distribution of GDNF in the five trophic factor recipients varied more than fivefold, from 59 to 325 mm3, and significantly correlated with motor function improvements. Significant increases were evident in the number of midbrain dopamine neurons immunopositive for tyrosine hydroxylase in both the substantia nigra and ventral tegmental area. Based on neurochemical and quantitative morphological measures, GDNF administration promoted recovery of both the nigrostriatal and ventral tegmental area-nucleus accumbens dopaminergic pathways without producing evident side effects. Increasing the dose threefold did not increase efficacy, suggesting that after achieving a critical threshold, GDNF tissue distribution is more important than dose for trophic stimulation of dopamine neurons.     

Acetylcholine–Dopamine Interactions in the Pathophysiology and Treatment of CNS Disorders

Dopaminergic neurons in the substantia nigra pars compacta and ventral tegmental area of the midbrain form the nigrostriatal and mesocorticolimbic dopaminergic pathways that, respectively, project to dorsal and ventral striatum (including prefrontal cortex). These midbrain dopaminergic nuclei and their respective forebrain and cortical target areas are well established as serving a critical role in mediating voluntary motor control, as evidenced in Parkinson's disease, and incentive‐motivated behaviors and cognitive functions, as exhibited in drug addiction and schizophrenia, respectively. Although it cannot be disputed that excitatory and inhibitory amino acid‐based neurotransmitters, such as glutamate and GABA, play a vital role in modulating activity of midbrain dopaminergic neurons, recent evidence suggests that acetylcholine may be as important in regulating dopaminergic transmission. Midbrain dopaminergic cell tonic and phasic activity is closely dependent upon projections from hindbrain pedunculopontine and the laterodorsal tegmental nuclei, which comprises the only known cholinergic inputs to these neurons. In close coordination with glutamatergic and GABAergic activity, these excitatory cholinergic projections activate nicotinic and muscarinic acetylcholine receptors within the substantia nigra and ventral tegmental area to modulate dopamine transmission in the dorsal/ventral striatum and prefrontal cortex. Additionally, acetylcholine‐containing interneurons in the striatum also constitute an important neural substrate to provide further cholinergic modulation of forebrain striatal dopaminergic transmission. In this review, we examine neurological and psychopathological conditions associated with dysfunctions in the interaction of acetylcholine and dopamine and conventional and new pharmacological approaches to treat these disorders.     

Selective loss of subpopulations of ventral mesencephalic dopaminergic neurons in the monkey following exposure to MPTP

Tyrosine hydroxylase immunohistochemical examination of the mesencephalon of severely parkinsonian MPTP-treated macaque fascicularis monkeys revealed a marked loss of substantia nigra pars compacta (SNc) neurons in both medial and central portions of the nucleus with a relative sparing of neurons in the dorsal-most portions of the substantia nigra. These animals also sustained 20–65% loss of neurons in the substantia nigra pars lateralis area, ventral tegmental area (A-10), and the retrorubral area (A-8 cell group, and the parabrachialis pigmentosus region). These animals all had extreme striatal dopamine depletions. A monkey which received several small doses of MPTP and yet remained asypptomatic for a motor disorder (although it had demonstrable behavioral performance deficits) had only a loss only ventral SNc neurons, with no appreciable cells in associated ventral mesencephalic dopamine areas and no loss of striatal dopamine. These data suggest that the effects of MPTP are not as selective as originally thought and, more importantly, indicate that MPTP-induced parkinsonism in the primate may be more analogous to idiopathic Parkinson's disease, where cells other than SNc cells are affected. Furthermore, the present findings suggest that only certain mesencephalic dopamine neurons are susceptible to MPTP-induced damage. The unique characteristics of these neurons need to be elucidated.     

Characterization of dopaminergic midbrain neurons in a DBH:BDNF transgenic mouse.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the survival and differentiation of central nervous system neurons, including dopaminergic cells in culture. To determine whether BDNF might play a role in the development of dopaminergic neurons in vivo, we used a previously characterized transgenic mouse (DBH:BDNF) that overexpresses BDNF in adrenergic and noradrenergic neurons as a result of fusion of the BDNF gene to the dopamine beta-hydroxylase (DBH) gene promoter. We quantified dopaminergic neuronal profiles at four midbrain coronal levels and compared DBH:BDNF transgenic animals with wild-type mice of the same genetic background. Analysis of sections immunostained with tyrosine hydroxylase (TH) showed that the mean number of dopaminergic neurons in the four selected midbrain sections was 52% greater (one-way analysis of variance, P < 0.0005) in transgenic mice (2,165 +/- 55 S. E.M., n = 4) than in control mice (1,428 +/- 71 S.E.M., n = 4). The increase in dopaminergic neuron profile count in DBH:BDNF transgenic animals was confirmed by analysis of the pars compacta of the substantia nigra on Nissl-stained sections. Surface area of the reference region of interest containing TH-immunoreactive neurons was similar in transgenic and control mice. Regional analysis of different midbrain areas containing dopaminergic neurons suggested that the increase in cell profile count occurs in a relatively homogeneous manner. Comparison of TH-immunoreactive cell size showed a tendency for smaller neurons in transgenic animals, but the difference was not statistically significant. We conclude that DBH:BDNF transgenic mice show increased number of TH-immunoreactive cells in the midbrain. We propose that BDNF rescues dopaminergic neurons from the perinatal period of developmental cell death as a consequence of increased anterograde transport of the neurotrophin via the coeruleonigral projection.     

Subsets of midbrain dopaminergic neurons in monkeys are distinguished by different levels of mRNA for the dopamine transporter: Comparison with the mRNA for the D2 receptor,tyrosine hydroxylase and calbindin immunoreactivity

The midbrain dopamine system can be divided into two groups of cells based on chemical characteristics and connectivity. The dorsal tier neurons, which include the dorsal pars compacta and the ventral tegmental area, are calbindin positive, and project to the shell of the nucleus accumbens. The ventral tier neurons are calbindin-negative and project to the sensonmotor striatum. This study examined the distribution of the mRNAs for the dopamine transporter molecule (DAT) and the D₂ receptor in the midbrain of monkeys by using in situ hybridization. The distribution patterns were compared to that of tyrosine hydroxylase and calbindin immunohistochemistry. The results show that high levels of hybridization for DAT and the D₂ receptor mRNA are found in the ventral tier, calbindin-negative neurons and relatively low levels are found in the dorsal, calbindin-positive tier. Within the dorsal tier, the dorsal substantia nigra pars compacta has the least amount of both messages. These results show that in monkeys, the ventral tegmental area and the dorsal pars compacta form a dorsal continuum of dopamine neurons which express lower levels of mRNA for DAT and D₂ receptor than the ventral tier. DAT has been shown to be involved in the selective neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Different levels of DAT mRNA and calbindin may explain the differential effects of MPTP neurotoxicity. © 1995 Wiley-Liss Inc.     

Anatomical and functional reconstruction of the nigrostriatal pathway by intranigral transplants

The main transplantation strategy in Parkinson's disease has been to place dopaminergic grafts not in their ontogenic site, the substantia nigra, but in their target area, the striatum with contrasting results. Here we have used green fluorescent protein transgenic mouse embryos as donors of ventral mesencephalic cells for transplantation into the pre-lesioned substantia nigra of an adult wild-type host. This allows distinguishing the transplanted cells and their projections from those of the host. Grafted cells integrated within the host mesencephalon and expressed the dopaminergic markers tyrosine hydroxylase, vesicular monoamine transporter 2 and dopamine transporter. Most of the dopaminergic cells within the transplant expressed the substantia nigra marker Girk2 while a lesser proportion expressed the ventral tegmental area marker calbindin. Mesencephalic transplants developed projections through the medial forebrain bundle to the striatum, increased striatal dopamine levels and restored normal behavior. Interestingly, only mesencephalic transplants were able to restore the nigrostriatal projections as dopamine neurons originating from embryonic olfactory bulb transplants send projections only in the close vicinity of the transplantation site that did not reach the striatum. Our results show for the first time the ability of intranigral foetal dopaminergic neurons grafts to restore the damaged nigrostriatal pathway in adult mice. Together with our previous findings of efficient embryonic transplantation within the pre-lesioned adult motor cortex, these results demonstrate that the adult brain is permissive to specific and long distance axonal growth. They further open new avenues in cell transplantation therapies applied for the treatment of neurodegenerative disorders such as Parkinson's disease.