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

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

Dopamine transporter messenger RNA in Parkinson's disease and control substantia nigra neurons

Dopamine transporter messenger RNA (mRNA) expression was assessed by in situ hybridization over individual pigmented neurons from the substantia nigra pars compacta in midbrain sections from 7 parkinsonian and 7 age-matched, neurologically normal patients. In the normal control brains, high levels of expression of dopamine transporter mRNA were noted over pigmented neurons in the substantia nigra pars compacta; neurons in the adjacent nucleus paranigralis of the ventral tegmental area displayed less hybridization. Nigra compacta neurons surviving in brains of patients with Parkinson's disease displayed only 57% of the dopamine transporter mRNA hybridization intensity displayed by nigral neurons in normal control brains. The disease-related decrease in the apparent level of dopamine transporter mRNA expression in remaining neurons could reflect neuronal dysfunction. Conceivably, it might also reflect differential vulnerability of those neurons that initially expressed higher levels of this transporter to the insult of parkinsonism.     

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.     

Dopamine transporter mRNA: dense expression in ventral midbrain neurons.

Oligonucleotides and a full-length cDNA encoding a functional dopamine transporter (DAT1) hybridize to a 3.7 kb mRNA that is concentrated in mRNA prepared from midbrain and absent in specimens from cerebellum or cerebral cortex. In situ hybridization reveals substantial hybridization densities overlying neurons of the substantia nigra, pars compacta, and the parabrachialis pigmentosus region of the ventral tegmental area (VTA). Neurons in the linear and paranigral VTA regions display lower levels of expression. Preliminary studies in arcuate neurons suggest modest hybridization. Different dopaminergic cell groups display different levels of DAT1 dopamine transporter expression.     

Differential expression patterns of mGluR1 alpha in monkey nigral dopamine neurons

The expression pattern of metabotropic glutamate receptor 1alpha (mGluR1alpha) was immunohistochemically investigated in substantia nigra dopaminergic neurons of the macaque monkey. In normal monkeys, mGluR1alpha immunoreactivity was weakly observed in the dorsal tier of the substantia nigra pars compacta (SNc-d) where calbindin-D28k-containing dopaminergic neurons invulnerable to parkinsonian degeneration are specifically located. On the other hand, mGluR1alpha was strongly expressed in the ventral tier of the substantia nigra pars compacta (SNc-v). In monkeys treated with the parkinsonism-inducing drug, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), mGluR1alpha expression was decreased in dopaminergic neurons in the SNc-v that were spared its toxic action. These results suggest that mGluR1alpha expression may be involved at least partly in the vulnerability of dopaminergic neurons to parkinsonian insults.     

Dopaminergic mRNA expression in the intact sebstantia nigra of unilaterally 6-OHDA-lesioned and grafted rats: an in situ hybridization study

Summary. The present study was performed to investigate the influence of intrastriatal fetal mesencephalic grafts on dopaminergic mRNA expression in the non-lesioned substantia nigra pars compacta of unilaterally 6- hydroxydopamine-lesioned rats. The expression of dopamine transporter mRNA, synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA was assessed in adjacent cryostat sections using in situ hybridization. Rotational behavior induced by apomorphine and amphetamine as well as hybridization of striatal sections cut at the grafting coordinates were used to prove the functional recovery and the presence of grafted cells, respectively. After grafting, the number of rotations was decreased and hybridization signals overlying cells in the grafted striatum were detected. Mean grain densities overlying labeled neurons in the substantia nigra pars compacta of grafted rats were compared to those of shamgrafted rats and revealed differential expression of dopamine transporter mRNA, whereas synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA expression showed no difference. The results will be discussed in relation to previous in vitro and in vivo studies suggesting a reduction of functional dopamine transporter molecules in the contralateral striatum. Received April 25, 2000; accepted August 17, 2000     

Neuropathological changes in the substantia nigra in schizophrenia but not depression

Schizophrenia is a chronic, disabling neuropsychiatric disorder characterised by positive, negative and cognitive symptoms. The aetiology is not known, although genetic, imaging and pathological studies have implicated both neurodevelopmental and neurodegenerative processes. The substantia nigra is a basal ganglia nucleus responsible for the production of dopamine and projection of dopaminergic neurons to the striatum. The substantia nigra is implicated in schizophrenia as dopamine has been heavily implicated in the dopamine hypothesis of schizophrenia and the prevalent psychotic symptoms and the monoamine theory of depression, and is a target for the development of new therapies. Studies into the major dopamine delivery pathways in the brain will therefore provide a strong base in improving knowledge of these psychiatric disorders. This post-mortem study examines the cytoarchitecture of dopaminergic neurons of the substantia nigra in schizophrenia (n = 12) and depression (n = 13) compared to matched controls (n = 13). Measures of nucleolar volume, nuclear length and nuclear area were taken in patients with chronic schizophrenia and major depressive disorder against matched controls. Astrocyte density was decreased in schizophrenia compared to controls (p = 0.030), with no change in oligodendrocyte density observed. Significantly increased nuclear cross-sectional area (p = 0.017) and length (p = 0.021), and increased nucleolar volume (p = 0.037) in dopaminergic neurons were observed in schizophrenia patients compared with controls, suggesting nuclear pleomorphic changes. No changes were observed in depression cases compared to control group. These changes may reflect pathological alterations in gene expression, neuronal structure and function in schizophrenia.     

Regional dopamine transporter gene expression in the substantia nigra from control and Parkinson's disease brains

OBJECTIVE—To test thehypothesis that differential regional dopamine transporter (DAT) geneexpression may underlie the selective vulnerability of certain nigraldopaminergic neurons in Parkinson's disease, DAT mRNA expression wasexamined in neuronal subpopulations of human postmortem ventralmesencephalon from patients with Parkinson's disease and controls.
METHODS—Radioactivein situ hybridisation histochemistry using a polymerase chain reactionderived ribonucleotide probe for DAT was performed on sections ofventral mesencephalon from the brains of five donors with no history ofneurological illness and from five patients with pathologicallyestablished Parkinson's disease. The number of silver grains overlyingmelanised neurons from the paranigral nucleus, dorsal and ventral tier,and pars lateralis of the substantia nigra pars compacta were comparedwith each other and to background labelling by using a one wayfactorial analysis of variance (ANOVA) with a significance level of5%.
RESULTS—In controlbrains, there was intense DAT mRNA expression in the ventral midbrainwith no significant difference in mRNA concentrations among the four regions studied. In theParkinson's disease brains, there was an overall decrease in theintensity of DAT mRNA expression in the surviving dopaminergic neurons.There were no significant differences in signal between regions ineither the control or parkinsonian brains.
CONCLUSION—Takentogether, these findings do not support the hypothesis thatdifferential regional DAT gene expression underlies the selectivevulnerability of certain nigral dopaminergic neurons in Parkinson'sdisease, as the vulnerable neurons of the substantia nigra parscompacta do not express more DAT mRNA than the resistant paranigral neurons.

     

Substantia nigra opiate receptors on basal ganglia efferents

Many behavioral effects of opiate narcotics and peptides have been linked to effects on dopamine neurons originating in the substantia nigra pars compacta and ventral tegmental area. Selective brain lesions were combined with quantitative autoradiography to determine whether opiate receptors are on dopaminergic somata and/or processes in the substantia nigra pars compacta and ventral tegmental area. 6-Hydroxydopamine lesions that eliminated dopamine neurons produced little change in the pattern or density of [³H]-naloxone binding in the substantia nigra pars compacta or ventral tegmental area. Radiofrequency lesions of the internal capsule or globus pallidus and kainic acid lesions of the striatum markedly decreased [³H]-naloxone binding in the pars compacta and pars reticulata. These results are consistent with a dense distribution of opiate receptors on pallido-nigral and/or striato-nigral fibers and strengthen the likelihood that local effects of opiates on dopamine function in the nigrostriatal pathway are mediated indirectly by actions on nondopaminergic processes.     

Correlation of striatal fluorodopa uptake in the MPTP Monkey with dopaminergic indices

Striatal ¹⁸F-6-fluorodopa (FD) uptake constants were measured by positron emission tomography in (1) normal cynomolgus monkeys and (2) a series of cynomolgus and rhesus monkeys that had received intracarotid infusions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). After the animals were killed, the number and average size of dopaminergic neurons in the substantia nigra pars compacta were measured. Striatal levels of dopamine and its metabolites, and the striatal activities of the dopaminergic synthetic enzymes, were also determined. The striatal FD uptake constants showed highly significant positive correlations with both number and size of dopaminergic neurons, indicating atrophy of surviving neurons in MPTP-treated animals. The uptake constants also showed significant positive correlations with striatal levels of dopamine, total catecholamines, and the activities of the synthetic enzymes. Both histochemical and biochemical data on tyrosine hydroxylase suggested some contralateral enzyme loss in these MPTP-treated monkeys, as well as decreased enzyme activity in surviving neurons on the lesioned side. However, residual enzyme activities were apparently not rate limiting to striatal FD uptake. It is concluded that PET-FD measurements by positron emission tomography provide a good index of the integrity of the nigrostriatal pathway.     

Does cholecystokinin colocalize with dopamine in the human substantia nigra?

In situ hybridization was used to examine the distribution of neurons containing cholecystokinin (CCK) mRNA in human, monkey and rat brain. In rat and monkey brain CCK mRNA was visualized in the substantia nigra pars compacta and in the ventral tegmental area. The dopaminergic cell bodies in the human substantia nigra did not however show detectable amounts of CCK mRNA. Low levels of CCK mRNA were observed in the nucleus paranigralis, the human equivalent of the rodent ventral tegmental area. High levels of CCK mRNA were seen in other regions of the same brains including the cortex and the hippocampus. Thus, the adult human substantia nigra dopaminergic cells, in contrast to primate and rodent substantia nigra, do not express CCK. These results question the hypothesis of an involvement of CCK in the regulation of dopaminergic neurons and help to explain the absence of decreased CCK levels in the caudate and putamen of Parkinson's disease victims.     

Glutamatergic pallidothalamic projections and their implications in the pathophysiology of Parkinson's disease

GABAergic projections emitted from the entopeduncular nucleus (ENT) and the substantia nigra pars reticulata (SNr) innervate different thalamic nuclei and they are known to be hyperactive after dopaminergic depletion. Here we show that isoform 2 of the vesicular glutamate transporter (VGLUT2) is expressed by neurons in the ENT nucleus but not in the SNr. Indeed, dual in situ hybridization demonstrated that the ENT nucleus contains two different subpopulations of projection neurons, one single-expressing GAD65/67 mRNAs and another one that co-expresses either of the GAD isoforms together with VGLUT2 mRNA. Unilateral dopaminergic depletion induced marked changes in pallidothalamic-projecting neuron gene expression, resulting in increased expression of GAD65/67 mRNAs together with a clear down-regulation of VGLUT2 mRNA expression. Our results indicate that the increased thalamic inhibition typical of dopamine depletion might be explained by a synergistic effect of increased GABA outflow coupled to decreased glutamate levels, both neurotransmitters coming from ENT neurons.     

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.     

Inhibiting BDNF expression by antisense oligonucleotide infusion causes loss of nigral dopaminergic neurons

Brain derived neurotrophic factor (BDNF) expression is significantly reduced in the Parkinson's disease substantia nigra. This neurotrophin has potent affects on dopaminergic neuron survival protecting them from the neurotoxins MPTP and 6-hydroxydopamine (6-OHDA) commonly used to create animal models of Parkinson's disease and also promoting dopaminergic axonal sprouting. In this study, we demonstrate that an antisense oligonucleotide infusion (200 nM for 28 days) to prevent BDNF production in the substantia nigra of rats mimics many features of the classical animal models of Parkinson's disease. 62% of antisense treated rats rotate (P < or = 0.05) in response to dopaminergic receptor stimulation by apomorphine. 40% of substantia nigra pars compacta tyrosine hydroxylase immunoreactive neurons are lost (P < or = 0.00001) and dopamine uptake site density measured by (3)H-mazindol autoradiography is reduced by 34% (P < or = 0.005). Loss of haematoxylin and eosin stained nigral neurons is significant (P < or = 0.0001) but less extensive (34%). These observations indicate that loss of BDNF expression leads both to down regulation of the dopaminergic phenotype and to dopaminergic neuronal death. Therefore, reduced BDNF mRNA expression in Parkinson's disease substantia nigra may contribute directly to the death of nigral dopaminergic neurons and the development of Parkinson's disease.     

Non-dopaminergic nigrostriatal pathway

The nigrostriatal projection was studied with a retrograde tracing method (Evans blue, EB) combined with a technique for dopamine histofluorescence. The study, realized in control rats and in animals with 6-hydroxydopamine-induced lesions of the dopaminergic pathway, yielded the following results.

(1) In 3 control rats injected with 0.2 μl of a 10% solution of EB in thecenter of the caudate-putamen 1 mm anterior to the globus pallidus, 96% of all substantia nigra neurons retrogradely labelled with the dye contained dopamine fluorescence. The remaining ones (average 350 per brain) were devoid of dopamine fluorescence and predominantly found in the posterior 75% of the substantia nuigra. These last cells were confined to the upper-half of the pars reticulata.

(2) In a series of 6 animals, the cytotoxic agent 6-hydroxydopamine was injected in various locations in the vicinity of either the substantia nigra ir the nigrostriatal tract 12–15 days prior to the injections of 0.2 μl of EB in the same striatal locations as in the controls. Despite a reduction of up to 85% in the number of dopaminergic cell bodies, the substantia nigra of these rats contained the same average number of EB-labelled neurons devoid of dopamine fluorescence.

(3) Eight rats received smaller injections (0.1 μl) of EB in various striatal sites and in tqo further cases such injections were placed in the globus pallidus to determine more accurately the anatomical location of the dopamine-negative nigral neurons retrogradely labelled with the dye. Following the striatal injections, these cells were found mostly in the upper-half of the pars reticulata and were arranged in longitudinally oriented clusters whose mediolateral location depended on the striatal injection site.

Following the pallidal injections, retrogradely labelled neruons devoid of dopamine fluorescence were found in greater numbers and were located in all areas of the pars reticulata. The possibility of retrograde labelling of some nigrothalamic neurons was not entirely ruled out in these two cases.

(4) Finally 6 rats received 0.1 μl injections of EB in various parts of the parietal cortex. In these cases the substantia nigra did not contain any EB-positivedopamine-negative neurons.

These results are interpreted as evidence in support of the existence of a topographically organized non-dopaminergic nigrostriatal projection.

GM1 ganglioside treatment partially reverses the nigrostriatal dopamine defect in the weaver mutant mouse

The weaver mutation in the mouse is a developmental disorder characterized by cerebellar atrophy as well as decreased numbers of substantia nigra dopaminergic neurons and a striatal dopamine loss. Since the nigrostriatal dopamine loss occurs postnatally, the present study was performed to determine whether early intervention with G_M1 ganglioside could alter the extent of this dopamine loss. Weaver mice that received injections of G_M1 ganglioside (30 mg/kg) daily, beginning at 7–10 days of age, had significantly higher striatal dopamine levels and significantly more tyrosine hydroxylase-positive substantia nigra pars compacta neurons than weaver mice that received only daily saline injections. These results show that G_M1 treatment can alter at least some aspects of this inherited developmental disorder. If the weaver defect is related to a deprivation of trophic support for certain midbrain dopaminergic neurons, the presence of G_M1 may be able to enhance the survival of these neurons.     

Absence of prostate apoptosis response-4 protein in substantia nigra of Parkinson's disease autopsies

The morphological evidence for apoptosis of dopaminergic neurons in Parkinson's disease (PD) remains a conflicting issue. The present study examined autopsy material containing substantia nigra and putamen of PD (n=7) and control subjects (n=5) for the expression of prostate apoptosis response-4 (Par-4) protein, a protein expressed by apoptotic cells. Par-4 was not detected in the substantia nigra pars compacta. By contrast, duodenal enterocytes, which served as positive controls and are known to be apoptotic, profoundly expressed Par-4. The present study is in agreement with previous studies of the substantia nigra pars compacta in PD, which failed to detect molecules expressed by apoptotic cells. The absence of Par-4 immunoreactivity suggests that death of dopaminergic neurons in PD follows a degenerative pathway that circumvents the induction of Par-4.     

Combination of alkaline phosphatase in situ hybridization with immunohistochemistry: colocalization of calretinin-mRNA with calbindin and tyrosine hydroxylase immunoreactivity in rat substantia nigra neurons

We describe a method to combine non-radioactive in situ hybridization using alkaline phosphatase (AP) labelled oligonucleotide-probes with immunohistochemistry on the same thin paraffin section. The simultaneous detection of calretinin-mRNA and calbindin- or tyrosine hydroxylase-like immunoreactivity in neurons of rat substantia nigra, pars compacta, was used as a test system to develop the method. Brains were fixed by perfusion with 4% paraformaldehyde and embedded in paraffin. Five-μm-thick sections were processed for non-radioactive in situ hybridization with a 33-base alkaline phosphatase conjugated synthetic oligonucleotide complementary to calretinin mRNA. After hybridization and colour reaction to visualize calretinin mRNA, sections were incubated with antibodies against calbindin D_28K or tyrosine hydroxylase. Immunoreaction was visualized using the avidin-biotin-complex-technique and diaminobenzidine. As the colour of both reaction products differ markedly, the distribution of calretinin mRNA-containing neurons (purple-blue, alkaline phosphatase product) and calbindin/tyrosine hydroxylase immunopositive cells (brown peroxidase product) could be differentiated easily on the same section. Calbindin- and tyrosine hydroxylase-like immunoreactivity was found in the majority of calretinin mRNA-containing cells within the substantia nigra, pars compacta, indicating that in this nucleus a proportion of the dopaminergic neurons contain both calcium binding proteins calbindin and calretinin. In conclusion, non-radioactive in situ hybridization using alkaline phosphatase labelled oligonucleotide probes can be readily combined with immunohistochemistry.