Substantia Nigra Compacta Research Articles

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons – inhibition results in a schizophrenia‐like syndrome in transgenic mice

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.

Dopamine D2 receptor binding, Drd2 expression and the number of dopamine neurons in the BXD recombinant inbred series: genetic relationships to alcohol and other drug associated phenotypes.

It has not been established to what extent the natural variation in dopamine systems contribute to the variation in ethanol response. The current study addresses this issue by measuring D2 dopamine (DA) receptor binding, the expression of Drd2, the number of midbrain DA neurons in the BXD recombinant inbred (RI) series and then compares these strain means with those previously reported for a variety of ethanol and other drug-related phenotypes. Data were collected for 21 to 23 of the BXD RI strains and the parental strains. D2 DA receptor autoradiography was performed using 125I-epidepride as the ligand [ Kanes S, Dains K, Cipp L, Gatley J, Hitzemann B, Rasmussen E, Sanderson S, Silverman S, Hitzemann R (1996) Mapping the genes for haloperidol-induced catalepsy. J Pharmacol Exp Ther 277:1016-1025]. Drd2 expression was measured using the Affymetrix oligoarray system. Immunocytochemical techniques were used to determine the number of midbrain DA neurons [Hitzemann B, Dains K, Hitzemann R (1994) Further studies on the relationship between dopamine cell density and haloperidol response. J Pharmacol Exp Ther 271:969-976]. The range of difference in receptor binding for the RI strains was approximately 2-fold in all regions examined, the core, the shell of the nucleus accumbens (NAc) and the dorsomedial caudate-putamen (CPu); heritability in all regions was moderate--(h2 approximately 0.35). Drd2 expression in forebrain samples from the RI and parental strains ranged 1.5- to h2-fold and was moderate-0.47. Variation in the number of tyrosine hydroxylase (TH) positive neurons was moderate, 41% and 26% and h2 was low--0.19 and 0.15 for the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Significant correlations were found between D2 DA receptor binding and the low dose (1.33 g/kg) ethanol stimulant response. (p < 0.002) and between expression and conditioned place preference (CPP) (p < 0.0005). No significant correlations were detected between ethanol preference and either receptor binding or Drd2 expression; however, a significant correlation was found between preference and Ncam expression. Ncam is approximately 0.2 Mb from Drd2. Overall, the data suggest ethanol preference and CPP are associated with the expression of Drd2 or closely linked genetic loci.

Abnormal expression of the G‐protein‐activated inwardly rectifying potassium channel 2 (GIRK2) in hippocampus, frontal cortex, and substantia nigra of Ts65Dn mouse: A model of Down syndrome

Ts65Dn, a mouse model of Down syndrome (DS), demonstrates abnormal hippocampal synaptic plasticity and behavioral abnormalities related to spatial learning and memory. The molecular mechanisms leading to these impairments have not been identified. In this study, we focused on the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) gene that is highly expressed in the hippocampus region. We studied the expression pattern of GIRK subunits in Ts65Dn and found that GIRK2 was overexpressed in all analyzed Ts65Dn brain regions. Interestingly, elevated levels of GIRK2 protein in the Ts65Dn hippocampus and frontal cortex correlated with elevated levels of GIRK1 protein. This suggests that heteromeric GIRK1-GIRK2 channels are overexpressed in Ts65Dn hippocampus and frontal cortex, which could impair excitatory input and modulate spike frequency and synaptic kinetics in the affected regions. All GIRK2 splicing isoforms examined were expressed at higher levels in the Ts65Dn in comparison to the diploid hippocampus. The pattern of GIRK2 expression in the Ts65Dn mouse brain revealed by in situ hybridization and immunohistochemistry was similar to that previously reported in the rodent brain. However, in the Ts65Dn mouse a strong immunofluorescent staining of GIRK2 was detected in the lacunosum molecular layer of the CA3 area of the hippocampus. In addition, tyrosine hydroxylase containing dopaminergic neurons that coexpress GIRK2 were more numerous in the substantia nigra compacta and ventral tegmental area in the Ts65Dn compared to diploid controls. In summary, the regional localization and the increased brain levels coupled with known function of the GIRK channel may suggest an important contribution of GIRK2 containing channels to Ts65Dn and thus to DS neurophysiological phenotypes.

Modulation of excitatory synaptic transmission by endogenous glutamate acting on presynaptic group II mGluRs in rat substantia nigra compacta

Excitatory synaptic inputs from the subthalamic nucleus (STN) have been proposed to underlie burst firing of substantia nigra pars compacta (SNc) dopamine (DA) neurons in Parkinson's disease. Given the potential importance of the STN-SNc synapse in health and disease, our goal was to study how transmission at this synapse is regulated. We tested the hypothesis that neurotransmission at STN-SNc synapses is tonically inhibited by endogenous glutamate acting on presynaptic group II metabotropic glutamate receptors (mGluRs). By using whole-cell recording techniques in brain slices, we examined the effect of LY341495, a mGluR antagonist that is most potent at group II mGluRs, on excitatory postsynaptic currents (EPSCs) that either were evoked in SNc DA neurons by stimulation of the STN or were spontaneously occurred in the presence of tetrodotoxin (miniature EPSCs; mEPSCs). LY341495 increased the evoked EPSC amplitude and mEPSC frequency without changing mEPSC amplitude. In contrast, the group III mGluR antagonist UBP1112 failed to increase the evoked EPSC amplitude. An elevation of extracellular glutamate concentration by a glutamate transporter inhibitor, TBOA, suppressed the evoked EPSCs. LY341495, but not UBP1112, partially reversed the TBOA action. The modulations of EPSCs by TBOA and LY341495 were associated with changes in paired-pulse facilitation ratio. Furthermore, TBOA decreased mEPSC frequency, which was partially reversed by LY341495, without affecting mEPSC amplitude. The results indicate that presynaptic group II mGluRs at STN-SNc synapses appear to be partially activated by a basal level of extracellular glutamate and able to sense the change in extracellular glutamate concentration, subsequently modulating synaptic glutamate release.

Molecular and cellular alterations in the Pitx3-deficient midbrain dopaminergic system

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra compacta (SNc). In order to provide insights into adaptive mechanisms of the mDA system in pathology, specific molecular and cellular parameters of the mDA system were studied in Pitx3-deficient Aphakia (ak) mice, which suffer from severe developmental failure of SNc mDA neurons. Here, we demonstrate differential changes in striatal gene expression, reflecting the specific neuronal loss in these mice. In addition, the neuronal activity of remaining mDA neurons in the ventral tegmental area (VTA) was significantly increased in ak mice. In conclusion, ak mice display specific molecular and cellular alterations in the mDA system that provide new insights in compensatory mechanisms present in mDA-associated disorders such as PD.

Role of nitric oxide on motor behavior.

The present review paper describes results indicating the influence of nitric oxide (NO) on motor control. Our last studies showed that systemic injections of low doses of inhibitors of NO synthase (NOS), the enzyme responsible for NO formation, induce anxiolytic effects in the elevated plus maze whereas higher doses decrease maze exploration. Also, NOS inhibitors decrease locomotion and rearing in an open field arena. These results may involve motor effects of this compounds, since inhibitors of NOS, NG-nitro-L-arginine (L-NOARG), N(G)-nitro-L-arginine methylester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), and 7-Nitroindazole (7-NIO), induced catalepsy in mice. This effect was also found in rats after systemic, intracebroventricular or intrastriatal administration. Acute administration of L-NOARG has an additive cataleptic effect with haloperidol, a dopamine D2 antagonist. The catalepsy is also potentiated by WAY 100135 (5-HT1a receptor antagonist), ketanserin (5HT2a and alfal adrenergic receptor antagonist), and ritanserin (5-HT2a and 5HT2c receptor antagonist). Atropine sulfate and biperiden, antimuscarinic drugs, block L-NOARG-induced catalepsy in mice. L-NOARG subchronic administration in mice induces rapid tolerance (3 days) to its cataleptic effects. It also produces cross-tolerance to haloperidol-induced catalepsy. After subchronic L-NOARG treatment there is an increase in the density NADPH-d positive neurons in the dorsal part of nucleus caudate-putamen, nucleus accumbens, and tegmental pedunculupontinus nucleus. In contrast, this treatment decreases NADPH-d neuronal number in the substantia nigra compacta. Considering these results we suggest that (i) NO may modulate motor behavior, probably by interfering with dopaminergic, serotonergic, and cholinergic neurotransmission in the striatum; (ii) Subchronic NO synthesis inhibition induces plastic changes in NO-producing neurons in brain areas related to motor control and causes cross-tolerance to the cataleptic effect of haloperidol, raising the possibility that such treatments could decrease motor side effects associated with antipsychotic medications. Finally, recent studies using experimental Parkinson's disease models suggest an interaction between NO system and neurodegenerative processes in the nigrostriatal pathway. It provides evidence of a protective role of NO. Together, our results indicate that NO may be a key participant on physiological and pathophysiological processes in the nigrostriatal system.

Melatonin protects against MPTP/MPP+‐induced mitochondrial DNA oxidative damage in vivo and in vitro

The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine ion (MPP(+)) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA oxidative damage. In SH-SY5Y cells, MPP(+) (1 mm) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 microm) preincubation significantly attenuated MPP(+)-induced mtDNA oxidative damage. Furthermore, MPP(+) time-dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Psim) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP(+) caused cell death in 49% of the control. However, melatonin prevented MPP(+)-induced mtOFR generation and Psim collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP(+)-induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Psim collapse.

Mitochondrial associated metabolic proteins are selectively oxidized in A30P α-synuclein transgenic mice—a model of familial Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra compacta. alpha-Synuclein is strongly implicated in the pathophysiology of PD because aggregated alpha-synuclein accumulates in the brains of subjects with PD, mutations in alpha-synuclein cause familial PD, and overexpressing mutant human alpha-synuclein (A30P or A53T) causes degenerative disease in mice or drosophila. The pathophysiology of PD is poorly understood, but increasing evidence implicates mitochondrial dysfunction and oxidative stress. To understand how mutations in alpha-synuclein contribute to the pathophysiology of PD, we undertook a proteomic analysis of transgenic mice overexpressing A30P alpha-synuclein to investigate which proteins are oxidized. We observed more than twofold selective increases in specific carbonyl levels of three metabolic proteins in brains of symptomatic A30P alpha-synuclein mice: carbonic anhydrase 2 (Car2), alpha-enolase (Eno1), and lactate dehydrogenase 2 (Ldh2). Analysis of the activities of these proteins demonstrates decreased functions of these oxidatively modified proteins in brains from the A30P compared to control mice. Our findings suggest that proteins associated with impaired energy metabolism and mitochondria are particularly prone to oxidative stress associated with A30P-mutant alpha-synuclein.

Differential expression of striatal synaptotagmin mRNA isoforms in hemiparkinsonian rats

Synaptotagmins (Syts) constitute a multi-gene family of 15 putative membrane trafficking proteins. The expression of some of the Syts in the brain might be dopaminergically controlled and thus affected by dopamine depletion in Parkinson’s disease. We used hemiparkinsonian rats to investigate the effects of chronic striatal dopamine depletion and the acute effects of antiparkinsonic drug l-DOPA or D1 agonist (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide (SKF82958) on the levels of striatal Syt I, II, IV, VI, VII, X, XI mRNA isoforms. On the 6-hydroxydopamine (6-OHDA)-lesioned side we observed a nearly total loss of tyrosine hydroxylase (TH), synaptotagmin I, Syt IV, Syt VII and Syt XI mRNA levels in the substantia nigra compacta (SNc). In dopamine-depleted striatum we also found a significant down-regulation Syt II and up-regulation of Syt X mRNA levels that could not be reversed by the acute treatment either with l-DOPA or SKF82958. By contrast, these two drugs induced an increase of Syt IV and Syt VII mRNA levels. A time-course study revealed the highest levels of Syt IV and VII mRNAs to occur at two hours and 12 hours after the treatment with SKF82958, respectively. D1 antagonist (±)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) but not D2 antagonist haloperidol prevented the l-DOPA-driven increase of Syt IV and VII mRNAs. These results imply that synaptic plasticity in response to chronic striatal dopamine depletion involves a complex pattern of changes in striatal Syt mRNA expression. The l-DOPA treatment does not reverse the changes in Syt II and Syt X gene expression, but recruits additional, D1 receptor-mediated changes in Syt IV and Syt VII gene expression. Whether these D1 receptor-mediated changes play a role in the alterations of synaptic transmission that results in the unwanted side effects of chronic l-DOPA treatment in Parkinson’s disease remains to be determined.

Specific pathophysiological functions of JNK isoforms in the brain

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c-JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6-hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment-specific expression of JNK isoforms with 46-kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6-hydroxydopamine was transiently improved in JNK3 ko and c-JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c-JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid-induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c-Jun irrespective on the experimental set-up. This comprehensive study provides novel insights into the context-dependent physiological and pathological functions of JNK isoforms.

Retrograde double‐labeling study of common afferent projections to the dorsal raphe and the nuclear core of the locus coeruleus in the rat

Common afferent projections to the dorsal raphe (DR) and locus coeruleus (LC) nuclei were analyzed in the rat by making paired injections of retrograde tracers, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold) and Fluorogold (FG), into the DR and the nuclear core of the LC. Our results demonstrate that the largest number of double-labeled neurons was located at various preoptic regions including medial preoptic area, lateral preoptic nucleus, and ventrolateral preoptic nucleus. The majority of labeled cells were also observed at the lateral hypothalamus, where the number of labeled cells was comparable to that of neurons at the medial preoptic area or lateral preoptic nucleus. A few double-labeled cells were observed at various hypothalamic regions including anterior, medial tuberal, posterior, and arcuate nuclei, as well as mesencephalic areas including substantia nigra compacta and ventrolateral/lateral periaqueductal gray matter. Cells were also observed at prelimbic/infralimbic prefrontal cortices, diagonal band of Broca, bed nucleus of stria terminalis, and pontine/medullary regions including various raphe nuclei, Barrington's nucleus, gigantocellularis, paragigantocellularis, prepositus hypoglossi, subcoeruleus, and dorsomedial tegmental area. Although electrophysiological studies need to be performed, a large number of double-labeled neurons located at preoptic regions as well as lateral hypothalamus might have their major role in simultaneous control over these monoaminergic nuclei as a means of influencing various sleep and arousal states of the animal. Double-labeled cells at the other locations might be positioned to influence a variety of other functions such as analgesia, cognition, and stress responses.

Effects of repeated inhalation of toluene on ionotropic GABA A and glutamate receptor subunit levels in rat brain

Toluene is a commonly abused solvent found in many industrial and commercial products. The neurobiological effects of toluene remain unclear, but many of them, like those of ethanol, may be mediated by γ-aminobutyric acid (GABA) and glutamate receptors. Chronic ethanol administration has been shown to alter levels of specific subunits for GABA type A (GABA A), N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. However, little is known about the effects of toluene on subunit levels of these receptors. To examine this, rats were exposed to toluene vapors (8000 ppm) or air for 10 days (30 min/day), and afterwards GABA A α1, NR1 and NR2B (NMDA) and GluR1 and GluR2/3 (AMPA) receptor subunit levels were determined in discrete brain regions of these animals by Western blotting. Toluene increased GABA A α1, NR1, NR2B and GluR2/3 subunits in the medial prefrontal cortex and decreased GABA A α1 and NR1 subunits in the substantia nigra compacta. Toluene inhalation produced modest increases in GABA A α1 subunits in the striatum, as well as slight decreases in this subunit in the ventral tegmental area. NR2B subunit levels were also slightly increased within the nucleus accumbens by toluene. These studies show that toluene differentially alters the levels of specific GABAergic and glutamatergic receptor subunits in a regionally selective manner.

Homeobox gene Pitx3 and its role in the development of dopamine neurons of the substantia nigra.

The homeobox gene Pitx3 plays an important part in the development and function of vertebrate midbrain dopaminergic neurons. Re-localization of the genetic defect in the mouse mutant aphakia to the Pitx3 locus, together with the subsequent identification of two deletions causing the gene to be silent, has been the hallmark of several studies into the role of Pitx3. In this review, we summarize the data and reflect on the role of Pitx3 in the development of dopamine neurons in the midbrain. The data indicate that Pitx3 is essential for the survival of dopamine neurons located in the substantia nigra compacta during development. Molecular analysis of the underlying mechanisms might provide new insights for understanding the selective degeneration observed in Parkinson patients.

Change in phase synchronization of local field potentials in anesthetized rats after chronic dopamine depletion

In order to investigate temporal characteristics of oscillatory neural activity and the effect of chronic dopamine depletion on it, local field potentials were measured in anesthetized rats without or with a 6-OHDA lesion at either the ventral tegmental area or the substantia nigra compacta, using a pair of electrodes that were separated by 120 microm. Coupling of neural activity in this mesoscopic scale was measured by a synchronization index that quantified the distribution of differences in instantaneous phase between the two potentials recorded. Phase synchrony was significantly stronger at deep basal ganglia sites, more so than at cortical sites, over a gamma range (30-120 Hz) in normal rats. After chronic dopamine depletion, this synchrony was no longer observable, especially after a substantia nigra lesion, while there was an increase in phase synchrony in the cortex at a lower frequency band (10 Hz). These findings are consistent with previously reported findings that the effect of dopaminergic innervation on oscillatory neural activity varies from synchronizing to desynchronizing, depending on the structure innervated and the frequency of the oscillation.

Alterations in the expressions of mRNA for GDNF and its receptors in the ventral midbrain of rats exposed to subchronic phencyclidine

Phencyclidine (PCP) produces schizophrenia-like symptoms in normal humans. This suggests that the dysfunction of glutamatergic neurotransmission may play an important role in the pathology of schizophrenia. However, PCP also exerts its effect on the mesolimbic dopamine (DA) system and modulates DA function in the brain, the abnormality of which is proposed to be a main pathology of schizophrenia. Recently, glial cell-line derived neurotrophic factor (GDNF) has been shown to play a protective role for DA neurons against neurotoxic injuries and maintaining DA function in the brain. We hypothesized that subchronic PCP may alter the function of GDNF in the ventral midbrain, where DA cell bodies are localized. Male Wistar rats were injected intraperitoneally with PCP daily for 10 days at 5 or 10 mg/kg, and their brains were removed 24 h after the last injection. The expressions of GDNF and its receptor (GFRα-1 and c-ret) mRNAs in the substantia nigra compacta (SNC) and ventral tegmental area (VTA) were determined by non-radioactive in situ hybridization, and those of GDNF and c-ret mRNA were found to be increased after the PCP subchronic administration. No significant changes, however, were observed in the expressions of GFRα-1 and basic fibroblast growth factor. These results suggest that subchronic PCP may modulate the function of the GDNF system, which exerts a trophic action on DA neurons in the ventral midbrain.

Electrophysiological and metabolic evidence that high-frequency stimulation of the subthalamic nucleus bridles neuronal activity in the subthalamic nucleus and the substantia nigra reticulata.

High-frequency stimulation (HFS) of the subthalamic nucleus (STN) has been shown to produce a dramatic alleviation of motor symptoms in patients with advanced Parkinson's disease. Its functional mechanism, however, remains obscure. We used extracellular recording and in situ cytochrome oxidase (CoI) mRNA hybridization to investigate the effects of HFS of the STN on neuronal activity of the STN and the substantia nigra reticulata (SNr) in normal rats and rats with 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra compacta (SNc). To allow detection of spikes and analysis of firing activity, artifacts recorded during stimulation were scaled down using a template subtraction method. In both normal and lesioned rats, the activity of a majority of STN neurons was inhibited during stimulation. In the SNr, HFS also induced an inhibition of the activity of a majority of neurons in normal and lesioned rats. In situ hybridization histochemistry confirmed these results in that it showed a significant decrease in levels of CoI mRNA expression in the STN and SNr in both normal and lesioned rats during stimulation. These data afford an interesting insight into the functional mechanism of deep brain stimulation and support the hypothesis that HFS exerts an inhibitory influence on STN neuronal firing.

Rapid increase of Nurr1 expression in the substantia nigra after 6-hydroxydopamine lesion in the striatum of the rat.

Nurr1 is a transcription factor essential for the genesis of ventral dopaminergic neurons. In this study, we investigated the expression of Nurr1 protein and mRNA in the adult rat brain by using immunohistochemistry and in situ hybridization, respectively. Another aim of our study was to investigate Nurr1 expression in substantia nigra after dopamine depletion induced by the injection of 6-hydroxydopamine in the striatum. We observed that Nurr1 mRNA and protein are expressed in several brain regions, including cortex, hippocampus, substantia nigra, and ventral tegmental area, in agreement with previous reports using in situ hybridization. Additionally, we found that Nurr1 is expressed in brain regions that have not been previously reported, such as striatum, septum, and superior colliculus. Highest levels of expression were found in cortex, medial septum, dentate gyrus, some hypothalamic nuclei, and substantia nigra. Interestingly, we observed that, in the superior colliculus, Nurr1 protein is localized in the cytoplasm of cells, whereas, in other regions, it was localized mainly in the nuclei, suggesting that Nurr1 subcellular localization is regulated and may have functional implications. Dopamine depletion induced by an injection of 6-hydroxydopamine into the striatum produced an increase in the number of cells expressing Nurr1 mRNA and protein in both substantia nigra compacta and substantia nigra reticulata, ipsilateral and contralateral to the lesioned side, measured 24 hr after the 6-hydroxydopamine injection. These results suggest that Nurr1 may be involved in many neuronal functions in the adult central nervous system and, in particular, might be related to the compensation processes that take place in dopaminergic cells in order to normalize extracellular dopamine levels in the striatum.

Midbrain Dopaminergic Neurons

Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system and are associated with one of the most prominent human neurological disorders, Parkinson's disease. During development, they are induced in the ventral midbrain by an interaction between two diffusible factors, SHH and FGF8. The local identity of this part of the midbrain is probably determined by the combinatorial expression of three transcription factors, Otx2, Pax2, and Pax5. After the last cell division, the neurons start to express transcription factors that control further differentiation and the manifestation of cellular properties characteristic for adult dopaminergic neurons of the substantia nigra compacta and the ventral tegmentum. The first to appear is the LIM-homeodomain transcription factor, Lmx1b. It is essential for the survival of these neurons, and it regulates the expression of another transcription factor, Pitx3, an activator of tyrosine hydroxylase. Lmx1b is followed by the orphan steroid receptor Nurr1. It is essential for the expression of the dopaminergic phenotype. Several genes involved in dopamine synthesis, transport, release, and reuptake are regulated by Nurr1. This requirement is specific to the midbrain dopaminergic neurons, since other populations of the same neurotransmitter phenotype develop normally in absence of the gene. A day after Nurr1, two homeodomain transcription factors, engrailed-1 and -2, are expressed. In animals deficient in the two genes, the midbrain dopaminergic neurons are generated, but then fail to differentiate and disappear very rapidly. Interestingly, alpha-synuclein, a gene recently linked to familial forms of Parkinson's disease, is regulated by engrailed-1 and -2.

Quantitative RT-PCR reveals a ubiquitous but preferentially neural expression of the KIS gene in rat and human

KIS is the only known protein kinase that possesses an RNA recognition motif. This original structure indicates a role for KIS in the maturation of RNAs possibly by phosphorylating and regulating the activities of RNA associated factors. Another function of KIS has recently been unravelled—it negatively regulates the cdk inhibitor p27 Kip1 and thus promotes cell cycle progression through G1. In order to explore the functional expression of this kinase, we quantified its mRNA in a wide range of rat and human tissues, during development and in tumors. In both species, the highest level of KIS gene expression was in adult neural tissues. Interestingly, within the adult rat brain, KIS mRNA is enriched in several areas including the substantia nigra compacta and nuclei of the brain stem. Furthermore, KIS gene expression increases dramatically during brain development. Altogether our results point to a ubiquitous function for KIS together with a particular implication during neural differentiation or in the function of mature neural cells. No dysregulation of KIS gene expression was detected in human tumors from breast, bladder, prostate, liver and kidney origins. On the other hand, the KIS gene was overexpressed in NF1-associated plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) as compared to dermal neurofibroma which suggests a possible implication of KIS in the genesis of NF1-associated tumors.

Differential Desensitization and Distribution of Nicotinic Acetylcholine Receptor Subtypes in Midbrain Dopamine Areas

Although many psychopharmacological factors contribute to nicotine addiction, midbrain dopaminergic systems have received much attention because of their roles in reinforcement and associative learning. It is generally thought that the mesocorticolimbic dopaminergic system is important for the acquisition of behaviors that are reinforced by the salient drives of the environment or by the inappropriate stimuli of addictive drugs. Nicotine, as obtained from tobacco, can activate nicotinic acetylcholine receptors (nAChRs) and excite midbrain neurons of the mesocorticolimbic system. Using midbrain slices from rats, wild-type mice, and genetically engineered mice, we have found differences in the nAChR currents from the ventral tegmental area (VTA) and the substantia nigra compacta (SNc). Nicotinic AChRs containing the alpha7 subunit (alpha7* nAChRs) have a low expression density. Electrophysiological analysis of nAChR currents, autoradiography of [125I]-alpha-bungarotoxin binding, and in situ hybridization revealed that alpha7* nAChRs are more highly expressed in the VTA than the SNc. In contrast, beta2* nAChRs are move evenly distributed at a higher density in both the VTA and SNc. At the concentration of nicotine obtained by tobacco smokers, the slow components of current (mainly mediated by beta2* nAChRs) become essentially desensitized. However, the minority alpha7* component of the current in the VTA/SNc is not significantly desensitized by nicotine in the range < or =100 nm. These results suggest that nicotine, as obtained from tobacco, can have multiple effects on the midbrain areas by differentially influencing dopamine neurons of the VTA and SNc and differentially desensitizing alpha7* and non-alpha7 nAChRs.