Substantia Nigra Pars Compacta Dopaminergic Neurons Research Articles

Tetrahydroberberine blocks ATP-sensitive potassium channels in dopamine neurons acutely-dissociated from rat substantia nigra pars compacta

Tetrahydroberberine (THB) exhibits neuroprotective effects but its targets and underlying mechanisms are largely unknown. Emerging evidence indicates that ATP-sensitive potassium (K ATP) channels in the substantia nigra pars compacta (SNc) promote Parkinson disease (PD) pathogenesis, thus blocking K ATP channels may protect neurons against neuronal degeneration. In the present study, we tested a hypothesis that THB blocks K ATP channels in dopaminergic (DA) neurons acutely dissociated from rat SNc. Using perforated patch-clamp recording in current-clamp mode, the functional K ATP channels can be opened by persistent perfusion of rotenone, an inhibitor of complex I of the mitochondrial respiratory chain. Bath-application of THB reversibly blocks opened K ATP channels in a concentration-dependent manner, which is comparable to a classical K ATP channel blocker, Tol. Compared to THB analogs, l-stepholidine ( l-SPD) or l-tetrahydropalmatine ( l-THP), THB exhibits more profound blockade in K ATP channels. In addition, exposure of THB alone to the recorded neuron increases action potential firing, and THB also restores rotenone-induced membrane hyperpolarization in the presence of dopamine D2 receptor antagonist (sulpiride), suggesting that THB exhibits an excitatory effect on SNc DA neurons through the block of K ATP channels. Collectively, the blockade of neuronal K ATP channels by THB in SNc DA neurons is a novel pharmacological mechanism of THB, which may contribute to its neuroprotective effects in PD.

Functional heterogeneity of NMDA receptors in rat substantia nigra pars compacta and reticulata neurones

The nigra substantia nigra pars compacta (SNc) and substantia pars reticulata (SNr) form two major basal ganglia components with different functional roles. SNc dopaminergic (DA) neurones are vulnerable to cell death in Parkinson's disease, and NMDA receptor activation is a potential contributing mechanism. We have investigated the sensitivity of whole-cell and synaptic NMDA responses to intracellular ATP and GTP application in the SNc and SNr from rats on postnatal day (P) 7 and P28. Both NMDA current density (pA/pF) and desensitization to prolonged or repeated NMDA application were greater in the SNr than in the SNc. When ATP levels were not supplemented, responses to prolonged NMDA administration desensitized in P7 SNc DA neurones but not at P28. At P28, SNr neurones desensitized more than SNc neurones, with or without added ATP. Responses to brief NMDA applications and synaptic NMDA currents were not sensitive to inclusion of ATP in the pipette solution. To investigate these differences between the SNc and SNr, NR2 subunit-selective antagonists were tested. NMDA currents were inhibited by ifenprodil (10 microM) and UBP141 (4 microM), but not by Zn(2+) (100 nm), in both the SNr and SNc, suggesting that SNc and SNr neurones express similar receptor subunits; NR2B and NR2D, but not NR2A. The different NMDA response properties in the SNc and SNr may be caused by differences in receptor modulation and/or trafficking. The vulnerability of SNc DA neurones to cell death is not correlated with NMDA current density or receptor subtypes, but could in part be related to inadequate NMDA receptor desensitization.

Functional expression of a large-conductance Ca 2+-activated K + channel in mouse substantia nigra pars compacta dopaminergic neurons

The existence of large-conductance Ca 2+-activated K + (BK) channels in substantia nigra pars compacta (SNc) has been a matter of debate. Using the patch-clamp technique in the inside-out configuration, we have recorded BK channel currents in SNc dopaminergic neurons. The channel has a conductance of 301 pS with a slight inward rectification and is both voltage- and calcium-dependent. Paxilline, a specific BK channel blocker, can completely block the channel, while tetraethylammonium (TEA), a nonspecific blocker of voltage-gated potassium channels, reduces its conductance and a high concentration of TEA (30 mM) inhibits its activity. ATP and GTP reduce the channel activity, while ADP is less potent, and AMP has no effect. The channel is also sensitive to changes in intracellular pH. Our results indicate that functional BK channels are expressed in SNc and suggest the possibility that the BK channel may be involved in the response of SNc dopaminergic neurons to metabolic stress.

Prostaglandin E receptor EP1 enhances GABA‐mediated inhibition of dopaminergic neurons in the substantia nigra pars compacta and regulates dopamine level in the dorsal striatum

Dopamine (DA) is a neuromodulator that is critical for sensory-motor, cognitive and emotional functions. We previously found that mice lacking prostaglandin E receptor EP1 showed impulsive emotional behaviors accompanied by enhanced DA turnover in the frontal cortex and striatum. Given that these behavioral phenotypes were corrected by DA receptor antagonists, we hypothesized that EP1 deficiency causes a hyperdopaminergic state for its behavioral phenotype. Here we tested this hypothesis by examining the EP1 action in the nigrostriatal dopaminergic system. We first used microdialysis and found an elevated extracellular DA level in the dorsal striatum of EP1-deficient mice compared with wild-type mice. Despite the EP1 expression in the striatum, neither deficiency nor activation of EP1 altered the intrastriatal control for DA release, uptake or degradation. Immunohistochemistry revealed punctate EP1 signals apposed with dopaminergic neurons in the substantia nigra pars compacta (SNc). Many EP1 signals were colocalized with a marker for GABAergic synapses. Further, an EP1 agonist enhanced GABA(A)-mediated inhibitory inputs to SNc dopaminergic neurons in midbrain slices. Therefore, the prostaglandin E(2)-EP1 signaling directly enhances GABAergic inputs to SNc dopaminergic neurons. The lack of this EP1 action may lead to a hyperdopaminergic state of EP1-deficient mice.

Influence of aging on the calbindin-D-28k immunoreactive positive dopaminergic neurons in the substantia nigra pars compacta of rats

We studied the relationship between aging and the vulnerability of substantia nigra pars compacta (SNc) calbindin-D-28k immunoreactive positive (CB +) dopaminergic (DA) neurons. Immunohistochemistry and cell counting were used to determine the number of CB + DA neuron in aged rats (24 mon) compared to adult rats (5 mon). Furthermore, the expression of CB mRNA and protein levels in SN was studied by semi-quantitative RT-PCR and Western blotting. An 11% loss of CB + DA neurons was detected in both the rostral (8.9%) and caudal (1.7%) segments but not in the intermedial segment of SNc in aged rats compared to adult rats ( P < 0.05). No difference was detected in CB mRNA and protein levels between aged and adult rats ( P > 0.05). These data suggest that expression levels of CB mRNA and protein may increase in the existing SNc DA neurons, which may compensate for the partial age dependent loss of CB + DA neurons in the SNc.

Chronic Nicotine Selectively Enhances α4β2* Nicotinic Acetylcholine Receptors in the Nigrostriatal Dopamine Pathway

These electrophysiological experiments, in slices and intact animals, study the effects of in vivo chronic exposure to nicotine on functional alpha4beta2* nAChRs in the nigrostriatal dopaminergic (DA) pathway. Recordings were made in wild-type and alpha4 nicotinic acetylcholine receptor (nAChR) subunit knock-out mice. Chronic nicotine enhanced methyllycaconitine citrate hydrate-resistant, dihydro-beta-erythroidine hydrobromide-sensitive nicotinic currents elicited by 3-1000 mum ACh in GABAergic neurons of the substantia nigra pars reticulata (SNr), but not in DA neurons of the substantia nigra pars compacta (SNc). This enhancement leads to higher firing rates of SNr GABAergic neurons and consequently to increased GABAergic inhibition of the SNc DA neurons. In the dorsal striatum, functional alpha4* nAChRs were not found on the neuronal somata; however, nicotine acts via alpha4beta2* nAChRs in the DA terminals to modulate glutamate release onto the medium spiny neurons. Chronic nicotine also increased the number and/or function of these alpha4beta2* nAChRs. These data suggest that in nigrostriatal DA pathway, chronic nicotine enhancement of alpha4beta2* nAChRs displays selectivity in cell type and in nAChR subtype as well as in cellular compartment. These selective events augment inhibition of SNc DA neurons by SNr GABAergic neurons and also temper the release of glutamate in the dorsal striatum. The effects may reduce the risk of excitotoxicity in SNc DA neurons and may also counteract the increased effectiveness of corticostriatal glutamatergic inputs during degeneration of the DA system. These processes may contribute to the inverse correlation between tobacco use and Parkinson's disease.

Impact of serotonin 2C receptor null mutation on physiology and behavior associated with nigrostriatal dopamine pathway function.

The impact of serotonergic neurotransmission on brain dopaminergic pathways has substantial relevance to many neuropsychiatric disorders. A particularly prominent role has been ascribed to the inhibitory effects of serotonin 2C receptor (5-HT(2C)R) activation on physiology and behavior mediated by the mesolimbic dopaminergic pathway, particularly in the terminal region of the nucleus accumbens. The influence of this receptor subtype on functions mediated by the nigrostriatal dopaminergic pathway is less clear. Here we report that a null mutation eliminating expression of 5-HT(2C)Rs produces marked alterations in the activity and functional output of this pathway. 5-HT(2C)R mutant mice displayed increased activity of substantia nigra pars compacta (SNc) dopaminergic neurons, elevated baseline extracellular dopamine concentrations in the dorsal striatum (DSt), alterations in grooming behavior, and enhanced sensitivity to the stereotypic behavioral effects of d-amphetamine and GBR 12909. These psychostimulant responses occurred in the absence of phenotypic differences in drug-induced extracellular dopamine concentration, suggesting a phenotypic alteration in behavioral responses to released dopamine. This was further suggested by enhanced behavioral responses of mutant mice to the D(1) receptor agonist SKF 81297. Differences in DSt D(1) or D(2) receptor expression were not found, nor were differences in medium spiny neuron firing patterns or intrinsic membrane properties following dopamine stimulation. We conclude that 5-HT(2C)Rs regulate nigrostriatal dopaminergic activity and function both at SNc dopaminergic neurons and at a locus downstream of the DSt.

Nicotine modulates GABAergic transmission to dopaminergic neurons in substantia nigra pars compacta

Dopaminergic neurons in the substantia nigra pars compacta (SNc) play important roles in motor control and drug addiction. As the major afferent, GABAergic innervation controls the activity of SNc dopaminergic neurons. Although it is clear that nicotine modulates SNc dopaminergic neurons by activating subtypes of somatodendritic nicotinic acetylcholine receptors (nAChRs), the detailed mechanisms of this activation remain to be addressed. In the current study, we recorded GABA(A) receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) from dissociated SNc dopaminergic neurons that were obtained using an enzyme-free procedure. These neurons preserved some functional terminals after isolation, including those that release GABA. We found that both extra- and intra-cellular calcium modulates sIPSCs in these neurons. Furthermore, both nicotine and endogenous acetylcholine enhance the frequency of sIPSCs. Moreover, endogenous acetylcholine tonically facilitates sIPSC frequency, primarily by activating the alpha4beta2* nAChRs on the GABAergic terminals. Nicotine facilitates GABA release onto SNc dopaminergic neurons mainly via the activation of presynaptic alpha4beta2* nAChRs.

Modulation of firing activity by ATP in dopamine neurons of the rat substantia nigra pars compacta

ATP acts as a neurotransmitter or co-neurotransmitter in many areas of the CNS and peripheral nervous systems; however, little is known about the expression and functional role of purinoceptors (P2) in midbrain dopaminergic neurons. Therefore, we investigated P2X receptor expression and regulation of spontaneous firing activity in dopaminergic neurons of the substantia nigra pars compacta (SNc) in rats using patch-clamp and Ca2+-imaging techniques. In most neurons, application of ATP (1 μM–1 mM) increased firing rate dose-dependently (EC50=1.26±0.26 μM, n=45). When the P2-receptor agonists such as 2-methylthio-adenosine 5′-triphosphate (2-MeSATP) or ATPγS were applied or pressure-applied to the neuron, the firing activity increased together with a rise in cytosolic Ca2+ concentration ([Ca2+]c), but application of beta,gamma-methylene ATP (P2X1, 3 agonist) or methylthio-adenosine 5′-diphosphate (P2Y1 agonist) had no effect. In many neurons, the effect of ATP was abolished by the application of the P2-receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). When ATP was applied in a Ca2+-free solution, there was no detectable change in [Ca2+]c, suggesting that ATP does not release Ca2+ from intracellular stores. In the single-cell reverse transcription polymerase chain reaction (RT-PCR), we found that 65% of dopaminergic neurons expressed mRNAs for P2X receptors; positive amplifications of P2X6 (57.1%), P2X2/6 (25.0%), and P2X4 mRNA (17.9%), respectively. From the above results, we could conclude that ATP modulates firing activities in the rat SNc dopaminergic neurons, possibly via P2X2, P2X2/6, and/or P2X4 receptors.

The involvement of NF-κB p65/p52 in the effects of GDNF on DA neurons in early PD rats

Glial cell line-derived neurotrophic factor (GDNF) can exert neuroprotective effects on the substantia nigra pars compacta (SNc) dopaminergic (DA) neurons that are undergoing degeneration in Parkinson's disease (PD). In an attempt to investigate the molecular signaling mechanisms underlying GDNF protection the DA neurons from degeneration, we established early PD rat models in which the DA neurons in SNc were degenerating. Whether the cytoplasmic NF-κB signaling pathway was involved in the protection of GDNF on the degenerating DA neurons was examined in the present study. The results showed that the nuclear NF-κB p65 levels in the DA neurons increased when GDNF was injected into SNc of early PD rat models. Immunoprecipitation assays showed that the nuclear NF-κB p65/p52 complex levels increased after GDNF administration, while the p65/p50 complex levels decreased. These results indicated that GDNF could activate the NF-κB signaling pathway in the degenerating DA neurons. And it was the noncanonical NF-κB signaling pathway, which contained the NF-κB p65/p52 complex that was involved in the effects of GDNF on DA neurons.

NR2B‐ and NR2D‐containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones

NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA-EPSCs with an estimated IC(50) of 0.36 microm and a maximum inhibition of 73.5 +/- 2.7% (10 microm), consistent with a substantial population of NR1/NR2B-containing diheteromeric receptors. UBP141, a novel NR2D-preferring antagonist, inhibited NMDA-EPSCs with an estimated IC(50) of 6.2 microm. During postnatal development, the maximum inhibitory effect of 10 microm ifenprodil significantly decreased. However, NMDA-EPSCs were not inhibited by Zn(2+) (200 nM) or potentiated by the Zn(2+) chelator TPEN (1 microm), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA-EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil-resistant component of the NMDA-EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D-containing triheteromeric NMDA receptors at a brain synapse.

Differential localization of GABAA receptor subunits within the substantia nigra of the human brain: An immunohistochemical study

Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.

Food for Thought?

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) of the brain. The reduced production and function of neurotrophins (proteins that promote neuronal survival) in PD brains compared to that in normal brains may be a contributing factor in PD. Leptin is a hormone that functions in the hypothalamus to reduce appetite. Leptin receptors are also abundant in dopaminergic neurons in the SNc, leading Weng et al . to investigate whether leptin might play a role in neuronal survival. Immunohistochemical analyses showed that degeneration of mouse dopaminergic neurons in the SNc caused by injection into the brain of the neurotoxin 6-OHDA (a mouse model of PD) was less severe if the mice were pre-injected with leptin. Leptin was also protective against 6-OHDA-induced toxicity in mouse MN9D cells, a dopaminergic cell line. Western blotting assays and treatments with pharmacological inhibitors and short hairpin (sh) RNAs showed that the ability of leptin to block 6-OHDA-induced apoptosis was dependent on leptin receptor-mediated activation of Janus kinase 2 (JAK2), mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and the transcription factor cAMP response element-binding protein (CREB), a known neuronal survival factor. Leptin also stimulated the phosphorylation and nuclear localization of CREB in dopaminergic SNc neurons and increased the abundance of brain-derived neurotrophic factor (BDNF) in the brain compared with that in untreated animals. Together, these data suggest that treatment with leptin may be useful in therapies to combat PD. Z. Weng, A. P. Signore, Y. Gao, S. Wang, F. Zhang, T. Hastings, X.-M. Yin, J. Chen, Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling. J. Biol. Chem. 282 , 34479-34491 (2007). [Abstract] [Full Text]

Microglial activation mediates neurodegeneration related to oligodendroglial α‐synucleinopathy: Implications for multiple system atrophy

The role of microglial activation in multiple system atrophy (MSA) was investigated in a transgenic mouse model featuring oligodendroglial alpha-synuclein inclusions and loss of midbrain dopaminergic neurons by means of histopathology and morphometric analysis. Our findings demonstrate early progressive microglial activation in substantia nigra pars compacta (SNc) associated with increased expression of iNOS and correlating with dopaminergic neuronal loss. Suppression of microglial activation by early long-term minocycline treatment protected dopaminergic SNc neurons. The results suggest that oligodendroglial overexpression of alpha-synuclein may induce neuroinflammation related to nitrosive stress which is likely to contribute to neurodegeneration in MSA. Further, we detected increased toll-like receptor 4 immunoreactivity in both transgenic mice and MSA brains indicating a possible signaling pathway in MSA which needs to be further studied as a candidate target for neuroprotective interventions.

Death in the substantia nigra: a motor tragedy

It is well known that the death of dopaminergic neurons of the substantia nigra pars compacta (SNc) is the pathological hallmark of Parkinson's disease (PD), the second most common and disabling condition in the expanding elderly population. Nevertheless, the intracellular cascade of events leading to dopamine cell death is still unknown and, consequently, treatment is largely symptomatic rather than preventive. Moreover, the mechanisms whereby nigral dopaminergic neurons may degenerate still remain controversial. Hitherto, several data have shown that the earlier cellular disturbances occurring in dopaminergic neurons include oxidative stress, excitotoxicity, inflammation, mitochondrial dysfunction and altered proteolysis. These alterations, rather than killing neurons, trigger subsequent death-related molecular pathways, including elements of apoptosis. In rare incidences, PD may be inherited; this evidence has opened a new and exciting area of research, attempting to shed light on the nature of the more common idiopathic PD form. In this review, the characteristics of the SNc dopaminergic neurons and their lifecycle from birth to death are reviewed. In addition, of the mechanisms by which the aforementioned alterations cause neuronal dopaminergic death, particular emphasis will be given to the role played by inflammation, and the relevance of the possible use of anti-inflammatory drugs in the treatment of PD. Finally, new evidence of a possible de novo neurogenesis in the SNc of adult animals and in PD patients will also be examined.

Effects of gender on nigral gene expression and parkinson disease.

To identify gene expression patterns in human dopamine (DA) neurons in the substantia nigra pars compacta (SNc) of male and female control and Parkinson disease (PD) patients, we harvested DA neurons from frozen SNc from 16 subjects (4 male PDs, 4 female PDs, 4 male and 4 female controls) using Laser Capture microdissection and microarrays. We assessed for enrichment of functional categories with a hypergeometric distribution. The data were validated with QPCR. We observed that gender has a pervasive effect on gene expression in DA neurons. Genes upregulated in females relative to males are mainly involved in signal transduction and neuronal maturation, while in males some of the upregulated genes (alpha-synuclein and PINK1) were previously implicated in the pathogenesis of PD. In females with PD we found alterations in genes with protein kinase activity, genes involved in proteolysis and WNT signaling pathway, while in males with PD there were alterations in protein-binding proteins and copper-binding proteins. Our data reveal broad gender-based differences in gene expression in human dopaminergic neurons of SNc that may underlie the predisposition of males to PD. Moreover, we show that gender influences the response to PD, suggesting that the nature of the disease and the response to treatment may be gender-dependent.

Iptakalim Modulates ATP-Sensitive K+ Channels in Dopamine Neurons from Rat Substantia Nigra Pars Compacta

Iptakalim, a novel cardiovascular ATP-sensitive K(+) (K(ATP)) channel opener, exerts neuroprotective effects on dopaminergic (DA) neurons against metabolic stress-induced neurotoxicity, but the mechanisms are largely unknown. Here, we examined the effects of iptakalim on functional K(ATP) channels in the plasma membrane (pm) and mitochondrial membrane using patch-clamp and fluorescence-imaging techniques. In identified DA neurons acutely dissociated from rat substantia nigra pars compacta (SNc), both the mitochondrial metabolic inhibitor rotenone and the sulfonylurea receptor subtype (SUR) 1-selective K(ATP) channel opener (KCO) diazoxide induced neuronal hyperpolarization and abolished action potential firing, but the SUR2B-selective KCO cromakalim exerted little effect, suggesting that functional K(ATP) channels in rat SNc DA neurons are mainly composed of SUR1. Immunocytochemical staining showed a SUR1-rather than a SUR2B-positive reaction in most dissociated DA neurons. At concentrations between 3 and 300 microM, iptakalim failed to hyperpolarize DA neurons; however, 300 microM iptakalim increased neuronal firing. In addition, iptakalim restored DA neuronal firing during rotenone-induced hyperpolarization and suppressed rotenone-induced outward current, suggesting that high concentrations of iptakalim close neuronal K(ATP) channels. Furthermore, in human embryonic kidney 293 cells, iptakalim (300-500 microM) closed diazoxide-induced Kir6.2/SUR1 K(ATP) channels, which were heterologously expressed. In rhodamine-123-preloaded DA neurons, iptakalim neither depolarized mitochondrial membrane nor prevented rotenone-induced mitochondrial depolarization. These data indicate that iptakalim is not a K(ATP) channel opener in rat SNc DA neurons; instead, iptakalim is a pm-K(ATP) channel closer at high concentrations. These effects of iptakalim stimulate further pharmacological investigation and the development of possible therapeutic applications.

Functional NR2B‐ and NR2D‐containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 nm) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 nm or 200 microm NMDA was not affected by zinc or TPEN (N,N,N',N'-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 microm ifenprodil reduced open probability, while 10 microm reduced channel open time but had no effect on open probability of channels activated by 100 nm NMDA. When the concentration of NMDA was increased to 200 microm, ifenprodil (10 microm) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits.

Tumor necrosis factor‐α receptor 1 (p55) knockout only transiently decreases the activation of c‐Jun and does not affect the survival of axotomized dopaminergic nigral neurons

The activation of the c-Jun N-terminal kinases and their substrate transcription factor c-Jun is central to the death of dopaminergic neurons of the substantia nigra pars compacta (SNC) but the underlying signal cascades are poorly understood. We have studied the impact of the p55 tumor necrosis factor-alpha receptor (TNF-R) 1 on the N-terminal phosphorylation of c-Jun and the survival of the dopaminergic SNC neurons after transection of the medial forebrain bundle. The axotomy raised the immunoreactivities of tumor necrosis factor-alpha, p75 TNF-R2 and ED1 (ectodysplasin A) in the substantia nigra equally in wildtype and knockout (ko) mice and of TNF-R1 in wildtype mice. Importantly, TNF-R1 ko significantly reduced the early phosphorylation of c-Jun between 18 h and 3 d post-axotomy but the functional deficiency of TNF-R1 did not affect the survival of the dopaminergic neurons up to day 30. These findings demonstrate that: (i) TNF-R1 is involved in the early cell body response after axon transection; (ii) TNF-R1 operates upstream of c-Jun N-terminal kinase/c-Jun, the central signal system of nerve fiber injury, and (iii) the failure of persistent reduction of activated c-Jun is linked to the failure of protection of dopaminergic SNC neurons by TNF-R1 ko.

Altered Mesencephalic Dopaminergic Populations in Adulthood as a Consequence of Brief Perinatal Glucocorticoid Exposure

Early exposure to stressors is strongly associated with enduring effects on central nervous system function, but the mechanisms and neural substrates involved in this biological 'programming' are unclear. This study tested the hypothesis that inappropriate exposure to glucocorticoid stress hormones (GCs) during critical periods of development permanently alters the mesencephalic dopaminergic populations in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Using a rat model, the synthetic GC dexamethasone was added to the maternal drinking water during gestational days 16-19 or over the first week of postnatal life. In adulthood, the effects upon tyrosine hydroxylase immunopositive (TH+) cell numbers in the midbrain, and monoamine levels in the forebrain, of the adult offspring were assessed and compared with control offspring whose dams received normal drinking water. In the VTA, both prenatal and postnatal dexamethasone treatment increased TH+ cell numbers by approximately 50% in males and females. Although prenatal dexamethasone treatment also increased TH+ cell numbers in the SNc by 40-50% in males and females, postnatal treatment affected females only by increasing TH+ cell numbers by approximately 30%. In comparison, similar changes were not detected in the monoamine levels of the dorsolateral striatum, nucleus accumbens or infralimbic cortex of either males or females, which is a feature likely to reflect adaptive changes in these pathways. These studies demonstrate that the survival or phenotypic expression of VTA and SNc dopaminergic neurones is profoundly influenced by brief perinatal exposure to GCs at times when endogenous levels are normally low. These findings are the first to demonstrate permanent changes in the cytoarchitecture within midbrain dopamine nuclei after perinatal exposure to stress hormones and implicate altered functionality. Thus, they have significance for the increasing use of GCs in perinatal medicine and indicate potential mechanisms whereby perinatal distress may predispose to the development of a range of psychiatric conditions in later life.