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.     

7 tesla magnetic resonance imaging: A closer look at substantia nigra anatomy in Parkinson's disease

A hallmark of Parkinson's disease (PD) is the progressive neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Dopaminergic denervation is commonly imaged using radiotracer imaging in target structures such as the striatum. Until recently, imaging made only a modest contribution to detecting neurodegenerative changes in the substantia nigra (SN) directly. Histologically, the SN is subdivided into the ventral pars reticulata and the dorsal pars compacta, which is composed of dopaminergic neurons. In humans, dopaminergic neurons, which are known to accumulate neuromelanin, form clusters of cells (nigrosomes) that penetrate deep into the SN pars reticulata (SNr). The SNr contains higher levels of iron than the SNc in normal subjects. Neuromelanin and T2*‐weighted imaging therefore better detect the SNc and the SNr, respectively. The development of ultra‐high field 7 Tesla (7T) magnetic resonance imaging (MRI) provided the increase in spatial resolution and in contrast that was needed to detect changes in SN morphology. 7T MRI allows visualization of nigrosome‐1 as a hyperintense signal area on T2*‐weighted images in the SNc of healthy subjects and its absence in PD patients, probably because of the loss of melanized neurons and the increase of iron deposition. This review is designed to provide a better understanding of the correspondence between the outlines and subdivisions of the SN detected using different MRI contrasts and the histological organization of the SN. The recent findings obtained at 7T will then be presented in relation to histological knowledge. © 2014 International Parkinson and Movement Disorder Society     

The subcellular localization of GABA(B) receptor subunits in the rat substantia nigra

The inhibitory effects of GABA within the substantia nigra (SN) are mediated in part by metabotropic GABA(B) receptors. To better understand the mechanisms underlying these effects, we have examined the subcellular localization of the GABA(B) receptor subunits, GABA(B1) and GABA(B2), in SN neurons and afferents using pre-embedding immunocytochemistry combined with anterograde or retrograde labelling. In both the SN pars compacta (SNc) and pars reticulata (SNr), GABA(B1) and GABA(B2) showed overlapping, but distinct, patterns of immunolabelling. GABA(B1) was more strongly expressed by putative dopaminergic neurons in the SNc than by SNr projection neurons, whereas GABA(B2) was mainly expressed in the neuropil of both regions. Immunogold labelling for GABA(B1) and GABA(B2) was localized in presynaptic and postsynaptic elements throughout the SN. The majority of labelling was intracellular or was associated with extrasynaptic sites on the plasma membrane. In addition, labelling for both subunits was found on the presynaptic and postsynaptic membranes at symmetric, putative GABAergic synapses, including those formed by anterogradely labelled striatonigral and pallidonigral terminals. Labelling was also observed on the presynaptic membrane and at the edge of the postsynaptic density at asymmetric, putative excitatory synapses. Double immunolabelling, using the vesicular glutamate transporter 2, revealed the glutamatergic nature of many of the immunogold-labelled asymmetric synapses. The widespread distribution of GABA(B) subunits in the SNc and SNr suggests that GABA(B)-mediated effects in these regions are likely to be more complex than previously described, involving presynaptic autoreceptors and heteroreceptors, and postsynaptic receptors on different populations of SN neurons.     

Subpopulations of neurons in rat substantia nigra display GABABR2 receptor immunoreactivity

A functional gamma-aminobutyric acid (GABA) B receptor is the first metabotropic receptor known to be composed of two heteromeric subunits, GABA_BR1 and GABA_BR2. Our previous report [Neuroscience 99 (2000) 65] has demonstrated that subpopulations of neurons in the rat substantia nigra display distinct patterns of distribution of GABA_BR1 receptor immunoreactivity. A robust level of GABA_BR1 receptor is only found in the dopaminergic neurons of the substantia nigra pars compacta (SNc). The objective of the present study was to determine the precise cellular localization of GABA_BR2 subunit in the rat substantia nigra using double immunofluorescence. Neuropilar elements in the SNc and the substantia nigra pars reticulata (SNr) were found to display GABA_BR2 immunoreactivity. In addition, the tyrosine hydroxylase-immunoreactive dopaminergic neurons and the parvalbumin-immunoreactive GABAergic neurons in the SNr were also found to display GABA_BR2 immunoreactivity. The present results thus demonstrate that a functional GABA_B receptor may be expressed by the dopaminergic neurons in the SNc. It is less clear whether neurons in the SNr express a functional GABA_B receptor. The present findings have important functional implications in GABA neurotransmission in the substantia nigra.     

Basal ganglia and thalamic input from neurons located within the ventral tier cell cluster region of the substantia nigra pars compacta in the rat

The most caudally located dopaminergic (DA) ventral tier neurons of the substantia nigra pars compacta (SNc) form typical cell clusters that are deeply embedded in the substantia nigra pars reticulata (SNr). Here we examine the efferent projections of 35 neurons located in the SNr region where these SNc cell clusters reside. The neuronal cell body was injected with biotinylated dextran amine so as to trace each complete axon in the sagittal or the coronal plane. Electrophysiological guidance guaranteed that the tracer was ejected among neurons displaying a typical SNc discharge pattern. Furthermore, double immunofluorescence and immunohistochemical labeling ensured that the tracer deposits were placed within the DA cell clusters. Three types of projection neurons occurred in the SNc ventral tier cell cluster region: type I neurons, projecting to basal ganglia; type II neurons, targeting both the basal ganglia and thalamus; and type III neurons, projecting only to the thalamus. The striatum was targeted by most of the type I and II neurons and the innervation reached both the striosome/subcallosal streak and matrix compartments. Many nigrostriatal fibers provided collaterals to the globus pallidus and, less frequently, to the subthalamic nucleus. At a thalamic level, type II and III neurons preferentially targeted the reticular, ventral posterolateral, and ventral medial nuclei. Our results reveal that the SNr region where DA ventral tier cell clusters reside harbors neurons projecting to the basal ganglia and/or the thalamus, thus suggesting that neurodegeneration of nigral neurons in Parkinson′s disease might affect various extrastriatal basal ganglia structures and multiple thalamic nuclei. J. Comp. Neurol. 518:1283–1300, 2010. © 2009 Wiley‐Liss, Inc.     

Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca2+ homeostasis

Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 μm ) effects on SNc neurons in acute rat midbrain slices, using whole‐cell patch‐clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide‐sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca²⁺] ([Ca²⁺]_i) (73.8 ± 7.7 nm ) and intracellular [Na⁺] (3.1 ± 0.6 mm ) (all with 1 μm ). The outward current was not affected by a high ATP level (10 mm ) in the patch pipette but was decreased by Trolox. The [Ca²⁺]_i rise was abolished by removing extracellular Ca²⁺, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N‐(p‐amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine‐123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm ) that, by itself, did not evoke a [Ca²⁺]_i rise resulted in a large (46.6 ± 25.3 nm ) Ca²⁺ response when baseline [Ca²⁺]_i was increased by a ‘priming’ protocol that activated voltage‐gated Ca²⁺ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca²⁺]_i and the rotenone‐induced [Ca²⁺]_i rise. This correlation was not seen in non‐dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP‐gated K⁺ channels and TRPM2‐like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone‐induced [Ca²⁺]_i rise by a small increase in baseline [Ca²⁺]_i.     

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 E₂‐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.     

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 μm ) and UBP141 (4 μm ), but not by Zn²⁺ (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.     

The dendritic dopamine projection of the substantia nigra: phenotypic denominator of weaver gene action in hetero- and homozygosity

While cerebellar granule cell migration and survival are affected by the weaver (wv) mutation both in the heterozygous and homozygous states, the dopamine (DA) deficit of the nigrostriatal projection has been shown to involve only midbrain DA cell bodies and nigrostriatal DA axons of homozygous mutants. We have identified a cellular site which is defective in the mesencephalic DA system of mice both heterozygous and homozygous for the wv gene. That deficit involves the dendritic DA projection which extends from the substantia nigra pars compacta (SNc) into the pars reticulata (SNr). In the midbrain of heterozygotes, dopaminergic dendrites are reduced by 60% at 20 days of age, when DA neurone number in the midbrain, DA content in the neostriatum and pattern of synaptic connectivity of nigrostriatal axon terminals are normal. At the same age, the deficit of dopaminergic dendrites in the SNr of homozygotes (76%) is disproportionate to the loss of DA cell bodies (42%). These findings: (a) may provide clues to the aetiopathogenetic mechanisms of wv gene operation; and (b) may explain the generalised convulsions intermittently manifested by weaver heterozygotes, as the SN has been implicated in the pathophysiology of experimental seizures.     

Microglia may compensate for dopaminergic neuron loss in experimental Parkinsonism through selective elimination of glutamatergic synapses from the subthalamic nucleus

Parkinson's disease (PD) symptoms do not become apparent until most dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate, suggesting that compensatory mechanisms play a role. Here, we investigated the compensatory involvement of activated microglia in the SN pars reticulata (SNr) and the globus pallidus (GP) in a 6‐hydroxydopamine‐induced rat hemiparkinsonism model. Activated microglia accumulated more markedly in the SNr than in the SNc in the model. The cells had enlarged somata and expressed phagocytic markers CD68 and NG2 proteoglycan in a limited region of the SNr, where synapsin I‐ and postsynaptic density 95‐immunoreactivities were reduced. The activated microglia engulfed pre‐ and post‐synaptic elements, including NMDA receptors into their phagosomes. Cells in the SNr and GP engulfed red fluorescent DiI that was injected into the subthalamic nucleus (STN) as an anterograde tracer. Rat primary microglia increased their phagocytic activities in response to glutamate, with increased expression of mRNA encoding phagocytosis‐related factors. The synthetic glucocorticoid dexamethasone overcame the stimulating effect of glutamate. Subcutaneous single administration of dexamethasone to the PD model rats suppressed microglial activation in the SNr, resulting in aggravated motor dysfunctions, while expression of mRNA encoding glutamatergic, but not GABAergic, synaptic elements increased. These findings suggest that microglia in the SNr and GP become activated and selectively eliminate glutamatergic synapses from the STN in response to increased glutamatergic activity. Thus, microglia may be involved in a negative feedback loop in the indirect pathway of the basal ganglia to compensate for the loss of dopaminergic neurons in PD brains.     

Immunohistochemical localization of voltage-gated calcium channels in substantia nigra dopamine neurons

The rhythmic firing of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) is thought to be mediated by nifedipine-sensitive Ca(2+) channels, although an involvement of omega-conotoxin-sensitive Ca(2+) channels is also suggested. In an attempt to localize such Ca(2+) channels at both the regional and cellular levels, their expression and distribution patterns were immunohistochemically investigated in the rat SNc. The three distinct subtypes of voltage-gated Ca(2+) channels were tested: the class B N-type alpha 1 subunit (CNB1), the class C L-type alpha 1 subunit (CNC1) and the class D L-type alpha 1 subunit (CND1). A large number of SNc neurons showed intense immunoreactivity against CND1 and they were distributed throughout the entire extent. By contrast, many fewer neurons displayed less intense CNC1 immunoreactivity and many of them were located in the lateral aspect of the SNc. No immunoreactivity against CNB1 was detected in the SNc. Moreover, double immunofluorescence analysis in combination with tyrosine hydroxylase staining revealed that virtually all DA neurons were CND1-immunoreactive whereas many DA neurons especially in the medial SNc exhibited only faint or no immunoreactivity against CNC1. Both CNC1 and CND1 were expressed in cell bodies and proximal dendrites of SNc DA neurons, whilst their distal dendrites that penetrated into the substantia nigra pars reticulata expressed CND1 alone. Thus, the ubiquitously and intensely expressed class D alpha 1 subunit of L-type Ca(2+) channels that is sensitive to both nifedipine and omega-conotoxin may be responsible for the pacemaker activity of SNc DA neurons.     

Prolonged blockade of NMDA or mGluR5 glutamate receptors reduces nigrostriatal degeneration while inducing selective metabolic changes in the basal ganglia circuitry in a rodent model of Parkinson's disease

We compared the neuroprotective and metabolic effects of chronic treatment with ionotropic or metabotropic glutamate receptor antagonists, in rats bearing a unilateral nigrostriatal lesion induced by 6-hydroxydopamine (6-OHDA). The ionotropic, N-methyl-D-aspartate receptor antagonist MK-801 increased cell survival in the substantia nigra pars compacta (SNc) and corrected the metabolic hyperactivity (increased cytochrome oxidase activity) of the ipsilateral substantia nigra pars reticulata (SNr) associated with the lesion, but showed no effects on the 6-OHDA-induced hyperactivity of the subthalamic nucleus (STN). Significant-although less pronounced-protection of SNc neurons was also observed following treatment with the metabotropic glutamate receptor (mGluR5) antagonist 2-methyl-6-(phenylehtynyl)-pyridine (MPEP). As opposed to MK-801, MPEP abolished the STN metabolic hyperactivity associated with the nigrostriatal lesion, without affecting SNr activity. Specific modulation of STN hyperactivity obtained with mGluR5 blockade may, therefore, open interesting perspectives for the use of this class of compounds in the treatment of Parkinson's disease.     

Distribution of substance p and enkephalin-like immunoreactivity in the substantia nigra of rat, cat and monkey

A comparative study of the distribution of substance P (SP) and enkephalin (ENK) immunoreactivity in the substantia nigra (SN) of the rat, cat and squirrel monkey (Saimiri sciureus) was undertaken by means of the indirect immunofluorescence technique. In the rat a dense neuronal network composed of fine fibers displaying SP immunoreactivity is uniformely distributed throughout the rostrocaudal extent of the substantia nigra pars reticulata (SNr) and in the ventral part of substantia nigra pars compacta (SNc). Some coarse SP-positive fibers also occur in SNc. In addition, ENK-immunoreactive fibers are scattered amongst SNc neurons but abound particularly in the caudolateral part of SNr. In cat innumerable fine SP-positive fibers are distributed in SNr according to a pattern similar to that found in rat. ENK-immunoreactive fine fibers are densely packed in the ventromedial part of SNr whereas coarse ENK fibers are scattered in both SNc and SNr but abound particularly in the caudolateral portion of SNr. In monkey fine SP and ENK-immunoreactive fibers occur in very large number in SNr. These two types of fibers are distributed according to a similar but strikingly complex and heterogeneous pattern. In addition, coarse fibers displaying either SP or ENK immunoreactivity are scattered amongst the SNc neurons in monkey. These findings reveal that SP immunoreactive fibers are present in large number and are distributed according to a somewhat similar pattern in rat, cat and monkey. In contrast, the number of ENK-positive fibers and the complexity of their organizational feature in SN increase strikingly from rodent to primate.     

An altered histaminergic innervation of the substantia nigra in Parkinson's disease

The central histaminergic system is one of the subcortical aminergic projection systems involved in several regulatory functions. The central dopaminergic and histaminergic systems interact extensively, but little is known about the histaminergic system in diseases affecting the dopaminergic neurons. The distribution of histaminergic fibers in the substantia nigra (SN) in postmortem brain samples from patients suffering from Parkinson's disease (PD) and normal controls was examined with a specific immunohistochemical method. Direct connections between dopaminergic neurones and histaminergic fibers were observed. Histamine in human SN was stored in fibers and varicosities. Sites of histamine formation were examined by l-histidine decarboxylase in situ hybridization. In both normal and PD brains HDC mRNA was found only in posterior hypothalamus and not in SN. The presence of histaminergic innervation of the human substantia nigra pars compacta (SNc) and reticulata (SNr), paranigral nucleus, radix of oculomotor nerve, and parabrachial pigmented nucleus was demonstrated. The density of histaminergic fibers in the middle portion of SNc and SNr was increased in brains with PD. In PD the morphology of histaminergic fibers was also altered; they were thinner than in controls and had enlarged varicosities. An increase of histaminergic innervation may reflect a compensatory event due to deficiency of, e.g., dopamine or a putative fiber growth inhibitory factor. Whether the changes seen in histaminergic fibers in PD are primary or secondary remains to be investigated.     

Heterogeneous distribution of cytochrome oxidase activity in the rat substantia nigra: correlation with tyrosine hydroxylase and dynorphin immunoreactivities

Cytochrome oxidase (COase) activity, an endogenous marker of neuronal activity, was examined in the substantia nigra of the adult rat at the light-microscopic level. In addition, the pattern of histochemical staining observed for COase activity was correlated with immunohistochemistry for tyrosine hydroxylase (a marker of dopaminergic neurons) and for dynorphin (a peptide present in afferents from the striatum). Differential oxidative metabolic activity was revealed in subregions of the substantia nigra by COase histochemistry. Neurons of the substantia nigra pars compacta (SNc), ventral tegmental area, and ventrally displaced dopaminergic neurons were characterized by little or no staining for COase. In contrast, the substantia nigra pars reticulata (SNr) possessed a heterogeneous distribution of COase activity that was characterized by denser staining in the ventrolateral than the dorsomedial part of the nucleus throughout its rostrocaudal extent, with the exception of the most rostral levels. This pattern of COase activity was inversely correlated with the density of ventrally descending tyrosine hydroxylase-positive dendrites arising from the medial portion of the SNc, as well as with the density of dynorphin immunoreactivity. The results suggest that the SNc and SNr possess distinct levels of oxidative metabolic activity. Furthermore, within the SNr itself, different levels of COase activity characterize subpopulations of neurons which may be differentially regulated by both striatal and dopaminergic influences.     

Subpopulations of neurons in rat substantia nigra display GABA(B)R2 receptor immunoreactivity.

A functional gamma-aminobutyric acid (GABA) B receptor is the first metabotropic receptor known to be composed of two heteromeric subunits, GABA_BR1 and GABA_BR2. Our previous report [Neuroscience 99 (2000) 65] has demonstrated that subpopulations of neurons in the rat substantia nigra display distinct patterns of distribution of GABA_BR1 receptor immunoreactivity. A robust level of GABA_BR1 receptor is only found in the dopaminergic neurons of the substantia nigra pars compacta (SNc). The objective of the present study was to determine the precise cellular localization of GABA_BR2 subunit in the rat substantia nigra using double immunofluorescence. Neuropilar elements in the SNc and the substantia nigra pars reticulata (SNr) were found to display GABA_BR2 immunoreactivity. In addition, the tyrosine hydroxylase-immunoreactive dopaminergic neurons and the parvalbumin-immunoreactive GABAergic neurons in the SNr were also found to display GABA_BR2 immunoreactivity. The present results thus demonstrate that a functional GABA_B receptor may be expressed by the dopaminergic neurons in the SNc. It is less clear whether neurons in the SNr express a functional GABA_B receptor. The present findings have important functional implications in GABA neurotransmission in the substantia nigra.     

Alterations in nigral NMDA and GABAA receptor control of the striatal dopamine level after repetitive exposures to nitrogen narcosis

Nitrogen pressure exposure in rats results in decreased dopamine (DA) release at the striatal terminals of the substantia nigra pars compacta (SNc) dopaminergic neurons, demonstrating the narcotic potency of nitrogen. This effect is attributed to decreased excitatory and increased inhibitory inputs to dopaminergic neurons, involving a change in NMDA and GABA_A receptor function. We investigated whether repetitive exposures to nitrogen modify the excitatory and inhibitory control of the dopaminergic nigro-striatal pathway.We used voltammetry to measure dopamine levels in freely-moving rats, implanted with dopamine-sensitive electrodes in the striatum. NMDA/GABA_A receptor agonists (NMDA/muscimol) and antagonists (AP7/gabazine) were administered through a guide-cannula into the SNc, and their effects on striatal dopamine levels were measured under normobaric conditions, before and after five repetitive exposures to 1 MPa nitrogen.NMDA-mediated dopamine release was greater following repetitive exposures, AP7-mediated inhibition of glutamatergic input was blocked, suggesting that NMDA receptor sensitivity was increased and glutamate release reduced. Muscimol did not modify dopamine levels following repetitive exposures, whereas the effect of gabazine was greater after exposures than before. This suggested that interneuronal GABA_A receptors were desensitized, leading to an increased GABAergic input at dopaminergic cells. Thus, repetitive nitrogen exposure induced persistent changes in glutamatergic and GABAergic control of dopaminergic neurons, resulting in decreased activity of the nigrostriatal pathway.     

EphB1 null mice exhibit neuronal loss in substantia nigra pars reticulata and spontaneous locomotor hyperactivity

The molecular mechanisms that regulate basal ganglia development are largely unknown. Eph receptor tyrosine kinases are potential participants in this process as they regulate development of other CNS regions and are expressed in basal ganglia nuclei, such as the substantia nigra (SN) and striatum. To address the role of Eph receptors in the development of these nuclei, we analysed anatomical changes in the SN and striatum of mice with null mutations for EphB1. These mice express beta-galactosidase as a marker for cells normally expressing EphB1. In situ hybridization data and a direct comparison of SN neurons expressing tyrosine hydroxylase (TH) and/or the beta-gal marker for EphB1 revealed that EphB1 is not expressed in TH+ neurons of pars compacta (SNc), but is restricted to neurons in pars reticulata (SNr). Consistent with this, we find that EphB1 null mice exhibit a significant decrease in the volume and number of neurons (40% decrease) in SNr, whereas the volume and number of TH+ neurons in SNc is not significantly affected nor are there changes in the distribution of nigrostriatal dopamine neurons. Although EphB1 is expressed in the striatum, EphB1-/- mice exhibit no significant changes in striatal volume and TH fiber density, and have no obvious alterations in striatal patch/matrix organization. Behavioral evaluation of EphB1 null mice in an open-field environment revealed that these mice exhibited spontaneous locomotor hyperactivity. These results suggest that EphB1 is necessary for the proper formation of SNr, and that neuronal loss in SNr is associated with altered locomotor functions.