Golgi staining-like retrograde labeling of brain circuits using rabies virus: Focus onto the striatonigral neurons.

BACKGROUND: The introduction of viral transneuronal tracers in the toolbox of neural tract-tracing methods has been an important addition in the field of connectomics for deciphering circuit-level architecture of the nervous system. One of the added values of viral compared to conventional retrograde tracers, in particular of rabies virus, is to provide a Golgi staining-like view of the infected neurons, revealing the thin dendritic arborizations and the spines that are major post-synaptic seats of neuronal connections. NEWMETHOD: Here, we comparatively illustrate the characteristics of the labeling obtained in the same model system, the basal ganglia circuitry, by different retrograde viral tracing approaches, using the Bartha strain of pseudorabies virus, the SAD and CVS strains of rabies virus and by the conventional retrograde tracer cholera toxin B. To best contrast the differences in the capacity of these tracers to reveal the dendritic morphology in details, we focused on one population of first-order infected neurons in the striatum, which exhibit high spine density, after tracer injection in the substantia nigra. RESULTS AND CONCLUSION: None of the viruses tested allowed to detect as many neurons as with cholera toxin B, but the SAD and CVS strains of rabies virus had the advantage of enabling detailed Golgi-like visualisation of the dendritic trees, the best numerical detection being offered by the transneuronal rCVS-N2c-P-mCherry while poor labeling was provided by rCVS-N2c-M-GFP. Results also suggest that, besides different viral properties, technical issues about constructs and detection methods contribute to apparently different efficiencies among the viral approaches.

Role of glutamate transporters in corticostriatal synaptic transmission.

High-affinity glutamate transporters (GTs) play a major role in controlling the extracellular level of this excitatory neurotransmitter in the CNS. Here we have characterized, by means of in vitro patch-clamp recordings from medium spiny neurons (MSNs), the role of GTs in regulating corticostriatal glutamatergic synaptic transmission in the adult rat. Charge transfer and decay-time, but not amplitude, of excitatory postsynaptic currents (EPSCs) were enhanced by dl-threo-beta-benzyloxyaspartate (TBOA), a broad inhibitor of GTs. Moreover, TBOA also potentiated currents induced by high-frequency stimulation (HFS) protocols. Interestingly, the effect of TBOA on EPSCs was lost when MSNs were clamped at +40 mV, a condition in which neuronal GTs, that are voltage-dependent, are blocked. However, in this condition TBOA was still able to enhance HFS-induced currents, suggesting that glial GT's role is to regulate synaptic transmission when glutamate release is massive. These data suggest that neuronal GTs, rather than glial, shape EPSCs' kinetics and modulate glutamate transmission at corticostriatal synapse. Moreover, the control of glutamate concentration in the synaptic cleft by GTs may play a role in a number of degenerative disorders characterized by the hyperactivity of corticostriatal pathway, as well as in synaptic plasticity.

[Cerebral oxidative stress: are astrocytes vulnerable to low intracellular glutamate concentrations? Consequences for neuronal viability]. / Stress oxydatif cérébral : les astrocytes sont-ils vulnérables aux faibles concentrations intracellulaires de glutamate? Implications sur la survie neuronale.

This review describes reactive oxygen species (ROS), their production and effects on crucial biological molecules, the different lines of defense against oxidative stress, with particular attention to glutathione, the main antioxidant in the brain, which neuronal synthesis seems to be dependent on astrocytic precursors. It also focuses on the different ways by which glutamate may induce oxidative stress in the brain. The different mechanisms leading to ROS production, activated during the excitotoxic cascade, are described. Oxidative glutamate toxicity is also briefly described. A novel form of oxidative glutamate toxicity by depletion of transported glutamate that we recently evidenced is detailed. This toxicity induced by pharmacological reversal of glutamate transport, which mimics glutamate transport reversal occurring in ischemia, involves glutathione depletion and oxidative stress, leading to delayed death of cultured striatal astrocytes differentiated by dibutyryl-cAMP, probably through apoptotic processes. Evidence suggesting that this oxidative glutamate toxicity by depletion of transported glutamate is very likely occurring in vivo and its consequences on neuronal survival are discussed.

Chronic L-DOPA treatment increases extracellular glutamate levels and GLT1 expression in the basal ganglia in a rat model of Parkinson's disease.

There is growing experimental evidence for the implication of glutamate-mediated mechanisms both in the pathophysiology of Parkinson's disease and in the development of dyskinesias with long-term administration of L-3,4-dihydroxyphenylalanine (L-DOPA). However, the impact of this treatment on glutamate transmission in the basal ganglia has been poorly investigated. In this study, we examined the effects of 6-hydroxydopamine-induced lesion of nigral dopamine neurons with or without subsequent chronic L-DOPA treatment on several parameters of glutamate system function in the rat striatum and substantia nigra pars reticulata. All the lesioned animals treated with L-DOPA developed severe dyskinesias. Extracellular glutamate levels, measured by microdialysis in freely moving conditions, and gene expression of the glial glutamate transporter GLT1, assessed by in situ hybridization, were unaffected by dopamine lesion or L-DOPA treatment alone, but were both markedly increased on the lesion side of rats with subsequent L-DOPA treatment. No change in the expression of the vesicular glutamate transporters vGluT1 and vGluT2 was measured in striatum. These data show that chronic L-DOPA treatment leading to dyskinesias increases basal levels of glutamate function in basal ganglia. The L-DOPA-induced overexpression of GLT1 may represent a compensatory mechanism involving astrocytes to limit glutamate overactivity and subsequent toxic processes.

Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum.

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6-hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa-induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.

Impaired glutamate uptake in the R6 Huntington's disease transgenic mice.

Huntington's disease (HD) is a late-onset neurodegenerative disease for which the mutation is CAG/polyglutamine repeat expansion. The R6 mouse lines expressing the HD mutation develop a movement disorder that is preceded by the formation of neuronal polyglutamine aggregates. The phenotype is likely caused by a widespread neuronal dysfunction, whereas neuronal cell death occurs late and is very selective. We show that a decreased mRNA level of the major astroglial glutamate transporter (GLT1) in the striatum and cortex of these mice is accompanied by a concomitant decrease in glutamate uptake. In contrast, the expression of the glutamate transporters, GLAST and EAAC1, remain unchanged. The mRNA level of the astroglial enzyme glutamine synthetase is also decreased. These changes in expression occur prior to any evidence of neurodegeneration and suggest that a defect in astrocytic glutamate uptake may contribute to the phenotype and neuronal cell death in HD.

Differential effects of corticostriatal and thalamostriatal deafferentation on expression of the glutamate transporter GLT1 in the rat striatum.

This study compared the effects of the disruption of the two main presumably glutamatergic striatal inputs, the corticostriatal and thalamostriatal pathways, on GLT1 expression in the rat striatum, using in situ hybridization and immunohistochemistry. Unilateral ibotenate-induced thalamic lesion produced no significant changes in striatal GLT1 mRNA labeling and immunostaining as assessed at 5 and 12 days postlesion. In contrast, significant increases in both parameters were measured after bilateral cortical lesion by superficial thermocoagulation. GLT1 mRNA levels increased predominantly in the dorsolateral part of the striatum; there, the increases were significant at 5 (+84%), 12 (+101%), and 21 (+45%) but not at 35 days postlesion. GLT1 immunostaining increased significantly and homogeneously by 17-26% at 12 and 21 days postlesion. The increase in GLT1 expression at 12 days postlesion was further confirmed by western blot analysis; in contrast, a 36% decrease in glutamate uptake activity was measured at the same time point. These data indicate that striatal GLT1 expression depends on corticostriatal but not thalamostriatal innervation. Comparison of our results with previous data showing that cortical lesion by aspiration downregulates striatal GLT1 expression further suggests that differential changes in GLT1 expression, and thus presumably in glial cell function, may occur in the target striatum depending on the way the cortical neurons degenerate.

Characterization of striatal lesions produced by glutamate uptake alteration: cell death, reactive gliosis, and changes in GLT1 and GADD45 mRNA expression.

This study investigated the time course of the striatal lesions produced by continuous local injection of the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (PDC) at the rate of 25 nmol/h in rats. The extent of the neurodegeneration area (defined as the lesion area) did not significantly vary with the duration of the PDC treatment between 3 and 14 days, but was markedly reduced 3 months after cessation of the 14-day treatment, probably reflecting striatal atrophy. After the 3-day treatment, the lesion zone showed calcium precipitates and marked microglial reaction contrasting with the reduction of astroglial labeling and loss of the glutamate transporter GLT1 mRNA expression; however reactive astrocytes were observed around the lesion. After the 14-day treatment, the lesion zone presented reactive astrocytes and microglia without calcification, and a partial recovery of GLT1 mRNA expression. Interestingly, the growth arrest DNA damage-inducible GADD45 mRNA expression was induced around the lesion after 3 days but inside the lesion after 14 days of treatment. Three months after the 14-day treatment, the astroglial reactivity persisted within the lesion whereas most of the other markers examined tended to normalize. These data suggest that defective glutamate transport induces primary death of neurons and dysfunction of astrocytes. They strongly implicate reactive astrocytes with GLT1 and GADD45 transcripts in preventing secondary neuronal death.

Ultrastructural and metabolic changes in the neuropeptide Y-containing striatal neuronal network after thermocoagulatory cortical lesion in adult rat.

This study examined the effects of unilateral thermocoagulatory cortical lesion on the pattern of neuropeptide Y immunostaining in the rat ipsilateral striatum at 4 and 21 days post-lesion. Light microscopic analysis showed a significant increase in the number of neuropeptide Y-positive neurons vs. control at both time points; paradoxically, the intraneuronal level of labelling significantly decreased at 4 days post-lesion but increased at 21 days post-lesion. Ultrastructural analysis in control condition showed a higher proportion of dendritic versus axonal labelled processes (3.5 ratio); all the neuropeptide Y synaptic terminals formed symmetrical contacts, mostly onto unlabelled dendrites. At 4 days post-lesion, the neuropeptide Y-positive axon density dramatically increased (+576%) without significant change in the labelled dendrite density, vs. control values; the density of neuropeptide Y synaptic terminals increased in parallel by 233%. In addition, a significant proportion of large neuropeptide Y boutons forming asymmetrical synapses onto unlabelled spines were observed. At 21 days post-lesion, densities of neuropeptide Y dendrites, axons, and synaptic terminals increased by 68, 246 and 125%, respectively, vs. control. But, the morphological features of the neuropeptide Y axonal processes and synaptic specializations of the boutons were similar to those observed in control condition. These data (1) raise an important issue regarding the origin of the terminals forming asymmetrical synapses in the striatum, (2) suggest that adaptative changes in the neuropeptide Y neuronal network may be a main component of striatal remodelling resulting from the progressive loss of cortical inputs, and (3) reinforce the view that neuropeptide Y and excitatory amino acid functions may be tightly linked in the striatum.

The "orphan" Na+/Cl(-)-dependent transporter, Rxt1, is primarily localized within nerve endings of cortical origin in the rat striatum.

Previous studies have shown that the striatum expresses very low levels of Na+/Cl(-)-dependent "orphan" transporter Rxt1 transcripts but contains high levels of protein. This study investigated the origin of Rxt1 expression in rat striatum. Striatal Rxt1 contents assessed by immunocytochemistry or western blotting were found to be significantly reduced after corticostriatal denervation but not after striatal or thalamic lesion with kainic acid or selective 6-hydroxydopamine-induced nigrostriatal deafferentation. Corticostriatal neurons retrogradely labeled by intrastriatal fluorogold injections were shown to express Rxt1 mRNA. Combination of anterograde biotin-dextran amine labeling of the corticostriatal pathway with Rxt1 immunogold detection at the ultrastructural level demonstrated the presence of Rxt1 in about one-third of the corticostriatal synaptic terminals and in numerous unidentified synaptic terminals. All the Rxt1-positive terminals formed asymmetrical contacts on spines. These data provide evidence that striatal Rxt1 immunoreactivity is mainly of extrinsic origin and more specifically associated with the corticostriatal pathway. Rxt1 appears as a selective presynaptic marker of synapses formed by presumably excitatory amino acid afferents, but it segregates a subclass of these synapses, thereby revealing a functional heterogeneity among excitatory amino acid systems.

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex.

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Relationships between striatin-containing neurons and cortical or thalamic afferent fibres in the rat striatum. An ultrastructural study by dual labelling.

Striatin, a recently isolated rat brain calmodulin-binding protein belonging to the WD-repeat protein family, is thought to be part of a calcium signal transduction pathway presumably specific to excitatory synapses, at least in the striatum. This study was aimed to specify the cellular and subcellular localization of striatin, and to determine the possible synaptic relationships between the two main excitatory afferent pathways, arising from the cerebral cortex and the thalamus, and the striatin-containing elements, in the rat striatum. Anterograde tract-tracing by means of biotinylated dextran amine injection in the frontoparietal cerebral cortex or the parafascicular nucleus of the thalamus was combined with immunogold detection of striatin. Striatin-immunoreactivity was confined to the neuronal somatodendritic compartment, including spines. Whereas 90-95% of the striatal neurons were striatin-positive, only about 50% of the sections of dendritic spines engaged in asymmetrical synaptic contacts exhibited striatin labelling. Among the sections of striatin-immunopositive dendritic spines, the number of immunogold particles ranged from one to more than seven, indicating an heterogeneity of the spine labelling. Moreover, within each class of spines presenting at least two silver-gold particles, the distribution of the particles varied from a clear-cut alignment under the postsynaptic densities (24-33% of spines) to a location distant from the synaptic area. In the cell bodies and dendrites, striatin labelling was usually not associated with the cytoplasmic membrane nor with the postsynaptic densities. In the striatum ipsilateral to the tracer injections, only 34.8% of the synaptic contacts formed by corticostriatal afferents involved striatin-positive elements (slightly labelled dendritic spines), whereas 56.7% of the synaptic contacts formed by thalamostriatal boutons were made on striatin-positive targets (mostly dendrites). In both cases, striatin labelling was usually not associated with the postsynaptic density. Most of the immunoreactive dendritic spines were in contact with unidentified afferents. These data reveal that striatin is expressed in the vast majority of the cell bodies of striatal spiny neurons, but is heterogeneously distributed among the dendritic spines of those neurons. Data also indicate a preferential relationship between striatin-containing structures and afferents from the parafascicular thalamic nucleus with respect to the frontoparietal cerebral cortex. But, at the dendritic spine level, striatin may be involved in signal transduction mechanisms involving as yet unidentified excitatory afferents to striatal neurons.

Continuous administration of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate produces striatal lesion.

This study examined the effects of chronic intrastriatal infusion of L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a selective competitive inhibitor of high affinity glutamate transport systems, via osmotic minipumps in rats. Injection of PDC at the rate of 25 nmol/h for 14 days caused striatal lesion. Histological evaluation on frontal striatal sections showed that the lesion was circumscribed to a circular area showing a dramatic neuronal loss accompanied by gliosis and representing 30% of the whole striatal surface at the level of the injection site. A total loss of neurons expressing glutamate decarboxylase (GAD67), enkephalin or substance P mRNA was observed on a similar circular area, suggesting degeneration of the two populations of striatal efferent neurons. In the whole striatum outside the region devoided of hybridization signal, a selective 27% decrease in enkephalin mRNA expression occurred, suggesting a higher sensitivity of enkephalin neurons versus substance P neurons to glutamate uptake-mediated alterations. Injection of PDC at the rate of 25 nmol/h for 3 days produced striatal lesion of similar extent. In contrast, PDC at the rate of 5 nmol/h did not produce neuronal damage when administered over 14 days. This study provides new in vivo evidence that defective glutamate transport is one of the critical conditions that may give rise to toxicity of an endogenous transmitter system in the striatum, and may underlie neuronal death in neurodegenerative diseases.

Repeated injections of dizocilpine maleate (MK-801) do not suppress the effects of nigrostriatal dopamine deafferentation on glutamate decarboxylase (GAD67) mRNA expression in the adult rat striatum.

The present study examined the effects of glutamate transmission blockade through N-methyl-D-aspartate (NMDA) receptor subtype by repeated administration of dizocilpine maleate (0.2 mg/kg. i.p., twice a day for eight days) alone or in combination with unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic pathway on GABAergic neurons in the adult rat striatum. For this purpose, the expression of the messenger RNA encoding for the 67 kDa isoform of the GABA synthesizing enzyme, glutamate decarboxylase (GAD67 mRNA), was studied in the various conditions by quantitative in situ hybridization. The dizocilpine maleate treatment alone did not induce significant change of GAD67 mRNA levels in the striatum, indicating that NMDA receptors may not have a major role in the transcriptional regulation of GAD67 in the adult rat striatum. As reported previously, the unilateral dopaminergic lesion resulted in marked increases in GAD67 mRNA levels in the ipsilateral striatum. This up-regulation was not significantly affected by the treatment with dizocilpine maleate started 12 days after the unilateral intranigral 6-hydroxydopamine injection. Therefore, NMDA receptors are unlikely to contribute to the dopamine lesion-induced GAD67 mRNA up-regulation in striatal projection neurons. This result is of major interest in comparison with our previous finding that NMDA receptor activation is necessary to maintain the up-regulation of enkephalin expression in the striatum after dopamine lesion.

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats.

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (10(-3) or 4 x 10(-3) M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K+ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine) (L-CCG) (10(-3) M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1-1000 microM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.

Bilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopamine pathway reproduces the effects of unilateral lesion on substance P but not on enkephalin expression in rat basal ganglia.

This study compared the effects of unilateral and bilateral 6-hydroxydopamine-induced lesions of the nigrostriatal dopaminergic neurons on substance P and enkephalin expression in the rat striatum and its main target structures by means of quantitative in situ hybridization and immunocytochemistry. In animals with bilateral lesion, substance P mRNA levels were decreased in the striatum, and this was matched by parallel reductions in substance P immunoreactivity in the striatum and in the striatonigral terminals at substantia nigra level in both hemispheres. These changes were similar to those observed ipsilaterally to unilateral lesion. In contrast, whereas increased striatal enkephalin immunoreactivity and mRNA levels and decreased immunoreactivity in the globus pallidus were observed on the lesioned side after unilateral lesion, no significant change in these enkephalin markers occurred in animals with bilateral lesion. These data suggest that the effects of dopamine deafferentation on substance P expression in the striatonigral system may be due primarily to removal of direct dopamine influence, whereas the effects on enkephalin expression in the striatopallidal system may involve complex interhemispheric adaptive mechanisms. The present finding that bilateral dopamine lesion does not simply reproduce the effects of unilateral lesion but creates a new functional state may have a critical bearing on the understanding and treatment of Parkinson's disease.

Striatal neuropeptide Y neurones are not a target for thalamic afferent fibres.

This study examined at the ultrastructural level the putative relationships between afferent fibres coming from the parafascicular nucleus of the thalamus and neuropeptide Y (NPY)-containing neurones in the rat striatum. Experiments used a combination of anterograde transport of the biotin dextran amine to label the thalamo-striatal pathway and immunogold labelling to reveal the NPY-containing neurones at the electron microscopic level. Examination of sections from three animals failed to demonstrate thalamic terminals in synaptic contact with NPY-immunoreactive dendrites or cell bodies, although both types of labelled elements were frequently involved in synaptic complex with unlabelled profiles. These results strongly suggest that striatal NPY interneurones are not under the direct influence of the main component of the thalamo-striatal system.

Activation of the adenylate cyclase-dependent protein kinase pathway increases high affinity glutamate uptake into rat striatal synaptosomes.

This study examined the effects of various agents known to alter protein phosphorylation through protein kinase A or C on high affinity glutamate uptake measured in vitro on rat striatal homogenates. Incubation of synaptosomes with the phosphatase inhibitor, okadaic acid, dramatically increased glutamate uptake indicating that underlying phosphorylation mechanisms may be involved in the regulation of this transport process. The protein kinase C activator, phorbol-12,13-dibutyrate, or inhibitor, staurosporine, did not significantly modify glutamate uptake. In contrast, forskolin, which activates adenylate cyclase, induced a dose-dependent increase in glutamate uptake. Saturation kinetic analysis indicated that forskolin increased the Vmax without modifying the Km of the transport process as compared to control values. The effect of forskolin was mimicked by dibutyryl adenosine monophosphate, an analog of cAMP which activates protein kinase A, and counteracted by high doses of N-[2-(methylamino) ethy1]-5-isoquinoline sulfonamide, a protein kinase A inhibitor. These results suggest that an adenylate cyclase-dependent protein kinase may be involved in the post-translational regulation of glutamate transporters.

Chronic dizocilpine maleate (MK-801) treatment suppresses the effects of nigrostriatal dopamine deafferentation on enkephalin but not on substance P expression in the rat striatum.

The present study examined the effects of chronic treatment with dizocilpine maleate (0.2 mg/kg i.p., twice a day for 8 days) alone or in combination with unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic neurons on substance P and enkephalin expression in the rat striatum. This was done by means of quantitative in situ hybridization histochemistry and immunocytochemistry. As reported previously, the unilateral dopaminergic lesion resulted in marked decreases in substance P mRNA expression and immunoreactivity in the ipsilateral striatum while enkephalin mRNA expression and Met-enkephalin immunoreactivity were considerably increased in this structure. Blockade of NMDA receptors by chronic dizocilpine maleate treatment alone resulted in decreased levels of striatal substance P mRNA without significant change in substance P immunoreactivity versus controls. Enkephalin mRNA levels were also decreased in the striatum, matched by parallel reductions in Met-enkephalin immunoreactivity. These observations indicate that NMDA receptor activity may exert tonic excitatory effects on substance P and enkephalin expression in the striatum. The same chronic treatment with dizocilpine maleate started 12 days after the 6-hydroxydopamine injection suppressed the lesion-induced up-regulation of enkephalin expression without significantly affecting the down-regulation of substance P expression. These data provide evidence that NMDA receptor-mediated mechanisms contribute to the alteration of striatal enkephalin expression associated with dopaminergic depletion in hemiparkinsonian rat models.

Effects of ionotropic excitatory amino acid receptor antagonists on glutamate transport and transport-mediated changes in extracellular excitatory amino acids in the rat striatum.

This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or DL-2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10-500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the Vmax of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10(-5) M) in the dialysis probe diminished by approximately 30-40% the increases in the concentrations of glutamate and aspartate elicited by L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.