The nucleus accumbens 5-HTR4-CART pathway ties anorexia to hyperactivity.

In mental diseases, the brain does not systematically adjust motor activity to feeding. Probably, the most outlined example is the association between hyperactivity and anorexia in Anorexia nervosa. The neural underpinnings of this 'paradox', however, are poorly elucidated. Although anorexia and hyperactivity prevail over self-preservation, both symptoms rarely exist independently, suggesting commonalities in neural pathways, most likely in the reward system. We previously discovered an addictive molecular facet of anorexia, involving production, in the nucleus accumbens (NAc), of the same transcripts stimulated in response to cocaine and amphetamine (CART) upon stimulation of the 5-HT(4) receptors (5-HTR(4)) or MDMA (ecstasy). Here, we tested whether this pathway predisposes not only to anorexia but also to hyperactivity. Following food restriction, mice are expected to overeat. However, selecting hyperactive and addiction-related animal models, we observed that mice lacking 5-HTR(1B) self-imposed food restriction after deprivation and still displayed anorexia and hyperactivity after ecstasy. Decryption of the mechanisms showed a gain-of-function of 5-HTR(4) in the absence of 5-HTR(1B), associated with CART surplus in the NAc and not in other brain areas. NAc-5-HTR(4) overexpression upregulated NAc-CART, provoked anorexia and hyperactivity. NAc-5-HTR(4) knockdown or blockade reduced ecstasy-induced hyperactivity. Finally, NAc-CART knockdown suppressed hyperactivity upon stimulation of the NAc-5-HTR(4). Additionally, inactivating NAc-5-HTR(4) suppressed ecstasy's preference, strengthening the rewarding facet of anorexia. In conclusion, the NAc-5-HTR(4)/CART pathway establishes a 'tight-junction' between anorexia and hyperactivity, suggesting the existence of a primary functional unit susceptible to limit overeating associated with resting following homeostasis rules.

Plasma membrane expression of the neuronal glutamate transporter EAAC1 is regulated by glial factors: evidence for different regulatory pathways associated with neuronal maturation.

At the glutamatergic synapse the neurotransmitter is removed from the synaptic cleft by high affinity amino acid transporters located on neurons (EAAC1) and astrocytes (GLAST and GLT1), and a coordinated action of these cells is necessary in order to regulate glutamate extracellular concentration. We show here that treatment of neuronal cultures with glial soluble factors (GCM) is associated with a redistribution of EAAC1 and GLAST to the cell membrane and we analysed the effect of membrane cholesterol depletion on this regulation. In enriched neuronal culture (90% neurons and 10% astrocytes), GCM treatment for 10 days increases EAAC1 and GLAST cell surface expression with no change in total expression. In opposite, GLT1 surface expression is not modified by GCM but total expression is increased. When cholesterol is acutely depleted from the membrane by 10 mM methyl-beta-cyclodextrin (beta5-MCD, 30 min), glutamate transport activity and cell surface expressions of EAAC1 and GLAST are decreased in the enriched neuronal culture treated by GCM. In pure neuronal culture addition of GCM also increases EAAC1 cell membrane expression but surprisingly acute treatment with beta5-MCD decreases glutamate uptake activity but not EAAC1 cell membrane expression. By immunocytochemistry a modification in the distribution of EAAC1 within neurons was undetectable whatever the treatment but we show that EAAC1 was no more co localized with Thy-1 in the enriched neuronal culture treated by GCM suggesting that GCM have stimulated polarity formation in neurons, an index of maturation. In conclusion we suggest that different regulatory mechanisms are involved after GCM treatment, glutamate transporter trafficking to and from the plasma membrane in enriched neuronal culture and modulation of EAAC1 intrinsic activity and/or association with regulatory proteins at the cell membrane in the pure neuronal culture. These different regulatory pathways of EAAC1 are associated with different neuronal maturation stages.

The metabotropic glutamate receptor subtype 5 antagonist MPEP and the Na+ channel blocker riluzole show different neuroprotective profiles in reversing behavioral deficits induced by excitotoxic prefrontal cortex lesions.

Overactivation of excitatory amino acid receptors has been involved in several neurodegenerative diseases. The present study aims at investigating the potential neuroprotective action of 2-methyl-6-(phenylethylnyl)-pyridine (MPEP), a selective non-competitive antagonist of metabotropic glutamate receptor subtype 5, and 2-amino-6-trifluoro methoxy-benzothiole (riluzole), a Na+ channel blocker exhibiting anti-glutamatergic properties, on the ibotenate-induced damage to the rat medial prefrontal cortex. The neuroprotective efficacy of these compounds was assessed on the recovery from behavioral deficits induced by prefrontal cortical excitotoxic lesions in a reaction time task. MPEP (3, 10 or 30 mg/kg) or riluzole (2, 4 or 8 mg/kg) was administered i.p. 30 min before and after medial prefrontal cortex lesions. As previously found, lesions to the medial prefrontal cortex significantly altered the motor preparatory processes involved in the reaction time task. These deficits were prevented by MPEP 3 mg/kg and riluzole 2 mg/kg while higher doses of either compound were ineffective. Furthermore, the neuron-specific nuclear protein immunostaining of the lesioned cortical area in animals treated with the efficient dose of either compound revealed that MPEP reduced the volume of the lesion whereas riluzole reversed the decrease of neuronal density within the lesioned area. Altogether, these results suggest a neuroprotective action of MPEP as well as riluzole at both behavioral and cellular levels on excitatory amino acid-induced toxicity.

Neuroprotective effects of tacrolimus (FK506) in a model of ischemic cortical cell cultures: role of glutamate uptake and FK506 binding protein 12 kDa.

BACKGROUND: The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection. METHODS: Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation. RESULTS: When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50-500 pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects. CONCLUSION: The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.

[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.

Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures.

This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol.

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.

Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures.

The expression and activity of glutamate transporters (EAAC1, GLAST and GLT1) were examined during the development of cortical neuron-enriched cultures. Protein content and mitochondrial respiration both increased during the first 7 days, later stabilized and decreased from DIV14. Glutamate transport and extracellular concentration were relatively constant from DIV3 to 18. The kinetic parameters of glutamate transport were at DIV7: K(m)=19+/-3 microM and V(max)=1068+/-83 pmol/mg protein/min and at DIV14: K(m)=40.8+/-9.3 microM and V(max)=1060+/-235 pmol/mg protein/min. The shift in K(m) towards higher values suggest a more important participation of GLAST after DIV14. At DIV7 and 14, glutamate transport was poorly sensitive to dihydrokaïnate (DHK) suggesting a weak participation of GLT1 in glutamate transport. Western blot experiments and immunocytochemistry showed that EAAC1 was expressed by neurons whatever the stage of the culture. GLAST was found in astrocytes as soon as DIV3 and labeling increased during the development of the culture. There was little neuronal GLT1 immunoreactivity at DIV7, only detected by immunocytochemistry. From DIV10 to 18, an increasing astrocytic expression of GLT1 was observed, also detected by Western blotting. These results show that: (1) glutamate uptake remains stable all along the development of the cultures although the pattern of expression of the different transporters is changing, suggesting that glutamate transport is highly regulated; (2) neuronal EAAC1 may play a critical role during the early stages of the culture when it is expressed alone; and (3) the developmental expression pattern of glutamate transporters in cortical neuron-enriched cultures is quite similar to that observed in vivo during early postnatal development.

[Brain dopamine receptors: molecular aspects and functional implications] / Les récepteurs dopaminergiques: aspects structuraux et implications fonctionnelles.

During the last decade, molecular biology methods have primarily contributed to the fine characterisation of five subtypes of dopaminergic receptors leading to the classification of the D1-like (D1 and D5) and D2-like (D2, D3 and D4) subclasses. Biochemical methods have provided evidences for a large diversity of transduction mechanisms associated to these different receptors which can contribute to explain the multiple actions of dopamine at cellular level in the brain, from changes in membrane excitability to influences of gene expression. Moreover, the characteristic intracerebral distribution of these receptors subtypes supports the involvement of the neurotransmitter in different aspects of behaviour, from motor to limbic and cognitive aspects. In this respect, in Parkinson's disease dopamine depletion will favour adaptive responses at the cellular level which likely involve differentially the dopaminergic receptor subtypes. Such adaptive responses involving long-lasting processes of intercellular communication will either act to limit the symptoms of the disease or, more likely, to contribute to the expression of clinical signs. The development of appropriate agonists to selectively stimulate the different subsets of receptors still remains a challenge but, actually, already contributes to efficiently compensate for the motor signs of Parkinson's disease. In any case, such dopaminergic agonists are considered as an essential strategy to limit the motor side effects of L-DOPAtherapy. Thus, the development of more selective agonists of the different subsets of the dopamine receptors may contribute to compensate for the other clinical signs of the disease, and particularly for cognitive deficits.

[Dopaminergic receptors: structural features and functional implications]. / Les récepteurs dopaminergiques: aspects structuraux et implications fonctionnelles.

During the last decade, molecular biology methods have primarily contributed to the fine characterisation of five subtypes of dopaminergic receptors leading to the classification of the D1-like (D1 and D5) and D2-like (D2, D3 and D4) subclasses. Biochemical methods have provided evidences for a large diversity of transduction mechanisms associated to these different receptors which can contribute to explain the multiple actions of dopamine at cellular level in the brain, from changes in membrane excitability to influences of gene expression. Moreover, the characteristic intracerebral distribution of these receptors subtypes supports the involvement of the neurotransmitter in different aspects of behaviour, from motor to limbic and cognitive aspects. In this respect, in Parkinson's disease dopamine depletion will favour adaptive responses at the cellular level which likely involve differentially the dopaminergic receptor subtypes. Such adaptive responses involving long-lasting processes of intercellular communication will either act to limit the symptoms of the disease or, more likely, to contribute to the expression of clinical signs. The development of appropriate agonists to selectively stimulate the different subsets of receptors still remains a challenge but, actually, already contributes to efficiently compensate for the motor signs of Parkinson's disease. In any case, such dopaminergic agonists are considered as an essential strategy to limit the motor side effects of L-DOPAtherapy. Thus, the development of more selective agonists of the different subsets of the dopamine receptors may contribute to compensate for the other clinical signs of the disease, and particularly for cognitive deficits.

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.

[Cellular bases of neurodegenerative processes]. / Bases cellulaires des processus neurodégénératifs.

Neurodegenerative processes are generally characterized by the long-lasting course of neuronal death and the selectivity of the neuronal population or brain structure involved in the lesion. This is the case for Alzheimer's, Parkinson's or Huntington's diseases, or amyotrophic lateral sclerosis (ALS). The reasons for such a specificity are largely unknown as are generally the mechanisms of the diseases. One common feature of these diseases, however, is that the neuronal death is thought to involve apoptosis, at least partly. Interestingly, apoptosis in the brain would involve specific gene products similar to that identified in the nematode c. elegans, partly corresponding in mammals to ICE-related compounds and Bcl2 protein. The involvement of calcium as well as of oxydative stress mechanisms in such neuronal death is to be fully proved but putative modulation by external signals (such as those provided through trophic factors or even neurotransmitters) represents an interesting way to validate the current hypothesis of neuronal death in neurodegenerative diseases in humans.

Impaired performance in a conditioned reaction time task after thermocoagulatory lesions of the fronto-parietal cortex in rats.

The present study examined whether cortical damage in rats may disrupt the integrative processes and motor control involved in the performance of a reaction time (RT) task. To investigate the nature of the deficits in the conditioned task, rats were subjected, after learning, to a coagulation of pia brain surface of varying extent, including the frontal and parietal cortical areas. They were then tested daily for over one month. The behavioural task required the rats to hold a lever down during a variable and random delay and react quickly to the onset of a visual cue by releasing the lever within a RT limit for food reinforcement. Extensive bilateral cortical lesions had no effect on spontaneous motor activity, but severely impaired RT performance. Latencies to release the lever after the cue were dramatically increased during the first postoperative sessions and gradually returned to baseline levels within 3 weeks, whereas less dramatic but long-lasting increase in premature responding (anticipatory response before the visual cue) was observed throughout the testing sessions. More restricted lesions to the frontoparietal cortex produced a similar pattern of incorrect responding with a faster recovery of delayed responses and a strong deficit in premature responding. The major effects of lesions confined to the rostral pole of the frontal cortex were observed on premature responding, however. The present results demonstrate that the impairment in movement initiation is rapidly recovered within 2-3 weeks even after extensive thermocoagulatory lesions of the frontal and parietal areas. This recovery suggests the involvement of adaptive processes developing progressively and probably reflecting the remarkable synaptic plasticity of the extrapyramidal motor output. In contrast, the long-lasting increase in premature responding, supposed to reflect some attentional deficits, may produce anatomofunctional long-term disorganization of subcortical structures such as the basal ganglia. Interestingly enough, these results show that the rat neocortex supports functions very similar to those of primates and provide a good model for studying these higher functions in operant motor procedures that require prior associative learning and appropriate motor coordination.

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.

Differential regional effects of long-term L-DOPA treatment on preproenkephalin and preprotachykinin gene expression in the striatum of 6-hydroxydopamine-lesioned rat.

The present study examined the effects of prolonged L-DOPA treatment (6 months) alone or in combination with unilateral 6-hydroxydopamine-induced lesion of the mesostriatal dopaminergic pathway on substance P and enkephalin mRNA expression in the rat neostriatum. This was done by means of quantitative in situ hybridization histochemistry. As reported previously, the unilateral dopaminergic lesion induced a significant and homogeneous decrease in striatal substance P mRNA expression and a marked increase in enkephalin mRNA expression in the ipsilateral neostriatum which was more pronounced in the dorsolateral than ventromedial part of the structure. Long-term L-DOPA treatment alone had no significant effects on the two striatal peptide mRNA levels. The chronic L-DOPA treatment in 6-hydroxydopamine-lesioned rats was found to partially reverse the lesion-induced down-regulation of substance P mRNA expression, without significantly affect the up-regulation of enkephalin when considering the neostriatum as a whole. Topographical analysis revealed that long-term L-DOPA treatment reversed, in fact, both post-lesional enkephalin and substance P responses to 6-hydroxydopamine lesion, in the ventromedial neostriatum, without significantly modified these peptide responses in the dorsolateral neostriatum. These findings provide new evidence that prolonged L-DOPA treatment differentially affects the post-lesional peptide responses in the ventromedial and dorsolateral parts of the neostriatum, suggesting regional cellular mechanisms in the neostriatum underlying the benefit and/or side-effects of L-DOPA treatment in parkinsonian patients.

The contralateral cortex contributes to the effects of hemidecortication on neuropeptide Y immunoreactivity in the rat striatum.

We have previously shown that unilateral lesion by thermocoagulation of sensori-motor cortex which provides excitatory afferents to the striatum increases the number of neuropeptide Y (NPY)-immunoreactive neurons in the rat striatum. The present study examined whether this paradoxical effect is due to adaptive neuronal mechanisms involving the crossed projections from the contralateral spared cortex. To test this hypothesis, we compared the effects of unilateral and bilateral cortical lesions on the number of NPY-immunoreactive neurons in the striatum. Results showed that animals with bilateral lesion have no significant change in NPY immunoreactivity versus control suggesting that the contralateral intact cortex is responsible for the increase of NPY-immunoreactive neurons detected after unilateral 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.

Opposite changes in striatal neuropeptide Y immunoreactivity after partial and complete serotonergic depletion in the rat.

The aim of this study was to investigate the consequences of partial vs. complete serotonergic (5-HT) depletions on the immunoreactivity of striatal interneurons containing neuropeptide Y (NPY). Taking into account the plasticity of the monoaminergic neurons, the effects of various doses of 5,7-dihydroxytryptamine (5,7-DHT) injected into the anterior raphe nuclei and P-chlorophenylalanine (PCPA) administration were compared in the dorsal (caudate-putamen) and the ventral (nucleus accumbens) striatum. Twenty days after administering 5,7-DHT injections inducing a substantial but partial decrease in the striatal 5-HT concentrations (about 80%), we detected a significant decrease in the number of NPY immunoreactive cells. In contrast, the PCPA inhibition of serotonin synthesis in the neurons spared by the partial lesion or the near-complete neurotoxic lesion induced an increase in the number of striatal NPY neurons. These results suggest that complex adaptive mechanisms are probably responsible for the changes in striatal NPY reactivity observed after a partial lesion and that these neurons can adapt according to the extent of 5-HT depletion. Upon comparing the NPY responses in the dorsal and ventral components of the striatal complex, no main differences were observed; while in the caudate-putamen, the changes were primarily found to occur in the medial zone. This finding is discussed here with reference to the topographical effects of dopaminergic or glutamatergic deafferentation. Finally, these results suggest that a complete interruption of the 5-HT transmission may lead to an increase in the intracellular NPY level, which may be associated with a decrease in the release of the peptide. It can therefore be postulated that serotonergic neurons normally exert a positive influence on NPY striatal neurons.

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.