Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities.

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV's putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV(-/-)) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV(+/-)) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV(-/-) and PV(+/-) mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

A role for Sv2c in basal ganglia functions.

SV2C is an isoform of the synaptic vesicle 2 protein family that exhibits a particular pattern of brain expression with enriched expression in several basal ganglia nuclei. In the present study, we have investigated SV2C implication in both normal and pathological basal ganglia functioning with a peculiar attention to dopamine neuron containing regions. In SV2C-/- mice, the expression of tyrosine hydroxylase mRNA in midbrain dopaminergic neurons was largely and significantly increased and enkephalin mRNA expression was significantly decreased in the caudate-putamen and accumbens nucleus. The expression of SV2C was studied in two models of dopaminergic denervation (6-OHDA- and MPTP-induced lesions). In dopamine-depleted animals, SV2C mRNA expression was significant increased in the striatum. In order to further understand the role of SV2C, we performed behavioral experiments on SV2C-/- mice and on knock-down mice receiving an injection of adeno-associated virus expressing SV2C miRNA specifically in the ventral midbrain. These modifications of SV2C expression had little or no impact on behavior in open field and elevated plus maze. However, even if complete loss of SV2C had no impact on conditioned place preference induced by cocaine, the specific knock-down of SV2C expression in the dopaminergic neurons completely abolished the development of a CPP while the reaction to an acute drug injection remains similar in these mice compared to control mice. These results showed that SV2C, a poorly functionally characterized protein is strongly involved in normal operation of the basal ganglia network and could be also involved in system adaptation in basal ganglia pathological conditions.

Control of neuronal excitability by calcium binding proteins: a new mathematical model for striatal fast-spiking interneurons.

Calcium binding proteins, such as parvalbumin (PV), are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Notably, at the level of the striatum, where PV is only expressed in the fast-spiking (FS) interneurons. FS interneurons form an inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN). So far the existing conductance-based computational models for FS neurons did not allow the study of the coupling between PV concentration and electrical activity. In the present paper, we propose a new mathematical model for the striatal FS interneurons that includes apamin-sensitive small conductance Ca(2+)-dependent K(+) channels (SK) and the presence of a calcium buffer. Our results show that a variation in the concentration of PV can modulate substantially the intrinsic excitability of the FS interneurons and therefore may be involved in the information processing at the striatal level.

Distribution of SV2C mRNA and protein expression in the mouse brain with a particular emphasis on the basal ganglia system.

Synaptic vesicle 2 proteins (SV2), SV2A, SV2B and SV2C, are integral proteins localized on the surface of synaptic vesicles in all neurons. SV2 proteins appear to play an important, but not yet fully understood role in synaptic vesicle exocytosis and neurotransmitter release. Moreover, SV2 seems to be the receptor of the botulinum neurotoxin A. In the present study, using single and double-labeling fluorescent immunohistochemistry and in situ hybridization we have identified the brain pattern of SV2C mRNA and protein expression in mice. Our results indicated that SV2C protein was expressed in a small subset of brain regions including the olfactory bulb, olfactory tubercle, nucleus accumbens, caudate-putamen, ventral pallidum, globus pallidus, substantia nigra and the ventral tegmental area. These results were confirmed by means of in situ hybridization, except for the globus pallidus and the substantia nigra pars reticulata, in which no labeling was found, suggesting that SV2C-positive fibers in these areas are terminals of striatal projecting neurons. In the striatum, we found that, in addition to its presence in the projection neurons, SV2C was densely expressed in a fraction (around 45%) of cholinergic interneurons. In addition, our data also showed that SV2C was densely expressed in most dopaminergic neurons in the substantia nigra pars compacta and the ventral tegmental area (more than 70% of the dopaminergic neurons analyzed were SV2C-positive). Altogether, our results suggest that SV2C may contribute to the regulation of neurotransmitter release and synaptic transmission in the basal ganglia including cholinergic striatal interneurons and nigro-striatal/mesolimbic dopamine neurons.

[Changes in neural networks by conditional transgenic approach: a key to our comprehension of neuro-psychiatric disorders in the basal ganglia system]. / Modifications des réseaux neuronaux par transgénèse conditionnelle: une clé pour notre compréhension des pathologies neuro- psychiatriques du système des noyaux de la base.

The striatum, the first relay of the basal ganglia system, is critically involved in motor functions and motivational processes. The dorsal striatum is central to the motor control and motor learning and the ventral striatum or nucleus accumbens is essential for motivation, the reward system and reinforcement by drugs. This system is dysfunctional in movement disorders such as Parkinson's disease and Huntington's disease and in psychiatric disorders including drug addiction. The striatum consists of two populations of neurons projecting at the origin of two distinct paths in the circuit of basal ganglia, and of different populations of interneurons. These two populations of efferent neurons, striatopallidal and striatonigral neurons, are characterized by their projection sites and their differential expression in dopamine receptors and neuropeptides. Their roles in motor control and motivational processes and in the mechanisms of neuroadaptation in the system's pathologies remain mostly unknown. To identify these specific functions, we have developed new animal models wearing molecular or cell "lesions" by a conditional transgenic approach to target a specific population of neurons. By this approach, we demonstrated the inhibitory role of the population of striatopallidal neurons in the motor control and in the process of drug addiction, identified new genes selectively expressed by striatopallidal neurons that could be the target for future therapies and identified the potential role of this population of neurons disturbances in attention-deficit hyperactivity disorder (ADHD).

An update on adenosine A2A-dopamine D2 receptor interactions: implications for the function of G protein-coupled receptors.

Adenosine A(2A)-dopamine D(2) receptor interactions play a very important role in striatal function. A(2A)-D(2) receptor interactions provide an example of the capabilities of information processing by just two different G protein-coupled receptors. Thus, there is evidence for the coexistence of two reciprocal antagonistic interactions between A(2A) and D(2) receptors in the same neurons, the GABAergic enkephalinergic neurons. An antagonistic A(2A)-D(2) intramembrane receptor interaction, which depends on A(2A)-D(2) receptor heteromerization and G(q/11)-PLC signaling, modulates neuronal excitability and neurotransmitter release. On the other hand, an antagonistic A(2A)-D(2) receptor interaction at the adenylyl-cyclase level, which depends on G(s/olf)- and G(i/o)-type V adenylyl-cyclase signaling, modulates protein phosphorylation and gene expression. Finally, under conditions of upregulation of an activator of G protein signaling (AGS3), such as during chronic treatment with addictive drugs, a synergistic A(2A)-D(2) receptor interaction can also be demonstrated. AGS3 facilitates a synergistic interaction between G(s/olf) - and G(i/o)-coupled receptors on the activation of types II/IV adenylyl cyclase, leading to a paradoxical increase in protein phosphorylation and gene expression upon co-activation of A(2A) and D(2) receptors. The analysis of A(2)-D(2) receptor interactions will have implications for the pathophysiology and treatment of basal ganglia disorders and drug addiction.

Adenosine A2A receptors and basal ganglia physiology.

Adenosine A2A receptors are highly enriched in the basal ganglia system. They are predominantly expressed in enkephalin-expressing GABAergic striatopallidal neurons and therefore are highly relevant to the function of the indirect efferent pathway of the basal ganglia system. In these GABAergic enkephalinergic neurons, the A2A receptor tightly interacts structurally and functionally with the dopamine D2 receptor. Both by forming receptor heteromers and by targeting common intracellular signaling cascades, A2A and D2 receptors exhibit reciprocal antagonistic interactions that are central to the function of the indirect pathway and hence to basal ganglia control of movement, motor learning, motivation and reward. Consequently, this A2A/D2 receptors antagonistic interaction is also central to basal ganglia dysfunction in Parkinson's disease. However, recent evidence demonstrates that, in addition to this post-synaptic site of action, striatal A2A receptors are also expressed and have physiological relevance on pre-synaptic glutamatergic terminals of the cortico-limbic-striatal and thalamo-striatal pathways, where they form heteromeric receptor complexes with adenosine A1 receptors. Therefore, A2A receptors play an important fine-tuning role, boosting the efficiency of glutamatergic information flow in the indirect pathway by exerting control, either pre- and/or post-synaptically, over other key modulators of glutamatergic synapses, including D2 receptors, group I metabotropic mGlu5 glutamate receptors and cannabinoid CB1 receptors, and by triggering the cAMP-protein kinase A signaling cascade.

Modulation of neuronal excitability by intracellular calcium buffering: from spiking to bursting.

We have investigated the detailed regulation of neuronal firing pattern by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch-clamp recording in acute slice. Theoretical results show that a high calcium buffer concentration alters the characteristic regular firing of cerebellar granule cells and that a transition to various modes of oscillations occurs, including bursting. Using bifurcation analysis, we show that this transition from spiking to bursting is a consequence of the major slowdown of calcium dynamics. Patch-clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal dramatic alterations in their excitability as compared to cells loaded with 0.15 mM BAPTA. In high calcium buffering conditions, granule cells exhibit all bursting behaviors predicted by the model whereas bursting is never observed in low buffering. These results suggest that cytosolic calcium buffering capacity can tightly modulate neuronal firing patterns leading to generation of complex patterns and therefore that calcium-binding proteins may play a critical role in the non-synaptic plasticity and information processing in the central nervous system.

Mono- and dual-frequency fast cerebellar oscillation in mice lacking parvalbumin and/or calbindin D-28k.

Calbindin is a fast Ca2+-binding protein expressed by Purkinje cells and involved in their firing regulation. Its deletion produced approximately 160-Hz oscillation sustained by synchronous, rhythmic Purkinje cells in the cerebellar cortex of mice. Parvalbumin is a slow-onset Ca2+-binding protein expressed in Purkinje cells and interneurons. In order to assess its function in Purkinje cell firing regulation, we studied the firing behavior of Purkinje cells in alert mice lacking parvalbumin (PV-/-), calbindin (CB-/-) or both (PV-/- CB-/-) and in wild-type controls. The absence of either protein resulted in Purkinje cell firing alterations (decreased complex spike duration and pause, increased simple spike firing rate) that were more pronounced in CB-/- than in PV-/- mice. Cumulative effects were found in complex spike alterations in PV-/- CB-/- mice. PV-/- and CB-/- mice manifested approximately 160-Hz oscillation that was sustained by Purkinje cells firing rhythmically and synchronously along the parallel fiber axis. This oscillation was dependent on GABA(A), N-methyl-D-aspartate and gap junction transmission. PV-/- CB-/- mice exhibited a dual-frequency (110 and 240 Hz) oscillation. The instantaneous spectral densities of both components were inversely correlated. Simple and complex spikes of Purkinje cells were phase-locked to one of the two oscillation frequencies. Mono- and dual-frequency oscillations presented similar pharmacological properties. These results demonstrate that the absence of the Ca2+ buffers parvalbumin and calbindin disrupts the regulation of Purkinje cell firing rate and rhythmicity in vivo and suggest that precise Ca2+ transient control is required to maintain the normal spontaneous arrhythmic and asynchronous firing pattern of the Purkinje cells.

Role of calcium binding proteins in the control of cerebellar granule cell neuronal excitability: experimental and modeling studies.

Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Calretinin is expressed in cerebellar granule cells which provide the major excitatory input to Purkinje cells through parallel fibers. Calretinin deficient mice exhibit dramatic alterations in motor coordination and in Purkinje cell firing recorded in vivo through unknown mechanisms. In the present paper, we review the results obtained with the patch clamp recording techniques in acute slice preparation. This data allow us to investigate the effect of a null mutation of the calretinin gene on the intrinsic electroresponsiveness of cerebellar granule cells at a mature developmental stage. Calretinin deficient granule cells exhibit faster action potentials and generate repetitive spike discharge showing an enhanced frequency increase with injected currents. These alterations disappear when 0.15 mM of the exogenous fast calcium buffer BAPTA is infused in the cytosol to restore the calcium buffering capacity. Furthermore, we propose a mathematical model demonstrating that the observed alterations of granule cell excitability can be explained by a decreased cytosolic calcium buffering capacity due to the absence of calretinin. We suggest that calcium binding proteins modulate intrinsic neuronal excitability and may therefore play a role in the information processing in the central nervous system.

Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the post-natal subventricular zone.

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) where dopamine D3 receptors are expressed. Here, we demonstrate that addition of 1 microm apomorphine increases cell numbers in post-natal SVZ cell cultures. This effect was prevented by a co-treatment with haloperidol, sulpiride or U-99194A, a D3-preferring antagonist, and mimicked by the dopamine D3 receptor selective agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT). EC50 values were 4.04 +/- 1.54 nm for apomorphine and 0.63 +/- 0.13 nm for 7-OH-DPAT, which fits the pharmacological profile of the D3 receptor. D3 receptors were detected in SVZ cells by RT-PCR and immunocytochemistry. D3 receptors were expressed in numerous beta-III tubulin immunopositive cells. The fraction of apoptotic nuclei remained unchanged following apomorphine treatment, thus ruling out any possible effect on cell survival. In contrast, proliferation was increased as both the proportion of nuclei incorporating bromo-deoxyuridine and the expression of the cell division marker cyclin D1 were enhanced. These findings provide support for a regulatory role of dopamine over cellular dynamics in post-natal SVZ.

Tricyclic antidepressant imipramine reduces the insulin secretory rate in islet cells of Wistar albino rats through a calcium antagonistic action.

AIMS/HYPOTHESIS: Treatments with antidepressants have been associated with modifications in glucose homeostasis. The aim of this study was to assess the effect of imipramine, a tricyclic antidepressant, on insulin-secreting cells. METHODS: Insulin radioimmunoassay, radioisotopic, fluorimetric and patch-clamp methods were used to characterise the effects of imipramine on ionic and secretory events in pancreatic islet cells from Wistar albino rats. RESULTS: Imipramine induced a dose-dependent decrease in glucose-stimulated insulin output (IC(50)=5.2 micromol/l). It also provoked a concentration-dependent reduction in (45)Ca outflow from islets perifused in the presence of 16.7 mmol/l glucose. Moreover, imipramine inhibited the increase in (45)Ca outflow mediated by K(+) depolarisation. Patch-clamp recordings further revealed that imipramine provoked a marked and reversible decrease of the inward Ca(2+) current. In single islet cells, imipramine counteracted the rise in [Ca(2+)](i) evoked by glucose or high K(+) concentrations. CONCLUSIONS/INTERPRETATION: These data indicate that imipramine dose-dependently reduces the insulin secretory rate from rat pancreatic beta cells. Such an effect appears to be mediated by the inhibition of voltage-sensitive Ca(2+) channels with subsequent reduction in Ca(2+) entry. Thus, it is possible that some adverse effects of imipramine are related, at least in part, to its capacity to behave as a Ca(2+) entry blocker.

A2A receptor and striatal cellular functions: regulation of gene expression, currents, and synaptic transmission.

A2A receptor is highly coexpressed with enkephalin and D2 receptor in striatopallidal neurons. A2A antagonists acutely enhance motor behavior in animal models of Parkinson's disease (PD) and are therefore considered potential PD therapeutic agents. Analysis of gene expression regulation using pharmacologic tools or A2A receptor-deficient mice (A2A-/-) shows that the A2A receptor positively and tonically controls the expression of enkephalin and immediate early genes in striatopallidal neurons. Because this regulation strictly mirrors the effect of D2 receptor, these data strongly support the hypothesis that A2A antagonists reduce the activity of striatopallidal neurons in models of PD. However, analysis of A2A-/- mice suggests additional effects of A2A receptor in the control of striatal physiology. Unexpectedly, these animals exhibited a reduction in exploratory activity and a 50% reduction in substance P expression. This was associated with a 45% decrease in the striatal extracellular dopamine concentration, suggesting that chronic absence of A2A receptor results in a functional hypodopaminergic state in the striatum. The A2A receptor controls inhibitory synaptic transmission negatively in the striatum and positively in the globus pallidus; this further supports the efficacy of A2A antagonists in reducing the activity of striatopallidal neurons in PD. The A2A receptor does not modulate basal alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-mediated excitatory corticoaccumbal synaptic transmission during normal physiologic conditions. However, genetic inactivation or pharmacologic blockade of the A2A receptor significantly reduced long-term potentiation (LTP) at this synapse. Therefore, this receptor is implicated in the induction of corticoaccumbal LTP, an effect that could be related to its involvement in long-term behavioral sensitization to repeated dopaminergic treatment.

Inactivation of adenosine A2A receptor impairs long term potentiation in the accumbens nucleus without altering basal synaptic transmission.

The nucleus accumbens is considered to be critically involved in the control of complex motivated behaviors. By modulating its glutamatergic excitatory input, mesolimbic dopaminergic afferents have been implicated in the reinforcing properties of drugs of abuse. However, they might not represent the only path for influencing the accumbens output. The aim of this study was to investigate possible modulation of synaptic transmission at this glutamatergic synapse by adenosine receptors. The standard field potential recording technique was used on brain slices from wild-type and A2A receptor-deficient mice. Neither the stimulus-response relationship nor paired-pulse facilitation was altered in the mutant mice. In both genotypes, the activation of A1 receptors by 2-chloro-N6-cyclopentyladenosine reduced the field excitatory postsynaptic potential (fEPSP) slope to a similar extent. In wild-type slices, activation or blockade of A2A receptors by 2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine and 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, respectively, did not modify the synaptic transmission. Moreover, a long lasting pre-activation of these A2A receptors did not influence the A1 receptor-mediated reduction in fEPSP slope. Long term potentiation (LTP) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor-mediated synaptic transmission could be elicited in both wild-type and A2A receptor-deficient mice. However, LTP appeared to be quantitatively modulated by the A2A receptor pathway since the level of potentiation was reduced in A2A receptor-deficient mice as well as in slices of wild-type mice in which the A2A receptor pathway was blocked. The involvement of the cAMP-dependent protein kinase was supported by the reduction in potentiation level in slices of wild-type mice treated with adenosine 3',5'-cyclic monophosphorothiotate, 8-(4-chlorophenylthio)-Rp isomer, an inhibitor of this enzyme. These data provide evidence that the adenosine acting at the A2A receptor is implicated in events directly or indirectly related to LTP induction in the accumbens whereas it is not involved in the regulation of the basal AMPA receptor-mediated excitatory synaptic transmission.

Interstitial cells of Cajal in the striated musculature of the mouse esophagus.

Interstitial cells of Cajal (ICC) are important regulatory cells in the smooth muscle coats of the digestive tract. Expression of the Kit receptor tyrosine kinase was used in this study as a marker to study their distribution and development in the striated musculature of the mouse esophagus. Sections and whole-mounts were studied by immunohistochemistry. KitW-lacZ transgenic mice, which carry the lacZ reporter gene inserted in place of the first exon of the Kit gene, were processed for Xgal histochemistry, for quantitative analysis and for ultrastructural studies. Spindle-shaped ICC were scarce in both muscle layers of the thoracic esophagus, while their number increased steeply toward the cardia in the striated portion of the intraabdominal esophagus. They did not form networks and had no relationship with intrinsic myenteric ganglia and motor end-plates. They were often close to nerve fibers immunoreactive for neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) or neuropeptide Y (NPY), but not to fibers immunoreactive for substance P (SP), calcitonin gene related peptide (CGRP), enkephalin, or the capsaicin receptor VRI. They were present in the fetus but absent in adult ICC-deficient KitW-lacZ/KitWv mice. Interstitial cells of Cajal were identified by electron microscopy by their ultrastructure in the striated muscle of the esophagus and exhibited Xgal labeling, while fibroblasts and muscle cells were unlabeled. Interstitial cells of Cajal are scattered between striated muscle cells in the mouse esophagus. They are close to nerves with defined neurochemical coding and could possibly represent specialized esophageal spindle proprioceptors.

Comparative study of hippocampal neuronal loss and in vivo binding of 5-HT1a receptors in the KA model of limbic epilepsy in the rat.

A high density of 5-HT1a receptors is present in pyramidal hippocampal cells. Mapping of these receptors may be performed in vivo using the tracer no-carrier-added 4-(18)F-fluoro-N-2-(1-(2-methoxyphenyl)-1-piperazinyl)ethyl-N-2-pyridinyl-benzamide (MPPF). We tested the hypothesis of a relationship between MPPF binding and post-epileptic neuronal loss in the hippocampus. The model of limbic epilepsy induced by kainic acid (KA) in the rat was used. Rats were sacrificed at various times (1 h-240 days) after systemic injection of 10 mg/kg KA. Determination of MPPF binding in the brain was combined with a quantification of neuronal loss using DNA labeling with propidium iodide and confocal microscopy. Hippocampal MPPF binding varied according to time elapsed from KA injection. An initial decrease from day 1 to day 6 post injection was followed by a relative increase between day 6 and day 30. This effect was observed in rats which showed hippocampal neuronal loss but also in one rat which did not. In KA treated rats, statistically significant relationship between MPPF binding and neuronal count was found during the acute period (rats sacrificed 1 h-day 6 after KA injection) and the chronic phase (rats sacrificed beyond day 60 after KA injection). The late relative increase of MPPF binding suggests an epilepsy-induced increase of 5-HT1a receptors in the hippocampus. This effect needs to be further characterized before considering PET determination of hippocampal MPPF binding as a method of post-epileptic neuronal loss assessment.

Acute and chronic caffeine administration differentially alters striatal gene expression in wild-type and adenosine A(2A) receptor-deficient mice.

In order to assess for the respective involvement of adenosine A(1) and A(2A) receptors (A(2A)-R) in the consequences of short- and long-term caffeine exposure on gene expression, the effects of acute caffeine administration on striatal, cortical, and hippocampal expression of immediate early genes (IEG), zif-268 and arc, and the effects of long-term caffeine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) exposure (once daily for 15 days) on striatal gene expression of substance P, enkephalin, and glutamic acid decarboxylase isoforms, GAD65 and GAD67, were evaluated in wild-type and A(2A)-R-deficient (A(2A)-R(-/-)) mice. In situ hybridization histochemistry was performed using oligonucleotides followed by quantitative image analysis. Our results demonstrated that a biphasic response of IEG expression to acute caffeine observed in the wild-type striatum was resumed in a monophasic response in the mutant striatum. In the cerebral cortex and hippocampus, the effect of caffeine was weak in wild-type, whereas in mutant mice it induced a 2-3-fold increase in the IEG expression to restore a level similar to the wild-type basal expression. Chronic caffeine and DPCPX-mediated regulation in neuropeptide and GADs striatal gene expression typically showed the mimicking of alterations resulting from the A(2A)-R genetic deficiency in 25 mg/kg caffeine-treated wild-type mice as well as the dose-dependent normalization of substance P and enkephalin expression in A(2A)-R(-/-) mice. These results indicate that, depending on the dose, the blockade of A(2A)-R or A(1) receptors by caffeine is preferentially revealed leading to highly differential alterations in striatal gene expression and they also suggested the central role of these two receptors on the control of dopaminergic functions.

The SH2 domain-containing 5-phosphatase SHIP2 is expressed in the germinal layers of embryo and adult mouse brain: increased expression in N-CAM-deficient mice.

The germinative ventricular zone of embryonic brain contains neural lineage progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. The ability to generate neurons persists at adulthood in restricted brain areas. During development, many growth factors exert their effects by interacting with tyrosine kinase receptors and activate the phosphatidylinositol 3-kinase and the Ras/MAP kinase pathways. By its ability to modulate these pathways, the recently identified Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2, SHIP2, has the potential to regulate neuronal development. Using in situ hybridization technique with multiple synthetic oligonucleotides, we demonstrated that SHIP2 mRNA was highly expressed in the ventricular zone at early embryonic stages and subventricular zones at latter stages of brain and spinal cord and in the sympathetic chain. No significant expression was seen in differentiated fields. This restricted expression was maintained from embryonic day 11.5 to birth. In the periphery, large expression was detected in muscle and kidney and moderate expression in thyroid, pituitary gland, digestive system and bone. In the adult brain, SHIP2 was mainly restricted in structures containing neural stem cells such as the anterior subventricular zone, the rostral migratory stream and the olfactory tubercle. SHIP2 was also detected in the choroid plexuses and the granular layer of the cerebellum. The specificity of SHIP2 expression in neural stem cells was further demonstrated by (i) the dramatic increase in SHIP2 mRNA signal in neural cell adhesion molecule (N-CAM)-deficient mice, which present an accumulation of progenitor cells in the anterior subventricular zone and the rostral migratory stream, (ii) the abundant expression of 160-kDa SHIP2 by western blotting in proliferating neurospheres in culture and its downregulation in non-proliferating differentiated neurospheres. In conclusion, the close correlation between the pattern of SHIP2 expression in the brain and the proliferative and early differentiative events suggests that the phosphatase SHIP2 may have important roles in neural development.

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.

Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors.

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A(2A) receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A(2A) receptor-deficient mice (A(2A)-R(-/-)). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A(2A) receptor by striatopallidal neurones. To clarify the functions of A(2A) receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60-80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three-fold increase in extracellular glutamate concentration. This was associated with an up-regulation of D(1) and D(2) dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A(1) receptors expression were indistinguishable between A(2A)-R(-/-) and wild-type mice. Altogether these results pointed out that the lack of A(2A) receptors leads to a functional hypodopaminergic state and demonstrated that A(2A) receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.