François Conquet

The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. Here we report that DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP‐null allele display less locomotor activity and free‐run with a shorter period than otherwise isogenic wild‐type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP−/− mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock.

Disruption of retinoid‐related orphan receptor β changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice

The orphan nuclear receptor RORβ is expressed in areas of the central nervous system which are involved in the processing of sensory information, including spinal cord, thalamus and sensory cerebellar cortices. Additionally, RORβ localizes to the three principal anatomical components of the mammalian timing system, the suprachiasmatic nuclei, the retina and the pineal gland. RORβ mRNA levels oscillate in retina and pineal gland with a circadian rhythm that persists in constant darkness. RORβ−/− mice display a duck‐like gait, transient male incapability to sexually reproduce, and a severely disorganized retina that suffers from postnatal degeneration. Consequently, adult RORβ−/− mice are blind, yet their circadian activity rhythm is still entrained by light–dark cycles. Interestingly, under conditions of constant darkness, RORβ−/− mice display an extended period of free‐running rhythmicity. The overall behavioral phenotype of RORβ−/− mice, together with the chromosomal localization of the RORβ gene, suggests a close relationship to the spontaneous mouse mutation vacillans described >40 years ago.

Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP.

ABSTRACT To study the molecular mechanisms of circadian gene expression, we have sought to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. Here, we report that the Cyp2a4 gene, encoding the cytochrome P450 steroid 15α-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both theCyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver was significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5genes in the mouse liver.

Selective involvement of mGlu1 receptors in corticostriatal LTD.

Although metabotropic glutamate receptors (mGluRs) have been proposed to play a role in corticostriatal long-term depression (LTD), the specific receptor subtype required for this form of synaptic plasticity has not been characterized yet. Thus, we utilized a corticostriatal brain slice preparation and intracellular recordings from striatal spiny neurons to address this issue. We observed that both AIDA (100 microM) and LY 367385 (30 microM), two blockers of mGluR1s, were able to fully prevent the induction of this form of synaptic plasticity, whereas MPEP (30 microM), a selective antagonist of the mGluR5 subtype, did not significantly affect the amplitude and time-course of corticostriatal LTD. Both AIDA and LY 367385 were ineffective on LTD when applied after its induction. The critical role of mGluR1s in the formation of corticostriatal LTD was confirmed in experiments performed on mice lacking mGluR1s. In these mice, in fact, a significant reduction of the LTD amplitude was observed in comparison to the normal LTD measured in their wild-type counterparts. We found that neither acute pharmacological blockade of mGluR1s nor the genetic disruption of these receptors affected the presynaptic modulation of corticostriatal excitatory postsynapic potentials (EPSPs) exerted by DCG-IV and L-SOP, selective agonists of group II and III mGluRs, respectively. Our data show that the induction of corticostriatal LTD requires the activation of mGluR1 but not mGluR5. mGluR1-mediated control of this form of synaptic plasticity may play a role in the modulatory effect exerted by mGluRs in the basal ganglia-related motor activity.

Corticostriatal LTP requires combined mGluR1 and mGluR5 activation

Metabotropic glutamate receptors (mGluRs) have been demonstrated to play a role in synaptic plasticity. It has been recently shown that mGluR1 is involved in corticostriatal long-term depression, by means of pharmacological approach and by using mGluR1-knockout mice. Here, we report that both mGluR1 and mGluR5 are involved in corticostriatal long-term potentiation (LTP). In particular, the mGluR1 antagonist LY 367385, as well as the mGluR5 antagonist MPEP, reduce LTP amplitude. Moreover, blockade of both mGluR1 and mGluR5 by LY 367385 and MPEP co-administration fully suppresses LTP. Accordingly, group II and group III mGluRs antagonists fail to affect LTP induction. Interestingly, LTP amplitude is also significantly reduced in both mGluR1- and mGluR5-knockout mice. The differential function of mGluR1 and mGluR5 in corticostriatal synaptic plasticity may play a role in the modulation of the motor activity mediated by the basal ganglia, thus providing a substrate for the pharmacological treatment of motor disorders involving the striatum.

Immunohistochemical localization of the mGluR1β metabotropic glutamate receptor in the adult rodent forebrain: Evidence for a differential distribution of mGluR1 splice variants

Alternative splicing has been shown to occur at the metabotropic glutamate receptor 1 (mGluR1) gene. Three main isoforms that differ in their carboxy‐termini have been described so far and named mGluR1α, mGluR1β and mGluR1c. These variants when expressed in recombinant systems all activate phospholipase C, although the [Ca2+] signals generated have different kinetics. Tissue distribution studies of specific mGluR1 splice variants are limited to the mGluR1α isoform. In the present work, we examined the localization of mGluR1β in the adult rat and mouse forebrain by using a specific antipeptide antibody. Furthermore, the mGluR1β immunostaining was compared with that obtained with antibodies specific for mGluR1α or with a pan‐mGluR1 antibody which recognizes all isoforms. mGluR1β‐like immunoreactivity (LI) was found confined to the neuropil and neuronal perikarya and appeared discretely distributed in the rodent forebrain. Differential cellular distribution between mGluR1α and mGluR1β was observed. In the hippocampus, mGluR1α‐LI was restricted to non‐principal neurons in all fields, whereas mGluR1β‐LI was strongest in principal cells of the CA3 field and dentate granule cells but absent in CA1. We have also shown that the vast majority of neurons in the striatum express mGluR1. The predominant form appeared to be mGluR1β, with a distribution pattern reflecting the patch‐matrix organization of the striatum. The specificity of the immunoreactivity described for mGluR1 splice variants was confirmed in mGluR1‐deficient mice. The observation of a different cellular and regional distribution of mGluR1 splice variants, in particular in the hippocampus, suggests that they may mediate different roles in synaptic transmission. J. Comp. Neurol. 400:391–407, 1998.

The regulation of hippocampal LTP by the molecular switch, a form of metaplasticity, requires mGlu5 receptors.

The role of metabotropic glutamate (mGlu) receptors in long-term potentiation (LTP) in the hippocampus is controversial. In the present study, we have used mice in which the mGlu1, mGlu5 or mGlu7 receptor has been deleted, by homologous recombination, to study the role of these receptor subtypes in LTP at CA1 synapses. We investigated the effects of the knockouts on both LTP and the molecular switch, a form of metaplasticity that renders LTP insensitive to the actions of the mGlu receptor antagonist MCPG ((S)-alpha-methyl-4-carboxyphenylglycine). We find that LTP is readily induced in the three knockouts and in an mGlu1 and mGlu5 double knockout. In addition, the molecular switch operates normally in either the mGlu1 or mGlu7 knockout. In contrast, the molecular switch is completely non-functional in the mGlu5 knockout, such that MCPG invariably blocks the induction of additional LTP in an input where LTP has already been induced. The effect of the mGlu5 receptor knockout was replicated in wildtype mouse slices perfused with the specific mGlu5 receptor antagonist MPEP (2-methyl-6-(phenylethynyl)-pyridine). In addition, the mGlu5 selective agonist CHPG ((RS)-2-chloro-5-hydroxyphenylglycine) sets the molecular switch. These data demonstrate that the operation of the molecular switch requires activation of mGlu5 receptors.

Incomplete regression of multiple climbing fibre innervation of cerebellar Purkinje cells in mGluR1 mutant mice

RECENT reports have suggested the existence of a causal relationship between impaired regression of multiple climbing fibre innervation and impaired motor coordination in protein kinase Cγ subunit (PKCγ) mutant mice. In the present patch-clamp study, performed in thin cerebellar slices prepared from adult mutant mice deficient in metabotropic glutamate receptors of the mGluR1 subtype, only 15% of Purkinje cells remained multiply innervated by climbing fibres, but motor coordination was largely impaired in these animals. The present results do not preclude the existence of a causal relationship between impairement of regression of multiple innervation during development and improper motor coordination in the adult.

Immunocytochemical localization of the mGluR1b metabotropic glutamate receptor in the rat hypothalamus

The mGluR1 metabotropic glutamate receptor is a G‐protein‐coupled receptor that exists as different C‐terminal splice variants. When expressed in mammalian cells, the mGluR1 splice variants exhibit diverse transduction mechanisms and also slightly differ in their apparent agonist affinities. In the present study, we used an affinity‐purified antiserum, specifically reactive to the mGluR1b splice variant, in combination with a highly sensitive preembedding immunocytochemical method for light microscopy to investigate the distribution of this receptor in the rat hypothalamus.

Regulation of synaptic plasticity by mGluR1 studied in vivo in mGluR1 mutant mice.

The role of the metabotropic glutamate receptor 1 (mGluR1) in synaptic plasticity was investigated in vivo in the intact hippocampus of mutant mice lacking this receptor. In a previous study we showed reduced long-term potentiation (LTP) in the dentate gyrus of mGluR1 -/- mice in vivo, but not when LTP was studied in a slice preparation. A possible explanation of this difference is that dentate neurons receive more inhibitory synaptic drive in vivo than in slice preparation where many inhibitory axon collaterals are lost. We report here that another form of synaptic plasticity, paired-pulse depression of the population spike, is also abnormal in the dentate gyrus of mGluR1-deficient mice when tested in vivo. In wild-type mice, stimulation of the medial perforant path produced paired-pulse depression of inter-pulse intervals (IPIs) up to 30 ms. Mutant mGluR1, on the other hand, showed a significantly longer IPI depression, up to 50 ms. Paired-pulse depression results from the activation of inhibitory interneurons. The GABA(B) agonist baclofen, acting presynaptically on the GABA interneurons, attenuated paired-pulse depression and allowed for a normal and stable LTP in mGluR1 mutant mice. These findings suggest an indirect role for mGluR1 in synaptic plasticity via a regulation of GABA inhibition.