Metabotropic glutamate receptor 1 (mGluR1) is a member of a large family of G-protein-coupled glutamate receptors, the physiological functions of which are largely unknown. Mice deficient in mGluR1 have severe motor coordination and spatial learning deficits. They have no gross anatomical or basic electrophysiological abnormalities in either the cerebellum or hippocampus, but they show impaired cerebellar long-term depression and hippocampal mossy fibre long-term potentiation. mGluR1-deficient mice should therefore be valuable models for studying synaptic plasticity.

Retinoid-related orphan receptor ␣ (ROR␣) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme ␤-galactosidase. In heterozygous ROR␣ ؉/؊ mice we found ␤-galactosidase activity, indicative of ROR␣ protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the ROR␣ gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, ROR␣ is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express ROR␣. Recently, it was reported that the ataxic mouse mutant staggerer (sg͞sg) is caused by a deletion in the ROR␣ gene. The analysis of the cerebellar and the behavioral phenotype of homozygous ROR␣ ؊/؊ mice proves identity to sg͞sg mice. Although the absence of ROR␣ causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of ROR␣ ؊/؊ mice display a normal phenotype. However, the pelage hair of both sg͞sg and ROR␣ ؊/؊ is significantly less dense and when shaved shows reluctance to regrow.Nuclear receptors form a structurally related superfamily of ligand-activated transcription factors (1). They are involved in several aspects of vertebrate physiology, such as development and homeostasis. Important examples are the steroid hormone receptors that regulate, in a ligand-dependent manner, specific sets of responding genes. The retinoid-related orphan nuclear receptor (ROR) ␣ (2, 3), ROR␤ (4), and ROR␥ (5) constitute a subfamily of nuclear receptors that bind to DNA both as monomers and dimers. Distribution of ROR␣ mRNA suggests that this receptor is widely expressed and functions in several organs including brain, heart, liver, lung, and testis; highest levels were found in peripheral blood leukocytes and skin (M.B.-A., unpublished data). ROR␣ exists in four splicing isoforms: ROR␣1-4. They display different N-terminal domains causing different DNA binding site preferences (3), and they display differential expression profiles: in the thalamus there is only ROR␣1 mRNA; ROR␣4 (ϭRZR␣) (2) transcripts are predominant in leukocytes and skin; ROR␣2 and ROR␣3 transcripts are exclusively detected in testis; and in the remaining tissues including the cerebellum there is a mixture of ROR␣1 and ROR␣4 transcripts (M.B.-A., unpublished results). In the central nervous system (CNS) ROR␣ mRNA localizes to the cerebellar Purkinje cells (PCs), various thalamic nuclei, and, during development, to other brain areas (6, 7). To study the physiological role of this orphan receptor we generated ROR␣ null-mutant mice by gene targeting. In the course of this work the genetic basis of the staggerer (sg) mutation in mouse was identified by positional cloning as a deletion in the R...

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The amino acids GABA and glutamate (Glu) are thought to be the principal substances in the central nervous system responsible for neuronal inhibition and excitation. Their distributions among the different neurons in a defined pathway may thus be indicative of the contributions of the cells to pathway function. Examples of such neurons are those of the cerebellar nuclei which, while regulating output from the Purkinje cells of the cerebellar cortex, are also found to project back to the cerebellar cortex. Immunohistochemical experiments were done to identify GABA and glutamate (Glu) containing cells in the adult rat cerebellar nuclei. Consecutive semithin and serial vibratome sections were incubated with antisera raised in rabbit against GABA and Glu. In semithin sections, only small neurons were intensely GABA immunoreactive (GABA-IR) (31.7%), and the majority (80.5%) were Glu immunoreactive (Glu-IR) of different sizes. Consistent with Glu being a metabolic precursor for GABA, 75.4% of the GABA-IR population colocalized Glu. In vibratome sections GABA-IR neurons showed some local differences in number, whereas the Glu-IR were uniformly distributed in the three nuclei studied. Measured mean diameters for these neurons showed a distinct size difference for the GABA- and Glu-IR with little overlap. Cerebellar nuclei neurons projecting to the cortex (nucleocortical neurons, NCN) were identified by locally preinjecting the retrograde transported WGA-apoHRP-colloidal gold complex in the cerebellar cortex. Vibratome sections of these cerebellar were silver intensified for the retrograde tracer and double labeled for GABA and Glu. Of the total number of identified NCN, 8.7% were GABA-IR (10 animals) and 47.7% Glu-IR (5 animals). Many retrograde labeled NCN in the core of the thick sections were immunonegative for both amino acids due to poor antibody penetration, thus underestimating the proportions of cells containing GABA and Glu. The size distributions for the GABA-IR and Glu-IR NCN were similar to those measured in non-retrograde labeled nuclei in thick sections. The conclusions reached are that GABA-IR neurons of the cerebellar nuclei, including the NCN, use GABA as the presumed inhibitory neurotransmitter and that Glu-IR neurons may use Glu or another excitatory neurotransmitter.

In rat prefrontal cortex (the prelimbic area of medial frontal cortex), the induction of long-term depression (LTD) and long-term potentiation (LTP) of glutamatergic synapses is powerfully modulated by dopamine. The presence of dopamine in the bathing medium facilitates LTD in slice preparations, whereas in the anesthetized intact brain, dopamine released from dopaminergic axon terminals in the prefrontal cortex facilitates LTP. Dopaminergic facilitation of LTD is at least partly achieved by postsynaptic biochemical mechanisms in which enzymatic processes triggered by dopamine receptor activation cooperate with those triggered by glutamate metabotropic receptor activation. Evidence suggests that dopamine facilitates LTP also in the slice condition. In this case, dopamine receptors must be pre-stimulated ('primed') before the application of high-frequency stimuli in the presence of dopamine. This procedure may mimic baseline stimulation of dopamine receptors that occurs under physiological conditions. Dopamine Modulates Glutamatergic Transmission in the Prefrontal CortexDopamine plays important roles in the function of primate prefrontal cortex (PFC) Goldman-Rakic, 1991, 1994;Goldman-Rakic, 1995;William and Goldman-Rakic, 1995) and the rodent medial frontal cortex (Glowinski et al., 1984), which is believed by many to be the functional counterpart of primate dorsal/lateral prefrontal cortex (particularly the prelimbic area of the medial frontal cortex) (Floresco et al., 1997;Zahrt et al., 1997;Birrell and Brown, 2000). In monkeys, interruption of dopaminergic transmission in dorsolateral PFC by focal D1 antagonist application impairs spatial working memory Goldman-Rakic, 1991, 1994) and exaggerates or inhibits (depending on the dose) the delay period activity of PFC neurons during the working memory task (William and Goldman-Rakic, 1995). Dopamine neurons in substantia nigra and ventral tegmental area (VTA) show increases of firing in response to salient or appetitive stimuli (Ljungberg et al., 1992;Mirenowicz and Schultz, 1996) or to conditioned stimuli predicting reward (Schultz et al., 1993). In the latter case (Schultz et al., 1993), one of the task phases in which the dopamine neurons showed the increases is actually a delay period of a spatial task. These results therefore raise the possibility that at least in certain behavioral phases, a phasic dopamine release coincides with the activity of PFC neurons to tune neuronal transmission at these neurons. Similarly in rats, normal working memory requires optimal levels of dopamine in the prelimbic area (i.e. rat PFC) (Zahrt et al., 1997;Mizoguchi et al., 2000), and some VTA neurons and PFC deep layer neurons were shown to increase their activity at the same behavioral phases in an appetitive operant task (Kosobud et al., 1994).Modulation of PFC glutamatergic transmission by dopamine has been studied at the cellular level. For example, it was shown that at least D1 subtype of dopamine receptors co-localize with glutamate receptors on dendritic spines and sh...

Metabotropic glutamate (mGlu) receptors are promising targets to treat numerous brain disorders. So far, allosteric modulators are the only subtype selective ligands, but pure agonists still have strong therapeutic potential. Here, we aimed at investigating the possibility of developing subtype-selective agonists by extending the glutamate-like structure to hit a nonconsensus binding area. We report the properties of the first mGlu4-selective orthosteric agonist, derived from a virtual screening hit, LSP4-2022 using cell-based assays with recombinant mGlu receptors [EC(50): 0.11 ± 0.02, 11.6 ± 1.9, 29.2 ± 4.2 μM (n>19) in calcium assays on mGlu4, mGlu7, and mGlu8 receptors, respectively, with no activity at the group I and -II mGlu receptors at 100 μM]. LSP4-2022 inhibits neurotransmission in cerebellar slices from wild-type but not mGlu4 receptor-knockout mice. In vivo, it possesses antiparkinsonian properties after central or systemic administration in a haloperidol-induced catalepsy test, revealing its ability to cross the blood-brain barrier. Site-directed mutagenesis and molecular modeling was used to identify the LSP4-2022 binding site, revealing interaction with both the glutamate binding site and a variable pocket responsible for selectivity. These data reveal new approaches for developing selective, hydrophilic, and brain-penetrant mGlu receptor agonists, offering new possibilities to design original bioactive compounds with therapeutic potential.

Down syndrome (DS) is a genetic disorder arising from the presence of a third copy of human chromosome 21 (Hsa21). Recently, O'Doherty et al. [An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science 309 (2005) 2033–2037] generated a trans-species aneuploid mouse line (Tc1) that carries an almost complete Hsa21. The Tc1 mouse is the most complete animal model for DS currently available. Tc1 mice show many features that relate to human DS, including alterations in memory, synaptic plasticity, cerebellar neuronal number, heart development and mandible size. Because motor deficits are one of the most frequently occurring features of DS, we have undertaken a detailed analysis of motor behaviour in cerebellum-dependent learning tasks that require high motor coordination and balance. In addition, basic electrophysiological properties of cerebellar circuitry and synaptic plasticity have been investigated. Our results reveal that, compared with controls, Tc1 mice exhibit a higher spontaneous locomotor activity, a reduced ability to habituate to their environments, a different gait and major deficits on several measures of motor coordination and balance in the rota rod and static rod tests. Moreover, cerebellar long-term depression is essentially normal in Tc1 mice, with only a slight difference in time course. Our observations provide further evidence that support the validity of the Tc1 mouse as a model for DS, which will help us to provide insights into the causal factors responsible for motor deficits observed in persons with DS.

Extensive work has shown that activation of the cAMP-dependent protein kinase A (PKA) is crucial for long-term depression (LTD) of synaptic transmission in the hippocampus, a phenomenon that is thought to be involved in memory formation. Here we studied the role of an alternative target of cAMP, the exchange protein factor directly activated by cyclic AMP (Epac). We show that pharmacological activation of Epac by the selective agonist 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (8-pCPT) induces LTD in the CA1 region. Paired-pulse facilitation of synaptic responses remained unchanged after induction of this LTD, suggesting that it depended on postsynaptic mechanisms. The 8-pCPT-induced LTD was blocked by the Epac signalling inhibitor brefeldin-A (BFA), Rap-1 antagonist geranylgeranyltransferase inhibitor (GGTI) and p38 mitogen activated protein kinase (P38-MAPK) inhibitor SB203580. This indicated a direct involvement of Epac in this form of LTD. As for other forms of LTD, a mimetic peptide of the PSD-95/Disc-large/ZO-1 homology (PDZ) ligand motif of the AMPA receptor subunit GluR2 blocked the Epac-LTD, suggesting involvement of PDZ protein interaction. The Epac-LTD also depended on mobilization of intracellular Ca2+ , proteasome activity and mRNA translation, but not transcription, as it was inhibited by thapsigargin, lactacystin and anisomycin, but not actinomycin-D, respectively. Finally, we found that the pituitary adenylate cyclase activating polypeptide (PACAP) can induce an LTD that was mutually occluded by the Epac-LTD and blocked by BFA or SB203580, suggesting that the Epac-LTD could be mobilized by stimulation of PACAP receptors. Altogether these results provided evidence for a new form of hippocampal LTD.