GPR88 in D1R-Type and D2R-Type Medium Spiny Neurons Differentially Regulates Affective and Motor Behavior.

The orphan receptor GPR88 is highly expressed in D1 receptor (D1R)- and D2R-medium spiny neurons (MSNs) and has been associated to striatum-dependent functions in rodents. The total deletion of Gpr88 in mice was shown to decrease anxiety-like behaviors, increase stereotypies and locomotion, and impair motor coordination and motor learning. Knowing the opposing role of D1R- and D2R-MSNs, we here investigated the respective roles of GPR88 in the two MSN subtypes for these behaviors. To do so, we compared effects of a conditional Gpr88 gene knock-out (KO) in D1R-MSNs (D1R-Gpr88 mice) or D2R-MSNs (A2AR-Gpr88 mice) with effects of the total Gpr88 KO (CMV-Gpr88 mice). Overall, most phenotypes of CMV-Gpr88 mice were recapitulated in A2AR-Gpr88 mice, including reduced marble burying, increased social interactions, increased locomotor activity and stereotypies in the open field, and reduced motor coordination in the rotarod. Exceptions were the reduced habituation to the open field and reduced motor skill learning, which were observed in CMV-Gpr88 and D1R-Gpr88 mice, but not in A2AR-Gpr88 mice. D1R-Gpr88 mice otherwise showed no other phenotype in this study. Our data together show that GPR88 modulates the function of both D1R- and D2R-MSNs, and that GPR88 activity in these two neuron populations has very different and dissociable impacts on behavior. We suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning.

GPR88 in A2A receptor-expressing neurons modulates locomotor response to dopamine agonists but not sensorimotor gating.

The orphan receptor, GPR88, is emerging as a key player in the pathophysiology of several neuropsychiatric diseases, including psychotic disorders. Knockout (KO) mice lacking GPR88 throughout the brain exhibit many abnormalities relevant to schizophrenia including locomotor hyperactivity, behavioural hypersensitivity to dopaminergic psychostimulants and deficient sensorimotor gating. Here, we used conditional knockout (cKO) mice lacking GPR88 selectively in striatal medium spiny neurons expressing A2A receptor to determine neuronal circuits underlying these phenotypes. We first studied locomotor responses of A2A R-Gpr88 KO mice and their control littermates to psychotomimetic, amphetamine, and to selective D1 and D2 receptor agonists, SKF-81297 and quinpirole, respectively. To assess sensorimotor gating performance, mice were submitted to acoustic and visual prepulse inhibition (PPI) paradigms. Total knockout GPR88 mice were also studied for comparison. Like total GPR88 KO mice, A2A R-Gpr88 KO mice displayed a heightened sensitivity to locomotor stimulant effects of amphetamine and SKF-81297. They also exhibited enhanced locomotor activity to quinpirole, which tended to suppress locomotion in control mice. By contrast, they had normal acoustic and visual PPI, unlike total GPR88 KO mice that show impairments across different sensory modalities. Finally, none of the genetic manipulations altered central auditory temporal processing assessed by gap-PPI. Together, these findings support the role of GPR88 in the pathophysiology of schizophrenia and show that GPR88 in A2A receptor-expressing neurons modulates psychomotor behaviour but not sensorimotor gating.

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.

Targeting transcription to the neuromuscular synapse.

Concomitant with innervation, genes coding for components of the neuromuscular junction become exclusively expressed in subsynaptic nuclei. A six-base pair element, the N box, can confer synapse-specific transcription to the acetylcholine nicotinic receptor delta and epsilon subunit, utrophin, and acetylcholine esterase genes. N box-dependent synaptic expression is stimulated by the nerve-derived signal agrin and the trophic factor neuregulin, which triggers the MAPK and JNK signaling pathways, to ultimately allow activation by the N box binding Ets transcription factor GABP.

Diversity and distribution of nicotinic acetylcholine receptors in the locus ceruleus neurons.

The neurons of the locus ceruleus are responsible for most of the noradrenergic innervation in the brain and nicotine potentiates noradrenaline release from their terminals. Here we investigated the diversity and subcellular distribution of nicotinic acetylcholine receptors (nAChRs) in the locus ceruleus both somatically, by combining single-cell reverse transcription-PCR with electrophysiological characterization, and at the level of nerve terminals, by conducting noradrenaline efflux experiments. The proportion of neurons in the locus ceruleus expressing the nicotinic subunit mRNAs varied from 100% (beta2) to 3% (alpha2). Yet, two populations of neurons could be distinguished on the basis of the pattern of expression of nAChR mRNAs and electrophysiological properties. One population (type A) of small cells systematically expressed alpha3 and beta4 mRNAs (and often alpha6, beta3, alpha5, alpha4), and nicotinic agonists elicited large currents with a potency order of cytisine > nicotine. Another population (type B) of cells with large soma did not contain alpha3 and beta4 mRNAs but, systematically, alpha6 and beta3 (and often alpha4) and responded to nicotinic agonists in the order of nicotine > cytisine. The nicotinic modulation of noradrenaline release in the hippocampus displayed an order of potency nicotine > cytisine, suggesting that noradrenergic terminals in the hippocampus originate largely from type B cells of the locus ceruleus. Accordingly, immunocytochemical labeling showed that beta3 is present in hippocampal terminals. The alpha6beta3beta2(alpha4) heterooligomer thus behaves as the main nicotinic regulator of the ceruleo-hippocampal pathway.

Reduced antinociception in mice lacking neuronal nicotinic receptor subunits.

Nicotine exerts antinociceptive effects by interacting with one or more of the subtypes of nicotinic acetylcholine receptors (nAChRs) that are present throughout the neuronal pathways that respond to pain. To identify the particular subunits involved in this process, we generated mice lacking the alpha4 subunit of the neuronal nAChR by homologous recombination techniques and studied these together with previously generated mutant mice lacking the beta2 nAChR subunit. Here we show that the homozygous alpha4-/- mice no longer express high-affinity [3H]nicotine and [3H]epibatidine binding sites throughout the brain. In addition, both types of mutant mice display a reduced antinociceptive effect of nicotine on the hot-plate test and diminished sensitivity to nicotine in the tail-flick test. Patch-clamp recordings further reveal that raphe magnus and thalamic neurons no longer respond to nicotine. The alpha4 nAChR subunit, possibly associated with the beta2 nAChR subunit, is therefore crucial for nicotine-elicited antinociception.

Disruption and basic phenotypic analysis of 18 novel genes from the yeast Saccharomyces cerevisiae.

In the frame of the European Network for Functional Analysis (EUROFAN) we have deleted 18 yeast open reading frames (ORFs) from chromosomes II, X and XIV using the short flanking homology-PCR strategy. Two diploid strains were used: FY1679 and CEN.PK2. The deletion kanMX6 cassettes with long flanking homology and the cognate gene clones have also been constructed. Heterozygous diploid deletant strains have been sporulated. Tetrad analysis revealed that all the ORFs studied were non-essential. However, four deletant strains exhibited phenotypes. The YBL025w delta strain showed extremely slow cellular growth under all conditions tested. The YJL204c delta strain grew slower than wild-type at 30 degrees C and 37 degrees C, was cold-sensitive, and the homozygous diploids did not sporulate. The YNL213c delta strain did not grow on glycerol and had lost mitochondrial DNA. The deletion of YNL215w caused slower growth on all media but the defect was more pronounced on glucose-minimal and glycerol-rich media than on glucose-rich medium. All deletion mutants were complemented by the corresponding plasmid borne cognate gene. The YJL204w, YNL213c and YNL215w ORFs do not bear significant homology to proteins of known function. YBL025w has recently been identified as RRN10, a gene that encodes an RNA polymerase I-specific transcription initiator factor. The deletion of the remaining fourteen ORFs did not reveal any mutant phenotype in our basic growth tests.

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance.

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae.

Pseudomonas fuscovaginae produces the lipodepsipeptides syringotoxin, fuscopeptin A and fuscopeptin B concurrently. These phytotoxins inhibit acidification of the external medium by fusicoccin-treated rice leaf sheath discs. When tested in vitro on H+-ATPase of rice shoot plasma membranes, syringotoxin and its structural analogue syringomycin, produced by P. syringae pv. syringae, displayed a double effect. At low concentrations they stimulated the ATPase activity of native right-side-out membrane vesicles in a detergent-like manner. At higher concentrations, however, this stimulation was reversed. With membranes treated with the detergent Brij 58, inhibition of ATPase activity was observed at low concentrations of the nonapeptides. The latter effect required the presence of an intact lactone ring formed by the nonapeptide head of these molecules. In contrast, fuscopeptins A and B inhibited enzyme activity regardless of the orientation of the vesicles. These observations were confirmed using plasma membranes from a yeast strain whose own H+-ATPase had been replaced by a single plant H+-ATPase isoform, PMA2, from Nicotiana plumbaginifolia. The kinetics of inhibition induced by the most active compound fuscopeptin B, showed a non-competitive pattern, with a Ki of about 1 microM. The combination of syringotoxin (or syringomycin) with the more hydrophobic fuscopeptins, in amounts with little or no effect, resulted in strong inhibition of the enzyme activity of rice membranes, suggesting a synergistic effect for the two types of toxins.

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The plasma membrane H+-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H+-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae, even after deletion of the enzyme's carboxy terminus. Functional chimerical H+-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H+-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H+-ATPase. The reverse Ala-598-->Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H+-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K(m) values for MgATP and decreased Vmax compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H+-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.

The complete inventory of the yeast Saccharomyces cerevisiae P-type transport ATPases.

A total of sixteen open reading frames encoding for P-type ATPases have been identified in the complete genome sequence of Saccharomyces cerevisiae. Phylogenetic analysis distinguishes 6 distinct families. Topology predictions, identification of aminoacid sequence motifs and phenotype analysis of the available mutants suggest that these families correspond to ATPases transporting either H+ (2 members), Ca2+ (2 members), Na+ (3 members), heavy metals (2 members), possibly aminophospholipids (5 members including 4 new ones) or unknown substrates (2 new members). It is proposed that the latter family which has homologs in Tetrahymena thermophila, Plasmodium falciparum and Caenorhabditis elegans constitutes a new group called P4-ATPases with characteristic topology and aminoacid signatures.

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.

Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H(+)-ATPases. We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H(+)-ATPase isoform of Nicotiana plumbaginifolia. Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted. The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0. The N. plumbaginifolia PMA2 expressed in S. cerevisiae has unusual low Km for ATP (23 microM) and high pH optimum (6.8). Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium. Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker. These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions.

In-frame recombination between the yeast H(+)-ATPase isogenes PMA1 and PMA2: insights into the mechanism of recombination initiated by a double-strand break.

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 promoter, were constructed by in vivo recombination between a gapped linearized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was screened by the expression of the lacZ reporter gene fused to the PMA2 coding region. Restriction and sequencing analysis of 35 chimeras showed that in all cases, the hybrid sequences was obtained as fusions between continuous sequences specific to PMA1 and PMA2, separated by a region of identity. In all but three cases, the junction sequences were not located at regions of greatest identity. Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categories. In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity. Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distribution is not related to the size of the region of identity at the PMA1-PMA2 boundary but depends on the degree of identity of the PMA genes upstream of the region of identity, the accumulation of successive mismatches leading to a clustered distribution of the junctions. Moreover, the introduction of seven closely spaced mismatches near the end of a PMA1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end. These data show that a low level of identity in the vicinity of the common boundary stretch is a strong barrier to recombination. In contrast, consecutive mismatches or regions of overall moderate identity which are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.