Association of VSNL1 with schizophrenia, frontal cortical function, and biological significance for its gene product as a modulator of cAMP levels and neuronal morphology.

We report an association of single-nucleotide polymorphisms (SNPs) for the VSNL1 gene (visinin-like 1) with schizophrenia and frontal cortical function in a sample of patients with Diagnostic and Statistical Manual of Mental Disorder-IV (DSM-IV) diagnoses of schizophrenia, compared with healthy controls. Moreover, VSNL1 SNPs were associated with performance in the Wisconsin Card Sorting Test, a measure for the assessment of frontal cortical function. The VSNL1 gene product, Visinin-like-protein-1 (VILIP-1), is a member of the neuronal EF-hand Ca(2+)-sensor protein family. Previously, VILIP-1 mRNA and protein expression were shown to be altered in animal models and in schizophrenia patients. VILIP-1 influences cytosolic cyclic adenosine mono phosphate (cAMP) levels, cell migration, exocytotic processes and differentiation in the periphery. This raises the question, whether, similar to other potential schizophrenia susceptibility genes such as Disc1, PDE4B and Akt, VSNL1 may affect cAMP signaling and neurite outgrowth in neurons. In dissociated rat hippocampal neurons, VILIP-1 small interfering RNA knockdown decreased cAMP levels and reduced dendrite branching, compared with control-transfected cells. In contrast, VILIP-1 overexpression had the opposite effect. Similar results have been obtained in the human dopaminergic neuronal cell line SH-SY5Y, where the effect on neurite branching and length was attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and the protein kinase A inhibitor KT5720. These results show that the association of VSNL1 SNPs with the disease and cognitive impairments, together with previously observed pathological changes in VILIP-1 protein expression, possibly occurring during brain development, may contribute to the morphological and functional deficits observed in schizophrenia.

C-type natriuretic peptide modulates bidirectional plasticity in hippocampal area CA1 in vitro.

C-type natriuretic peptide (CNP) and the natriuretic peptide receptor B (NPR-B) are expressed throughout the hippocampus. We tested whether CNP affected long-term potentiation (LTP) or long-term depression (LTD) in area CA1. Field potentials (FP) were simultaneously recorded in stratum pyramidale (SP) and stratum radiatum (SR) of area CA1 in rat hippocampal slices. To induce LTD and LTP stimulation was applied to SR in area CA1 at 1 and 5 Hz and 30-100 Hz, respectively. CNP (100 nM) increased LTD magnitude while LTP induction was impeded. Thus, in the presence of CNP the threshold for LTP induction was shifted to higher stimulus frequencies, a modulation that showed layer-specific differences in area CA1. Effects of CNP were prevented by the NPR-B antagonist HS-142-1. In the presence of the GABA(A) receptor blocker bicuculline (BMI, 5 microM), CNP-mediated effects were attenuated in SP and SR. Intracellular recordings under this condition revealed that CNP significantly reduced number of action potentials generated during depolarizing current steps. The input resistance of CA1 cells and amplitude of isolated excitatory postsynaptic potential (EPSPs) were significantly increased by CNP whereas these changes were not observed in the absence of BMI. 100 Hz stimulation induced stable potentiation of the EPSP amplitude in CA1 pyramidal cells while this effect was strongly attenuated by CNP. This effect was prevented by BMI. Immunohistochemistry indicated that the peptide binds to receptors expressed on pyramidal cells and GAD(65/67)-immunopositive interneurons. 20 Hz stimulation, applied for 30 s, induced LTP in SR and SP. CNP attenuated LTP in SP and reversed LTP into LTD in SR. These effects were mimicked by low-dose dl-2-amino-5-phosphonopentanoic acid (dl-APV) (10 microM) suggesting partial N-methyl d-aspartate (NMDA) receptor dependency of CNP-mediated effects. Together, our data suggest that CNP is involved in the regulation of bidirectional plasticity in area CA1 potentially by modulating GABA(A)-mediated inhibition and NMDA receptors.

C-type natriuretic peptide decreases hippocampal network oscillations in adult rats in vitro.

C-type natriuretic peptide (CNP) is an abundant neuropeptide in the human brain and the cerebrospinal fluid. CNP is involved in anxiogenesis and exerts its effects through the natriuretic peptide receptor B (NPR-B), which is expressed in the hippocampus. Hippocampal network oscillations of distinct frequency bands like gamma (gamma)-oscillations and sharp wave-ripple complexes (SPW-Rs) are likely involved in various cognitive functions such as the storage of information and memory consolidation in vivo. Here, we tested the effects of CNP on distinct network oscillations in horizontal slices of rat hippocampus. We found that CNP decreased the power of stimulus- and ACh/physostigmine-induced gamma-oscillations. In contrast to stimulus-induced gamma-oscillations, CNP increased the frequency of ACh-induced, persistent network oscillations. Moreover, the peptide hormone reduced the incidence of LTP-associated SPW-Rs in area CA3 and CA1. Immunohistochemistry indicates that the peptide binds to receptors expressed on a subset of GAD 65-67-immunopositive cells in addition to binding to principal and other presumably non-neuronal cells. CNP caused a hyperpolarization of CA3 neurons increased their input resistance and decreased inhibitory conductance. Together, our data suggest that the effects of CNP on synchronized hippocampal network oscillations might involve effects on hippocampal interneurons.

Neuronal Ca2+ sensor VILIP-1 leads to the upregulation of functional alpha4beta2 nicotinic acetylcholine receptors in hippocampal neurons.

The neuronal Ca2+-sensor protein VILIP-1, known to affect clathrin-dependent receptor trafficking, has been shown to interact with the cytoplasmic loop of the alpha4-subunit of the alpha4beta2 nicotinic acetylcholine receptor (nAChR), which is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The alpha4beta2 nAChR is crucial for nicotine addiction and the beneficial effects of nicotine on cognition. Its dysfunction has been implicated in frontal lobe epilepsy, Alzheimer's disease and schizophrenia. Here we report that overexpression of VILIP-1 enhances ACh responsiveness, whereas siRNA against VILIP-1 reduces alpha4beta2 nAChR currents of hippocampal neurons. The underlying molecular mechanism likely involves enhanced constitutive exocytosis of alpha4beta2 nAChRs mediated by VILIP-1. The two interaction partners co-localize in a Ca2+-dependent manner with syntaxin-6, a Golgi-SNARE protein involved in trans-Golgi membrane trafficking. Thus, we speculate that regulation of VILIP-1-expression might modulate surface expression of ligand-gated ion channels, such as the alpha4beta2 nAChRs, possibly comprising a novel form of physiological up-regulation of ligand-gated ion channels.

Expression of the neuronal calcium sensor visinin-like protein-1 in the rat hippocampus.

Visinin-like protein-1 (VILIP-1) belongs to the neuronal calcium sensor (NCS) family of EF-hand Ca(2+)-binding proteins which are involved in a variety of Ca(2+)-dependent signal transduction processes in neurons. VILIP-1 has been implicated in the pathology of CNS disorders including Alzheimer's disease and schizophrenia, but its expression has also been found to be regulated following induction of hippocampal synaptic plasticity underlying learning and memory processes. VILIP-1 is strongly expressed in different populations of principal and non-principal neurons in the rat hippocampus. VILIP-1-containing interneurons are morphologically and neurochemically heterogeneous. On the basis of co-localizing markers, VILIP-1 is rarely present in perisomatic inhibitory parvalbumin containing cells. However, VILIP-1 is frequently expressed in mid-proximal dendritic inhibitory cells characterized by calbindin immunoreactivity, and most strongly co-expressed in calretinin-positive disinhibitory interneurons. Partial co-localization of the metabotropic glutamate receptor mGluR1alpha with VILIP-1 was often found in interneurons located in the stratum oriens of the hippocampal CA1 region and in hilar interneurons. Partial co-localization of alpha4beta2 nicotinic acetylcholine receptor with VILIP-1 was seen in stratum oriens interneurons and particularly at the border of the hilus in the dentate gyrus, where VILIP-1 also strongly co-localized with calretinin. We speculate that depending on the regulation of the expression of VILIP-1 in hippocampal pyramidal cells or defined types of interneurons, it may have different effects on hippocampal synaptic plasticity and network activity in health and disease.

Structural determinants of alpha4beta2 nicotinic acetylcholine receptor trafficking.

The structural determinants of nicotinic acetylcholine receptor (AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum (ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the alpha4beta2 AChR subunits were required for cell surface expression of alpha4beta2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the alpha4 AChR subunit attenuates cell surface expression of mutant alpha4beta2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the beta2 AChR subunit abolishes cell surface expression of mutant alpha4beta2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the beta2 AChR subunit only poorly inhibits trafficking of mutant alpha4beta2 AChR to the cell surface. All mutant alpha4beta2 AChRs exhibit high-affinity binding for [3H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type alpha4beta2 AChRs colocalize with the Golgi marker giantin, whereas mutant alpha4beta2 AChRs fail to do so. The striking difference between mutant alpha4 versus mutant beta2 AChR subunits on cell surface expression of mutant alpha4beta2 AChRs points to a cooperative or regulatory role for the alpha4 AChR subunit and an obligatory role for the beta2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of alpha4beta2 AChR ER export.

Group I mGlu receptors regulate the expression of the neuronal calcium sensor protein VILIP-1 in vitro and in vivo: implications for mGlu receptor-dependent hippocampal plasticity?

Metabotropic glutamate (mGlu) receptors are involved in several forms of synaptic plasticity in the rat hippocampus. Agonists which activate group I mGlu receptors induce slow-onset potentiation without prior tetanization in the hippocampal area CA1. Activation of group I mGlu receptors induces protein synthesis which may contribute to mGlu receptor-dependent forms of long-term plasticity. Calcium-binding proteins are widely considered to comprise key elements for synaptic plasticity. Therefore, we investigated whether the calcium sensor protein VILIP-1 is associated with group I mGlu receptor-mediated plasticity in the dentate gyrus (DG) in vivo.Application of either the group I and II mGlu agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) or the selective group I agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in slow-onset potentiation in the DG of adult rats. In hippocampal cell cultures both agonists elicited an enhanced expression of VILIP-1. In situ hybridization revealed strong hippocampal expression of VILIP-1 and intracerebral application of DHPG to adult rats significantly enhanced hippocampal VILIP-1 expression. The DHPG effects in both, hippocampal cultures and in vivo, were prevented by the group I mGlu receptor antagonist 4-Carboxyphenylglycine (4CPG). Calcium sensor proteins thus appear to be regulated by mGlu receptors in an activity-dependent manner. A specific role for group I mGlu receptors is evident. Furthermore, the sensor proteins may function as molecular switches for the long-term regulation of synaptic plasticity.

Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function.

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.

The neuronal calcium sensor protein VILIP-1 is associated with amyloid plaques and extracellular tangles in Alzheimer's disease and promotes cell death and tau phosphorylation in vitro: a link between calcium sensors and Alzheimer's disease?

To investigate whether the observed association of intracellular neuronal calcium sensor (NCS) proteins with amyloid plaques and neurofibrillar tangles in Alzheimer brains is linked to a possible neuroprotective or neurotoxic activity of the protein, we performed cytotoxicity tests in PC12 cells transfected with the calcium sensor protein VILIP-1 (visinin-like protein) and the calcium buffer protein calbindin-D28K. Whereas VILIP-1 expression enhanced the neurotoxic effect of ionomycin already at low ionophore concentrations, calbindin-D28K protected against ionomycin-induced cytotoxicity only at high ionomycin and therefore calcium concentrations. However, in double-transfected cells calbindin-D28K rescued VILIP-1-mediated cytotoxicity at low ionomycin concentrations. Since VILIP-1 was found to be associated with fibrillar tangles in Alzheimer brains, we tested whether VILIP-1 has an influence on tau hyperphosphorylation. VILIP-1 expression enhanced hyperphosphorylation of tau protein compared to nontransfected or calbindin-D28K-transfected cells. These results raise the possibility that the observed reduction in VILIP-1-expressing cells may indicate a selective vulnerability of these neurons and that the calcium sensor protein is involved in the pathophysiology of Alzheimer's disease. The calcium sensor protein may influence tau phosphorylation and have a role in calcium-mediated neurotoxicity opposed to the previously discovered protective effect of calcium buffer proteins.

Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones.

The family of intracellular neuronal calcium-sensors (NCS) belongs to the superfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in living neural cells. Visinin-like protein-1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP-1. In cells transfected with membrane-associated wild-type VILIP-1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic localization of VILIP-1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP-1 with the recombinant catalytic domain of particulate GCs-A, B and with native GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colocalization of GC-B, but not GC-A, with VILIP-1 in cerebellar granule cells, these results suggest that VILIP-1 may be a physiological regulator of GC-B.

Long-term depression: a cellular basis for learning?

Long-term depression (LTD) comprises a persistent activity-dependent reduction in synaptic efficacy which typically occurs following repeated low frequency afferent stimulation. Hippocampal LTD has been a subject of particular interest due to the established role of the hippocampus in certain forms of information storage and retrieval. Recently, it was reported that LTD in the CA1 region may be associated with novelty acquisition in rats. CA1 LTD expression may also be increased in stressful conditions. This suggests a more complex role for this form of plasticity than the oft-cited hypothesis that it simply serves to prevent synapse saturation, by means, for example, of enabling reversal of long-term potentiation (LTP). One possibility is that LTD may be directly involved in the creation of a memory trace. Alternatively, LTD may prime a synapse in readiness for the expression of LTP, thereby contributing indirectly to information storage. There is increasing evidence that LTD is not mechanistically the reverse of LTP. Although some common processes exist, molecular, biochemical, electrophysiological and pharmacological studies all point to several quite distinct induction and maintenance mechanisms for this form of synaptic plasticity. Taken together these findings suggest that hippocampal LTD must be considered in a new light. This review focuses on the interpretation of novel and established information with regard to LTD in the hippocampal CA1 region in terms of its possible role as a cellular basis for learning and memory.

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia.

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca(2+)-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca(2+)-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.

Ca2+-induced Ca2+ release supports the relay mode of activity in thalamocortical cells.

Ca2+ ions play an important role during rhythmic bursting of thalamocortical neurons within sleep. The function of Ca2+ during the tonic relay mode of these neurons during wakefulness is less clear. Here, we report that tonic activity in thalamocortical cells results in an increase in the intracellular Ca2+ concentration and subsequent release of Ca2+ from intracellular stores mediated via ryanodine receptors (RyRs). Blockade of Ca2+ release shifted the regular firing of single action potentials toward the generation of spike clusters. Regular spike firing and intracellular Ca2+ release thus appear to be functionally coupled in a positive feedback manner, thereby supporting the relay mode of thalamocortical cells during wakefulness. Regulatory influences may be coupled to this system via the cyclic ADP ribose pathway.

The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association.

Visinin-like protein-3 is a member of the family of intracellular neuronal calcium sensors belonging to the superfamily of EF-hand proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades. To gain insights into the characteristics of visinin-like protein-3, we have generated specific antibodies against visinin-like protein-3 and determined the developmental and tissue distribution of the protein and its exact cellular and subcellular localization. Expression of visinin-like protein-3 protein appeared late in development mainly in the cerebellum. It is strongly expressed in cerebellar Purkinje cells. The protein expression results were further confirmed by in situ hybridization and compared with hippocalcin messenger RNA localization. Native cerebellar visinin-like protein-3 was shown to bind calcium and to associate in a calcium-dependent manner with membrane fractions during subcellular fractionation. Recombinant wild-type visinin-like protein-3 was shown to be N-terminally myristoylated in transfected cells. The membrane association was strongly reduced for the non-myristoylated mutant of visinin-like protein-3 in transfected cells. These results suggest that visinin-like protein-3, which is mainly expressed in Purkinje cells in vivo, shows a calcium-dependent association with cell membranes which is mediated by a calcium-myristoyl switch.

Novelty acquisition is associated with induction of hippocampal long-term depression.

Homosynaptic long-term depression (LTD) consists of a persistent nonpathological decrease in synaptic transmission, which is induced by low-frequency stimulation. In vivo, low-frequency stimulation (1 Hz, 900 pulses) induces LTD in Wistar but not Hooded Lister rats. In this study, we investigated the influence of behavioral learning and behavioral state on the expression of LTD in both rat strains. Recordings were taken from freely moving animals that had undergone chronic implantation of a recording electrode in the hippocampal CA1 region and a bipolar stimulating electrode in the ipsilateral Schaffer collateral-commissural pathway. Exposure of the rat strains to stress induced a significant elevation in serum corticosterone levels but did not facilitate LTD expression. However, LFS given during exploration of a novel environment resulted in LTD expression in Hooded Lister, and LTD enhancement in Wistar, rats. Reexposure to the same environment did not result in new expression of LTD. Behavioral comparison between the first and second environmental exposure confirmed that the animals had habituated to the novel environment. These observations strongly implicate an association between novelty acquisition and LTD.

Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function.

Intracellular neuronal calcium sensors (NCS) constitute a rapidly growing family of calcium-binding proteins which belong to the superfamily of EF-hand proteins. The NCS family includes as subgroups the recoverins and GCAPs (guanylyl cyclase-activating proteins), which are primarily expressed in retinal photoreceptor cells, and the frequenins and VILIPs (visinin-like proteins), which are widely but differentially expressed in the nervous system. In this review the recent developments in elucidating the functional activities of NCS proteins on signal transduction pathways in neurons are surveyed and discussed. We will focus our attention on calcium-dependent membrane association by the so-called calcium-myristoyl switch as a possible mechanism of signal transduction and on the roles of NCS proteins in intraneuronal signaling cascades, which are best studied in the visual and olfactory systems.

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain.

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.

Subtype-specific involvement of metabotropic glutamate receptors in two forms of long-term potentiation in the dentate gyrus of freely moving rats.

In this study, the role of metabotropic glutamate receptors in N-methyl-D-aspartate receptor-dependent and voltage-gated calcium channel-dependent long-term potentiation in the dentate gyrus of freely moving rats was investigated. Antagonists for group 1 metabotropic glutamate receptors ((S)-4-carboxyphenylglycine), group 1/2 metabotropic glutamate receptors ((RS)-alpha-methyl-4-carboxyphenylglycine) and group 2 metabotropic glutamate receptors ((RS)-alpha-methylserine O-phosphate monophenylester) were used. The N-methyl-D-aspartate receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid, and the L-type voltage-gated calcium channel antagonist, methoxyverapamil were used to investigate the N-methyl-D-aspartate receptor and voltage-gated calcium channel contribution to the long-term potentiation recorded. Field excitatory postsynaptic potential slope and population spike amplitude were measured. Drugs were applied, prior to tetanus, via a cannula implanted into the lateral cerebral ventricle. 200 Hz tetanization produces a long-term potentiation which is inhibited by application of D(-)-2-amino-5-phosphonopentanoic acid and (RS)-alpha-methyl-4-carboxyphenylglycine. In this study, a dose-dependent inhibition of 200 Hz long-term potentiation expression was obtained with (S)-4-carboxyphenylglycine. Long-term potentiation induced by 400 Hz tetanization was not inhibited by D(-)-2-amino-5-phosphonopentanoic acid, although the amplitude of short-term potentiation was reduced. (RS)-alpha-methyl-4-carboxyphenylglycine and (S)-4-carboxyphenylglycine, both in the presence and absence of D(-)-2-amino-5-phosphonopentanoic acid, inhibited the development of 400 Hz long-term potentiation. (RS)-alpha-methylserine O-phosphate monophenylester had no significant effect on long-term potentiation induced by either 200 or 400 Hz tetanization. Application of methoxyverapamil significantly inhibited 400 Hz long-term potentiation, but had no effect on 200 Hz long-term potentiation. These data suggest that 400 Hz long-term potentiation, induced in the presence of D(-)-2-amino-5-phosphonopentanoic acid, requires activation of L-type calcium channels. Furthermore, these results strongly support a critical role for group 1 metabotropic glutamate receptors in both N-methyl-D-aspartate receptor- and voltage-gated calcium channel-dependent long-term potentiation.

Low level expression of calcium-sensor protein VILIP induces cAMP-dependent differentiation in rat C6 glioma cells.

Wild-type visinin-like-protein (VILIP) and a myristoylation-deficient VILIP mutant, when stably expressed at low levels in C6 cells, enhances or reduces the basal cAMP-level, respectively. The morphology of wild-type VILIP-transfected cells resembles that of differentiated astrocytes, whereas the myristoylation mutant shows a phenotype similar to parental cells, but with reduced cell growth. In both parental and myristoylation mutant cells a differentiated phenotype similar to that produced by wild-type VILIP-transfected cells is inducible with 8-bromo-cAMP. The changed morphology parallels an increase in the expression of the astrocytic differentiation marker glial fibrillary acidic protein (GFAP) in wild-type VILIP-transfected and cAMP-differentiated cells, but a decrease of GFAP in myristoylation mutant cells. These results suggest that depending on myristoylation, low level ectopic expression of VILIP affects basal cAMP homeostasis differentially, thereby influencing differentiation of C6 model cells.