GABAB receptor cell-surface export is controlled by an endoplasmic reticulum gatekeeper.

Endoplasmic reticulum (ER) release and cell-surface export of many G protein-coupled receptors (GPCRs) are tightly regulated. For gamma-aminobutyric acid (GABA)B receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell-surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gatekeepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function.

Calcium-dependent protein kinases in plants: evolution, expression and function.

Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in metabolism, osmosis, hormone response and stress signaling pathways. CPKs are a large multigene family of proteins that are present in all plants studied to date, as well as in protists, oomycetes and green algae, but are not found in animals and fungi. Despite the increasing evidence of the importance of CPKs in developmental and stress responses from various plants, a comprehensive genome-wide analysis of CPKs from algae to higher plants has not been undertaken. This paper describes the evolution of CPKs from green algae to plants using a broadly sampled phylogenetic analysis and demonstrates the functional diversification of CPKs based on expression and functional studies in different plant species. Our findings reveal that CPK sequence diversification into four major groups occurred in parallel with the terrestrial transition of plants. Despite significant expansion of the CPK gene family during evolution from green algae to higher plants, there is a high level of sequence conservation among CPKs in all plant species. This sequence conservation results in very little correlation between CPK evolutionary groupings and functional diversity, making the search for CPK functional orthologs a challenge.

The Potential of Molecular Indicators of Plant Virus Infection: Are Plants Able to Tell Us They Are Infected?

To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method (Bioanalyzer); and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.

Is the Glycoprotein Responsible for the Differences in Dispersal Rates between Lettuce Necrotic Yellows Virus Subgroups?

Lettuce necrotic yellows virus is a type of species in the Cytorhabdovirus genus and appears to be endemic to Australia and Aotearoa New Zealand (NZ). The population of lettuce necrotic yellows virus (LNYV) is made up of two subgroups, SI and SII. Previous studies demonstrated that SII appears to be outcompeting SI and suggested that SII may have greater vector transmission efficiency and/or higher replication rate in its host plant or insect vector. Rhabdovirus glycoproteins are important for virus-insect interactions. Here, we present an analysis of LNYV glycoprotein sequences to identify key features and variations that may cause SII to interact with its aphid vector with greater efficiency than SI. Phylogenetic analysis of glycoprotein sequences from NZ isolates confirmed the existence of two subgroups within the NZ LNYV population, while predicted 3D structures revealed the LNYV glycoproteins have domain architectures similar to Vesicular Stomatitis Virus (VSV). Importantly, changing amino acids at positions 244 and 247 of the post-fusion form of the LNYV glycoprotein altered the predicted structure of Domain III, glycosylation at N248 and the overall stability of the protein. These data support the glycoprotein as having a role in the population differences of LNYV observed between Australia and New Zealand.

Recombinant proteins and peptides as diagnostic and therapeutic reagents for arthropod allergies.

Domestic arthropods are chief sources of potent allergens that trigger sensitization and stimulate IgE-mediated allergies. Diagnosis and immunotherapy of arthropod allergies rely on the use of natural allergen extracts which are associated with low specificity and efficacy, the risk of anaphylactic reactions, and the extended period of treatment. Most of the problems associated with natural allergen extracts for allergy diagnosis and immunotherapy can be circumvented with the use of recombinant allergens and peptides. Recombinant allergens are recently developed for microarray-based multi-allergen tests which provide component-resolved diagnosis (CRD) of the patient's sensitization profile. Moreover, recombinant protein technology and peptide chemistry have been used to construct isoallergens, allergen mutants, allergoids, T and B cell peptides, hypoallergens, and mimotopes with reduced allergenicity but enhanced immunogenicity for allergen-specific immunotherapy (SIT) and vaccination. The basics of recombinant arthropod allergen technology are in place providing a lucid future for the advancement of diagnosis and immunotherapy of arthropod allergies.

Involvement of the Sst1 somatostatin receptor subtype in the intrahypothalamic neuronal network regulating growth hormone secretion: an in vitro and in vivo antisense study.

Five somatostatin (SRIH) receptors (sst1-5) have been cloned. Recent anatomical evidence suggests that sst1 and sst2 may be involved in the central regulation of GH secretion. Given the lack of specific receptor antagonists, we used selective antisense oligodeoxynucleotides (ODNs) to test the hypothesis that one or both of these subtypes are involved in the intrahypothalamic network regulating pulsatile GH secretion. In mouse neuronal hypothalamic cultures the proportion of GHRH neurons coexpressing sst1 or sst2 messenger RNAs (mRNAs) was identical. In contrast, sst1 mRNAs were more often present than sst2 in SRIH-expressing neurons. Firstly, sst1 antisense ODN in vitro treatment abolished sst1, but not sst2, receptor modulation of glutamate sensitivity and decreased sst1, but not sst2, mRNAs. The reverse was true after treatment with sst2 antisense. Sense ODNs did not alter the effects of SRIH agonists. In a second series of experiments, nonanaesthetized adult male rats were infused for 120 h intracerebroventricularly with ODNs. Only the sst1 antisense ODN diminished the amplitude of ultradian GH pulses without modifying their frequency. In parallel, sst1 antisense ODN strongly diminished sst1 immunoreactivity in the anterior periventricular nucleus and median eminence, as well as sstl periventricular nucleus mRNA levels. The effectiveness of the sst2 antisense ODN was attested by the inhibition of hypothalamic binding of [125I]Tyr0-D-Trp8-SRIH. Scrambled ODNs had no effect on GH secretion or on sst mRNAs or SRIH binding levels. These results favor a preferential involvement of sst1 receptors in the intrahypothalamic regulation of GH secretion by SRIH.

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. I. Development of reciprocal graft-host interactions.

In this paper, we have morphologically studied the developmental events underlying the neuronal replacement, 3-21 days after grafting. Despite their abnormal environment, Purkinje cell progenitors proceed with their proliferation in the grafted neuroepithelium, with a time window similar to that characterizing proliferation of this neuronal class in control mouse embryos. Only postmitotic Purkinje cells leave the grafts and migrate to the host molecular layer following stereotyped pathways. These neurons invade the host molecular layer, either through a tangential migration under the pial basal lamina from the graft/host interface or breaking locally the latter, and passing directly from the lateral swellings of the graft lying on the surface of the host folia. Whatever the pathway for host invasion, the migrating Purkinje cells penetrate radially and/or obliquely into the host molecular layer until their inward-oriented processes attain the molecular/granular layer interface, which occurs about 7 days after grafting. At the end of their migration, the grafted Purkinje cells with bipolar shapes and long and smooth processes begin to build up their ultimate dendritic trees. This dendritogenesis proceeds with constructive and regressive processes, passing through the same three developmental phases described by Ramón y Cajal (Trab. Lab. Invest. Biol. Univ. Madrid 24:215-251, 1926) for control Purkinje cells (phase of the fusiform cell, phase of the stellate cell with disoriented dendrons, and phase of orientation and flattening of the dendrites). In the grafted cerebella, the duration of the second and third phases is somewhat shorter than during normal cerebellar ontogenesis. Synaptogenesis between adult host axons and grafted Purkinje cells starts when the latter attain their second phase of dendritic development. Somatic filopodia emerging from grafted Purkinje cells begin, 10-11 days after grafting, to be synaptically contacted by axonal sprouts of the host climbing fibers resulting, 2 days later, in the formation of pericellular nests. Synaptogenesis between slender dendritic spines and host parallel fibers, together with that of axon terminals from host molecular layer interneurons and the smooth surface of the grafted Purkinje cell somata, begin earlier than in control mouse development, being almost simultaneous with climbing fiber/Purkinje cell synaptogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. II. Development of synaptic responses: an in vitro study.

Solid pieces of cerebellar primordia from 12-day-old C57Bl embryos were implanted in the cerebellar vermis of 3-4-month-old "Purkinje cell degeneration" mutant mice. Ten to 22 days after grafting, mutant mice were sacrificed, and synaptic responses of grafted Purkinje cells were studied by intracellular recordings performed in 400 microns thick sagittal slices in vitro. As early as 10 days after transplantation, grafted Purkinje cells have already completed their migration from the implant into the host molecular layer. Accordingly, inhibitory as well as excitatory responses were already elicited in these cells by electrical stimulation of the host subcortical white matter. Furthermore, a transient stage of multiple innervation of Purkinje cells by climbing fibers exists between 10 and 15 days after grafting, as revealed by the stepwise variation in amplitude of the climbing fiber-mediated excitatory postsynaptic potentials recorded before 15 days after grafting. Thirteen days after transplantation, typical all-or-none climbing fiber-mediated responses, parallel fiber-mediated excitatory postsynaptic potentials, and inhibitory postsynaptic potentials were also already present. Finally, normal adult-type synaptic responses were observed in all tested cells 15 to 17 days after grafting. Together with the companion paper (Sotelo et al., 1990), these results demonstrate that grafted Purkinje cells are able to impose on host afferents a pattern of synaptogenesis which closely follows that occurring during normal development, in particular, the transient stage of multiple innervation of Purkinje cells by climbing fibers.

Chemoresponsiveness of intracellular nuclei neurones to L-aspartate, L-glutamate and related derivatives in rat cerebellar slices maintained in vitro.

The sensitivity of intracerebellar nuclei neurones to pulse applications of L-aspartate, L-glutamate, N-methyl-D,L-aspartate and quisqualate was tested in rat cerebellar slices maintained in vitro. The responses of the nuclear neurones to the four agonists consisted of a transient and dose-dependent increase in their firing of simple spikes. When suprathreshold currents were used, quisqualate induced the highest increase in the spike discharge frequency of the cells. Quisqualate mediated responses were unaffected by steady applications of 2-amino-5-phosphonovalerate, whereas the sensitivity of the responses induced by the three other agonists was in the order N-methyl-D,L-aspartate, L-aspartate, L-glutamate. When the superfusing solution was devoid of Mg2+ ions, N-methyl-D,L-aspartate and L-aspartate mediated responses were much potentiated, while quisqualate induced responses were not enhanced. In such a medium, L-glutamate elicited responses were more or less potentiated depending on cells. These results suggest that rat intracerebellar nuclei neurones bear both N-methyl-D-aspartate and non-N-methyl-D-aspartate, probably quisqualate, receptors, and that L-aspartate and L-glutamate have a mixed action upon both types. L-Aspartate preferentially activates N-methyl-D-aspartate receptors, whereas L-glutamate predominantly acts via non-N-methyl-D-aspartate receptors. Furthermore, the potency of L-glutamate in activating N-methyl-D-aspartate receptors appears to vary as a function of the cells.

Differential effects of serotonin on the spontaneous discharge and on the excitatory amino acid-induced responses of deep cerebellar nuclei neurons in rat cerebellar slices.

The effects of steady iontophoretic applications of serotonin on the spontaneous discharge and on the excitatory responses induced in deep cerebellar nuclei neurons by iontophoretic pulse applications of L-glutamate, L-aspartate, N-methyl-D,L-aspartate and quisqualate were studied in rat cerebellar slices maintained in vitro. Serotonin increased the spontaneous firing rate of deep cerebellar nuclei neurons in 91% of the tested cells by 109% on the average and had no effect on the remaining recorded neurons. Conversely, the monoamine induced a depression of the excitatory responses induced by four agonists tested and the depressant potency of serotonin was in the order quisqualate, glutamate, aspartate, N-methyl-D,L-aspartate. These effects persisted in low calcium high magnesium solution, suggesting that the serotonin receptors involved in these phenomena were, at least partially, postsynaptically located. The serotonin-induced increase in the cell firing rate appeared to be methysergide-resistant whereas the serotonin-induced decrease in the responses elicited by excitatory amino acids was depressed by this antagonist, which could indicate that these differential effects of serotonin are mediated via different mechanisms and/or serotonin receptor subtypes.

Electrophysiological demonstration of a synaptic integration of transplanted Purkinje cells into the cerebellum of the adult Purkinje cell degeneration mutant mouse.

After implantation of solid pieces of cerebellar primordia from 12-day-old C57BL embryos into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice, Purkinje cells from the donor leave the implant and differentiate while migrating into the host molecular layer. Electrophysiological studies were performed using in vitro cerebellar slice preparations from "Purkinje cell degeneration" mutants 1-2 months after grafting, when grafted Purkinje cells have reached their final location in the host molecular layer and have completed their morphological differentiation. Intracellular recordings obtained from 45 Purkinje cells in mutant mice demonstrated that such grafted neurons have normal bioelectrical properties including sodium and calcium conductances and inward rectification. Moreover, all grafted Purkinje cells responded to electrical white matter stimulation by a typical all-or-none climbing fiber response. Responses mediated through the activation of mossy and parallel fibers, as well as inhibitory postsynaptic potentials, were also recorded in a significant number of grafted Purkinje cells. On the whole, all these excitatory and inhibitory responses in grafted "Purkinje cell degeneration" mutant mice have characteristics comparable to those in control mice. After electrophysiological studies, Purkinje cells were further characterized by their positive staining by calbindin antibody. Neurons of this class were dispersed throughout the molecular layer of the host folia in which the electrophysiological recordings had been performed. The ectopic location of their perikarya, the presence of dendritic trees spanning most of the molecular layer (without entering the granular layer), and the occasional presence of axons emerging from the ectopic neurons and forming loose bundles at the white matter axis of the folia, corroborate the grafted nature of the Purkinje cells studied. Therefore, these experiments demonstrate that embryonic Purkinje cells from the graft can complete differentiation in the adult host cerebellum, and establish specific synaptic contacts with the presynaptic elements previously impinging on the missing neurons of "Purkinje cell degeneration" mutants. This process leads to a qualitative functional synaptic restoration of the cortical cerebellar network.

Effect of excitatory amino acids on Purkinje cell dendrites in cerebellar slices from normal and staggerer mice.

The sensitivity of Purkinje cells to short pulse applications of L-aspartate, L-glutamate and related derivatives in their dendritic fields was tested in normal and staggerer mutant mice using cerebellar slices maintained in vitro. In normal mice, the response of Purkinje cells to L-aspartate and L-glutamate consisted of a transient and dose-dependent increase of their firing of simple spikes. The potency of L-aspartate in exciting Purkinje cells was lower than that of L-glutamate when the two drugs were released from adjacent barrels of the same iontophoretic electrode. Quisqualate was an even more potent excitant of these cells than L-aspartate and L-glutamate, whereas N-methyl-DL-aspartate had little or no effect. In staggerer mutant mice, the sensitivity of Purkinje cells to L-aspartate, L-glutamate and quisqualate was not significantly altered. On the contrary, N-methyl-DL-aspartate had a much stronger potency than normal in exciting Purkinje cells although this was still smaller than that of the other agonists tested. These results suggest that the sensitivity of Purkinje cells to L-aspartate and L-glutamate, i.e. the putative neurotransmitters of the climbing and parallel fibers respectively, remains largely normal in staggerer mice. In contrast, in the mutant, N-methyl-D-aspartate receptors are likely to be much more developed than normal.

Cortistatin affects glutamate sensitivity in mouse hypothalamic neurons through activation of sst2 somatostatin receptor subtype.

Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin. Even if it shares several biological properties with somatostatin, the effects of cortistatin on cortical electrical activity and sleep are opposite to those elicited by somatostatin. We recently demonstrated that somatostatin could modulate glutamate sensitivity, either positively through activation of the sstl receptor subtype, or negatively through activation of the sst2 receptor subtype in hypothalamic neurons in culture which express almost exclusively these two sst subtypes. Thus, in the present study we compared the effects of cortistatin and somatostatin in hypothalamic neurons in culture, in order to define the former peptide activity on both subtypes. We first determined that the affinities of cortistatin and somatostatin were similar on cloned rat sstl and sst2 receptor subtypes in transfected cells and hypothalamic neurons membranes. We then found that cortistatin, like somatostatin, depresses the glutamate response but, unlike somatostatin, never potentiates glutamate sensitivity in hypothalamic neurons. The observed effect of cortistatin is strongly suggestive of an activation of the somatostatin sst2 receptor subtype in hypothalamic neurons in culture.

NADPH-diaphorase colocalization with somatostatin receptor subtypes sst2A and sst2B in the retina.

PURPOSE: To investigate the differential localization of somatotropin release-inhibitory factor (SRIF) receptor subtypes (sst2A and sst2B) and their possible colocalization with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the rat and rabbit retina. METHODS: Polyclonal antibodies raised against sst2A and sst2B receptors were applied to 10- to 14-microm cryostat sections of rat and rabbit retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. Double labeling was performed for sst2A or sst2B receptors with NADPH-diaphorase, and with markers for the cell types present in the retina (protein kinase C [PKC], tyrosine hydroxylase; [TH], calbindin, and recoverin). RESULTS: sst2A immunoreactivity was detected in rod bipolar cells and colocalized with NADPH-diaphorase in the rabbit, but not the rat, retina. sst2B was present only in photoreceptor cells of the rat and colocalized with NADPH-diaphorase. CONCLUSIONS: These results suggest that SRIF, acting through sst2A receptors in bipolar cells and sst2B receptors in photoreceptor cells, may affect nitric oxide function in the rabbit and rat retina.

Automatic recognition of nervous structures by image analysis. A pattern recognition method applied to the study of mouse cerebellum.

An automatic image pattern analysis method based on differential densitometry is presented. It is applied to histological sections of mouse cerebellum. This method allows an automatic recognition of any cerebellar microscopic field. After recognition, histological images are decomposed into homogeneous parts with respect to cerebellar cellular layers, and both densitometric patterns and partial areas of each of these layers are estimated. This method is accurate to within 94% for a learning sample and 75% for a test sample. This score can be improved by adjunct algorithms presently under study.

Postnatal development of the chemosensitivity of rat cerebellar Purkinje cells to excitatory amino acids. An in vitro study.

In vitro sagittal slices of immature rat cerebellum were used to study the development of the sensitivity of Purkinje cells (PCs) to L-aspartate (L-Asp), L-glutamate (L-Glu) and related derivatives. As early as postnatal day 0 all PCs already displayed clear excitatory responses to short iontophoretic applications of L-Asp, L-Glu and quisqualate while in the same conditions no effect of N-methyl-D,L-aspartate (NMDLA) was detected. By postnatal day 5, i.e. after the onset of the synaptogenesis, the sensitivity of PCs to L-Asp, L-Glu and quisqualate significantly increased up to values similar to those recorded in adult rat cerebellum and surprisingly nearly all (87%) the recorded cells now also displayed excitatory responses to NMDLA. Although this sensitivity of PCs to NMDLA was significantly lower than that observed with the other drugs, it persisted until the end of the first postnatal month when the adult type of connectivity is already well established but at this stage only 30 per cent of the tested cells were still sensitive to the agonist. During this period, excitatory responses elicited by NMDLA were selectively antagonized by 2-amino-5-phosphonovalerate (2-APV), suggesting that during postnatal development, NMDA receptor types are transiently expressed on PCs membranes since in the adult, NMDLA no longer had an excitatory effect. Instead, this drug now exerted a preferential antagonistic action on the excitatory response elicited by L-Asp. Also in the adult, no major changes occurred in the sensitivity of PCs to L-Asp, L-Glu and quisqualate when these drugs were ejected at a dendritic site whereas, when ejected at the somatic level, the sensitivity of the cell appeared 2-3 times lower.

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro.

A voltage clamp analysis of the effects of L-aspartate, L-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications of L-aspartate and L-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl-D,L-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl-D,L-aspartate much more severely depressed L-aspartate than L-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due to L-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due to L-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl-D-aspartate and quisqualate receptors.

Bioelectrical properties of cerebellar Purkinje cells in reeler mutant mice.

Electroresponsive properties of intracellularly recorded Purkinje cells (PCs) in reeler mice were studied by using sagittal cerebellar slices maintained in vitro. In normally as well as in abnormally located PCs, fast spikes and slower rising multiphasic spikes were elicited by depolarizing currents, and they were abolished by tetrodotoxin and by cadmium respectively. This demonstrates that these neurons, as PCs in normal animals, do have sodium- and calcium-dependent spikes, thus indicating that these bioelectrical properties do not depend on the connectivity of PCs.

Olivocerebellar projections in control and staggerer mutant mice. An autoradiographic study.

Olivocerebellar projections were studied in normal and in staggerer mutant mice by means of autoradiographic methods following [3H]leucine injections in the inferior olivary complex. In normal mice, a sagittal banding pattern of labeled climbing fibers was apparent in the contralateral cerebellar cortex, thus confirming previous observations in rodents. In staggerer mice, olivocerebellar projections also displayed a clear sagittal zonation, thus indicating that this organization may be achieved in the mutant, in spite of pronounced abnormalities of the cerebellar cortex.

Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. II. Membrane conductances.

The development of membrane conductances of intracerebellar nuclei neurons was studied in the rat since birth up to the weaning period by the use of thick sagittal cerebellar slices maintained in vitro. Mature nuclear neurons express fast sodium and slowly inactivating sodium conductances, as well as calcium conductances. As early as birth, fast sodium and calcium conductances appear well developed whereas slowly inactivating sodium conductances mature within the first postnatal week.