Abstract:
The number and type of receptors present at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Because most neurons receive multiple and distinct synaptic inputs and contain several different subtypes of receptors stimulated by the same neurotransmitter, the assembly and trafficking of receptors in neurons is a complex process involving many levels of regulation. To investigate the mechanism that neurons use to regulate the assembly of receptor subunits, we studied a GluR2 knock-out mouse. GluR2 is a critical subunit that controls calcium permeability of AMPA receptors and is present in most native AMPA receptors. Our data indicate that in the absence of GluR2, aberrant receptor complexes composed of GluR1 and GluR3 are formed in the hippocampus, and that there is an increased number of homomeric GluR1 and GluR3 receptors. We also show that these homomeric and heteromeric receptors are less efficiently expressed at the synapse. Our results show that GluR2 plays a critical role in controlling the assembly of AMPA receptors, and that the assembly of subunits may reflect the affinity of one subunit for another or the stability of intermediates in the assembly process. Therefore, GluR1 may have a greater preference for GluR2 than it does for GluR3.

Abstract:
NMDA (N-methyl-D-aspartate) receptors (NMDARs) are targeted to dendrites and anchored at the post-synaptic density (PSD) through interactions with PDZ proteins. However, little is known about how these receptors are sorted from the endoplasmic reticulum and Golgi apparatus to the synapse. Here, we find that synapse-associated protein 102 (SAP102) interacts with the PDZ-binding domain of Sec8, a member of the exocyst complex. Our results show that interactions between SAP102 and Sec8 are involved in the delivery of NMDARs to the cell surface in heterologous cells and neurons. Furthermore, they suggest that an exocyst-SAP102-NMDAR complex is an important component of NMDAR trafficking.

Abstract:
BACKGROUND: The secreted frizzled related proteins (sFRP) are soluble proteins thought to interfere with the Wnt signaling. Our group previously demonstrated that one of these members, sFRP-1/FrzA, is strongly expressed during early phases of the vascularization process in embryonic vasculature and in the endothelium of arteries and capillaries in adults and modulated vascular cell proliferation. METHODS AND RESULTS: Analysis of the expression of sFRP-1 during cyclic ovarian angiogenesis revealed that sFRP-1 is expressed during the formation of neovessels and becomes undetectable when the vasculature is fully maturated. We then studied the role of FrzA in several distinct angiogenic models. FrzA induced angiogenesis in a chick chorioallantoic membrane model. Moreover, gene transfer of AdFrzA in grafted mesenchymal and glioma cells increased vessel density and tumor growth. FrzA induced formation of vessels, which were enlarged, longer, and appeared to be more mature compared with vessels formed under control treatments. In vitro, FrzA increased migration and tube formation of endothelial cells and seemed to protect them from apoptosis. FrzA-angiogenic effect in vitro was independent of vascular endothelial growth factor, fibroblast growth factor-2, or angiopiotin-1 induction and Akt activation. In contrast, FrzA decreased glycogen synthase kinase-3 phosphorylation. CONCLUSIONS: These results showed that FrzA has proangiogenic effects and suggest that Wnt signaling may be involved in normal differentiation as well as in the pathological development of vasculature.

Abstract:
We investigated whether three calcium-binding proteins, calretinin, parvalbumin, and calbindin, could identify specific aspects of the postnatal development of the rat lateral (LVN) and medial (MVN) vestibular nuclei and their vestibular and cerebellar connections. Calretinin levels in the vestibular nuclei, increased significantly between birth and postnatal day (P) 45. In situ hybridization and immunocytochemical staining showed that calretinin-immunoreactive neurons were mostly located in the parvocellular MVN at birth and that somatic and dendritic growth occurred between birth and P14. During the first week, parvalbumin-immunoreactive fibers and endings were confined to specific areas, i.e., the ventral LVN and magnocellular MVN, and identified exclusively the maturation of the vestibular afferents. Calbindin was located within the dorsal LVN and the parvocellular MVN and identified the first arrival of the corticocerebellar afferents. From the second week, in addition to labeling vestibular afferents in their specific target areas, parvalbumin was also found colocalized with calbindin in mature Purkinje cell afferents. Thus, the specific spatiotemporal distribution of parvalbumin and calbindin could correspond to two successive phases of synaptic remodeling involving integration of the vestibular sensory messages and their cerebellar control. On the basis of the sequence of distribution patterns of these proteins during the development of the vestibular nuclei, calretinin is an effective marker for neuronal development of the parvocellular MVN, parvalbumin is a specific marker identifying maturation of the vestibular afferents and endings, and calbindin is a marker of the first appearance and development of Purkinje cell afferents.

Abstract:
The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.

Abstract:
Balance epithelia in birds closely resemble their mammalian counterparts, but their cells turnover rapidly and they quickly regenerate hair cells, leading to functional recovery from damage that would be permanent for a mammal. We isolated and cultured sheets of the chicken's utricular epithelium in bromo-deoxyuridine and specific inhibitors of different intracellular signalling pathways to identify signals that influence turnover and regeneration. Synthesis (S-phase) entry was effectively blocked by inhibition of PI3-K, TOR or MAPK, and significantly decreased by inhibitors of PKC. Comparisons indicate that activated PI3-K and TOR are required for S-phase entry in both avian and mammalian balance epithelia, but activation of the MAPK pathway appears to have a more significant role in avian utricles than in mammals. The dissimilarities in the requirements for these signalling pathways do not appear sufficient to explain the marked difference in regenerative capacity between the ears of birds and mammals.

Abstract:
Targeting of glutamate receptors (GluRs) to synapses involves rapid movement of intracellular receptors. This occurs in forms of synaptic upregulation of receptors, such as long-term potentiation. Thus, many GluRs are retained in a cytoplasmic pool in dendrites, and are transported to synapses for upregulation, presumably via motor proteins such as myosins travelling along cytoskeletal elements that extend up into the spine. In this ultrastructural immunogold study of the cerebellar cortex, we compared synapses between normal rats/mice and dilute lethal mutant mice. These mutant mice lack myosin Va, which has been implicated in protein trafficking at synapses. The postsynaptic spine in the cerebellum lacks the inositol trisphosphate receptor (IP3R) -laden reticular tubules that are found in normal mice and rats (Takagishi et al., Neurosci. Lett., 1996, 215, 169). Thus, we tested the hypothesis that myosin Va is necessary for transport of GluRs and associated proteins to spine synapses. We found that these spines retain a normal distribution of (i) GluRs (delta 1/2, GluR2/3 and mGluR1alpha), (ii) at least one associated MAGUK (membrane-associated guanylate kinase) protein, (iii) Homer (which interacts with mGluR1alpha and IP3Rs), (iv) the actin cytoskeleton, (v) the reticulum-associated protein BiP, and (vi) the motor-associated protein, dynein light chain. Thus, while myosin Va may maintain the IP3R-laden reticulum in the spine for proper calcium regulation, other mechanisms must be involved in the delivery of GluRs and associated proteins to synapses. Other possible mechanisms include diffusion along the extrasynaptic membrane and delivery via other motors running along the spine's actin cytoskeleton.

Abstract:
The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.

Abstract:
Changes in the expression of the AMPA receptor subunits GluR1-4 and of the NMDA receptor subunits NR1, NR2A-D were investigated in the developing rat medial and lateral vestibular nuclei. Analyses were performed using nonradioactive in situ hybridization and immunoblotting with subunit-specific antibodies. During the postnatal development, glutamatergic receptor subunits were differentially expressed in the vestibular nuclei. The level of expression of GluR1, GluR4 and NR1 subunits was higher in the developing brain as compared to the adult. We observed a gradual increase in GluR2/3, NR2A, NR2B and NR2C levels of expression in the medial and lateral vestibular nuclei during the first 3 weeks of postnatal development. In situ hybridization results were consistent with immunoblot analyses. The differential expression of AMPA and NMDA receptor subunits in immature vestibular neurons is consistent with changes in glutamate receptor properties. This may be related to the postsynaptic regulation of receptor subunits associated with the synaptic plasticity of the vestibular neuron connections during specific sequences of postnatal development.

Abstract:
The aim of this study was to investigate the potential plasticity of the vestibular system, in structural and biochemical terms, at the level of the gravity receptors (the sensory hair cells), the primary neurons relaying the sensory signals (the vestibular ganglion neurons) and their projections into the vestibular nuclei. We studied the biochemical differentiation of the sensory cells and of the vestibular ganglion by investigating which calcium-binding proteins were present. We studied the development of peripheral synaptic connections of the efferent system by investigating the distribution of CGRP (calcitonin-gene related-peptide) and we also studied the cerebellar synaptic connections in the vestibular nuclei, as identified by the presence of calbindin. Putative changes were studied after a 17-day episode of microgravity (Neurolab STS-90), in developing rats between postnatal days 8 and 25. The extent to which these changes could be caused by alterations in gravity was determined by examining sensory and nervous structures not involved in gravity detection, the cochlea and the cochlear nuclei.

Abstract:
The changes in expression and the subcellular localization of brain-derived neurotrophic factor (BDNF) protein in the rat vestibular nuclear complex (VNC), have been investigated at different postnatal stages. Immunoblotting and ELISA analyses showed a down-regulation of BDNF protein expression in VNC with age. In addition, observations by confocal microscopy revealed that BDNF is mainly located in neuronal somata at postnatal day 8 (P8) and restricted to processes by P15. These results support the idea that BDNF could have different roles in the VNC according to the stage of development The protein could act as a neurotrophic factor in embryonic and early postnatal stages whereas in later developmental stages of the VNC it could be involved in neuronal maturation and regulation of neuronal circuitry.

Abstract:
The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

Abstract:
The early development of the inner ear is largely determined by two members of the neurotrophic family: brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3). Little information is available on the role of these neurotrophins during the late stages of vestibular development in the rat which take place during the first postnatal weeks. At this period where terminal synaptogenesis and maturation occur, we have investigated the expression and the activity of BDNF, the most important neurotrophin in the vestibular system. Using different experimental approaches, we show that BDNF is released by vestibular epithelia on postnatal day 3 (P3) and continues to have a trophic effect on vestibular neurones in vitro. Immunocytochemistry coupled to confocal microscopy revealed a remarkable evolution in BDNF localization during later stages of development. Whereas BDNF is present in both supporting cells and hair cells at P3, its distribution gradually changed and is highly compartmentalized within the upper part of supporting cells at P8 and P15. In parallel, we observed the presence of a truncated form of the BDNF receptor in sensory hair cells. These results suggest an original role for supporting cells, which could be involved in the release of BDNF during the late stages of synaptogenesis in mammalian vestibular epithelia. In particular, BDNF could participate to the set up of the calyx, a specific nerve structure surrounding type I vestibular hair cells.

Abstract:
The effects of the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) neurotrophins on the intracellular calcium level ([Ca2+]i) were studied in vestibular ganglion neurons (VGNs) from postnatal day 3 (P3) rats cultured for 50 hr. We first assessed the expression of trkB and trkC mRNA receptors in cultured VGNs. Immunobloting and immunocytochemistry confirmed the presence of the neurotrophin receptors on neurons. Both neurotrophins induced transient [Ca2+]i elevations in VGNs: BDNF-treated neurons responded in 65% and NT-3-treated neurons in 56%. The responses could be inhibited by anti-BDNF or anti-NT-3 antibodies. The [Ca2+]i elevation was dependent on extracellular calcium since it was abolished in calcium-free medium but also implicates the release of calcium from intracellular stores as tested by prior depletion with thapsigargin. Our results suggest the implication of a short-term calcium regulation in VGNs, which could reflect specific fast effects of neurotrophins in the early postnatal rat vestibular system.

Abstract:
The localization of neurons expressing mRNAs for the NR1 and NR2A-D subunits of the glutamatergic NMDA receptor was examined by non-radioactive in situ hybridization throughout the guinea pig vestibular nuclei. After deafferentation of the vestibular nuclei by unilateral labyrinthectomy, modifications of the mRNA distributions were followed for 30 days. A quantitative analysis was performed in the medial vestibular nucleus by comparison of the labelled neurons in the ipsi- and contra-lateral nuclei. In vestibular nuclei, the NR1 subunit mRNA was found in various populations of neurons. The NR2A and NR2C subunit mRNAs were less widely distributed, whereas little NR2D mRNA was detected and only rare cells contained NR2B mRNA. NR1 and NR2A-D mRNAs were colocalized in some but not other neuronal types. Twenty hours after the lesion, there was a transient ipsilateral increase of NR1 mRNA level in the medial vestibular nucleus, followed by a decrease 48 h after the lesion and, at 3 days, by recovery to the control level. An ipsilateral increase in the mRNA level of NR2C subunit was detected 20 h after lesion and maintained at 48 h. No significant changes were apparent in NR2A, NR2B and NR2D mRNA levels. The distributions and the differential signal intensities of NR2A-D mRNAs suggest various subunit organizations of the NMDA receptors in different neurons of the vestibular nuclei. Neuronal plasticity reorganizations in the vestibular nuclei following unilateral labyrinthectomy appear to include only changes in NR1 and NR2C mRNA levels modifying the functional diversity of the NMDA receptor in the ipsilateral medial vestibular nucleus neurons. The transient changes in NR1 and the NR2C subunit mRNA expressions in response to sensory deprivation are consistent with an active role for NMDA receptors in the appearance and development of the vestibular compensatory process.

Abstract:
We examined the localization of neurons expressing mRNA for calretinin, a cytosolic EF hand calcium-binding protein, throughout the vestibular nuclei of rat and guinea pig by non-radioactive in situ hybridization, using an alkaline phosphatase labeled oligonucleotide probe. Labeled cells were particularly numerous in the medial vestibular nucleus (mVN) and their distribution was similar in rat and guinea pig, and presented a characteristic rostrocaudal and mediolateral pattern. The effects of hemilabyrinthectomy were assessed at various times post lesion from 10 h to 30 days by comparison of the pattern of labeling in the ipsi- and contra-lateral vestibular nuclei of guinea pig. After up to 48 h no modification in the calretinin mRNA distribution was detected. After 3 to 30 days of survival, there was a decrease (about 30%) of the calretinin expressing neurons in the nucleus on the side of the lesion. The unilateral sensory deprivation seemed to induce a permanent asymmetry in the expression of calretinin which was not abolished after vestibular compensation. These results suggested that the calretinin expression in these neurons depends upon the integrity and activity of sensorineuronal peripheral vestibular influences.