Genetic evidence for the adhesion protein IgSF9/Dasm1 to regulate inhibitory synapse development independent of its intracellular domain.

Normal brain function requires balanced development of excitatory and inhibitory synapses. An imbalance in synaptic transmission underlies many brain disorders such as epilepsy, schizophrenia, and autism. Compared with excitatory synapses, relatively little is known about the molecular control of inhibitory synapse development. We used a genetic approach in mice to identify the Ig superfamily member IgSF9/Dasm1 as a candidate homophilic synaptic adhesion protein that regulates inhibitory synapse development. IgSF9 is expressed in pyramidal cells and subsets of interneurons in the CA1 region of hippocampus. Electrophysiological recordings of acute hippocampal slices revealed that genetic inactivation of the IgSF9 gene resulted in fewer functional inhibitory synapses; however, the strength of the remaining synapses was unaltered. These physiological abnormalities were correlated with decreased expression of inhibitory synapse markers in IgSF9(-/-) mice, providing anatomical evidence for a reduction in inhibitory synapse numbers, whereas excitatory synapse development was normal. Surprisingly, knock-in mice expressing a mutant isoform of IgSF9 lacking the entire cytoplasmic domain (IgSF9(ΔC/ΔC) mice) had no defects in inhibitory synapse development, providing genetic evidence that IgSF9 regulates synapse development via ectodomain interactions rather than acting itself as a signaling receptor. Further, we found that IgSF9 mediated homotypic binding and cell aggregation, but failed to induce synapse formation, suggesting that IgSF9 acts as a cell adhesion molecule (CAM) to maintain synapses. Juvenile IgSF9(-/-) mice exhibited increased seizure susceptibility indicative of an imbalance in synaptic excitation and inhibition. These results provide genetic evidence for a specific role of IgSF9 in inhibitory synapse development/maintenance, presumably by its CAM-like activity.

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary.

BACKGROUND: Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca2+-activated K+ channel (K(Ca)) of big conductance (BK(Ca)), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of K(Ca) channels (including BK(Ca) beta-subunits), and 2. biophysical properties of BK(Ca) channels. METHODS: GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique. RESULTS: We identified two K(Ca) types in human GCs, the intermediate- (IK) and the small-conductance K(Ca) (SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by K(Ca) blockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single K(Ca) channels with distinctive features. Both, IK and BK(Ca) channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BK(Ca) channel revealed the presence of mRNAs encoding several BK(Ca) beta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BK(Ca) channels which we observed in electrophysiological recordings. CONCLUSION: Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK(Ca), they express all three K(Ca) types known. We suggest that the plurality and co-expression of different K(Ca) channels and BK(Ca) beta-subunits might allow differentiated responses to Ca2+ signals over a wide range caused by various intraovarian signalling molecules (e.g. acetylcholine, ATP, dopamine). The knowledge of ovarian K(Ca) channel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.

Electrical activity suppresses axon growth through Ca(v)1.2 channels in adult primary sensory neurons.

BACKGROUND: Primary sensory neurons of the dorsal root ganglia (DRG) regenerate their spinal cord axon if the peripheral nerve axon has previously been cut. This conditioning lesion confers axon growth competence to the neurons. However, the signal that is sensed by the cell upon peripheral lesion to initiate the regenerative response remains elusive. RESULTS: We show here that loss of electrical activity following peripheral deafferentiation is an important signal to trigger axon regrowth. We first verified that firing in sensory fibers, as recorded from dorsal roots in vivo, declined after peripheral lesioning but was not altered after central lesioning. We found that electrical activity strongly inhibited axon outgrowth in cultured adult sensory neurons. The inhibitory effect depended on the L-type voltage-gated Ca(2+) channel current and involved transcriptional changes. After a peripheral lesion, the L-type current was consistently diminished and the L-type pore-forming subunit, Ca(v)1.2, was downregulated. Genetic ablation of Ca(v)1.2 in the nervous system caused an increase in axon outgrowth from dissociated DRG neurons and enhanced peripheral nerve regeneration in vivo. CONCLUSIONS: Our data indicate that cessation of electrical activity after peripheral lesion contributes to the regenerative response observed upon conditioning and might be necessary to promote regeneration after central nervous system injury.

Serine phosphorylation of ephrinB2 regulates trafficking of synaptic AMPA receptors.

Plasticity in the brain is essential for maintaining memory and learning and is associated with the dynamic membrane trafficking of AMPA receptors. EphrinB proteins, ligands for EphB receptor tyrosine kinases, are transmembrane molecules with signaling capabilities that are required for spine morphogenesis, synapse formation and synaptic plasticity. Here, we describe a molecular mechanism for ephrinB2 function in controlling synaptic transmission. EphrinB2 signaling is critical for the stabilization of AMPA receptors at the cellular membrane. Mouse hippocampal neurons from conditional ephrinB2 knockouts showed enhanced constitutive internalization of AMPA receptors and reduced synaptic transmission. Mechanistically, glutamate receptor interacting proteins bridge ephrinB ligands and AMPA receptors. Moreover, this function involved a regulatory aspect of ephrinB reverse signaling that involves the phosphorylation of a single serine residue in their cytoplasmic tails. In summary, our findings uncover a model of cooperative AMPA receptor and ephrinB reverse signaling at the synapse.