Rett syndrome is a neurodevelopmental disorder caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2) and represents the leading genetic cause for mental retardation in girls. MeCP2-mutant mice have been generated to study the molecular mechanisms of the disease. It was suggested that an imbalance between excitatory and inhibitory neurotransmission is responsible for the behavioral abnormalities, although it remained largely unclear which synaptic components are affected and how cellular impairments relate to the time course of the disease. Here, we report that MeCP2 KO mice present an imbalance between inhibitory and excitatory synaptic transmission in the ventrolateral medulla already at postnatal day 7. Focusing on the inhibitory synaptic transmission we show that GABAergic, but not glycinergic, synaptic transmission is strongly depressed in MeCP2 KO mice. These alterations are presumably due to both decreased presynaptic gamma-aminobutyric acid (GABA) release with reduced levels of the vesicular inhibitory transmitter transporter and reduced levels of postsynaptic GABA(A)-receptor subunits alpha2 and alpha4. Our data indicate that in the MeCP2 -/y mice specific synaptic molecules and signaling pathways are impaired in the brain stem during early postnatal development. These observations mandate the search for more refined diagnostic tools and may provide a rationale for the timing of future therapeutic interventions in Rett patients.
Neurexins constitute a large family of highly variable cell-surface molecules that may function in synaptic transmission and/or synapse formation. Each of the three known neurexin genes encodes two major neurexin variants, ␣-and -neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three ␣-neurexins in mice recently demonstrated their essential role at synapses. In multiple ␣-neurexin knock-outs, neurotransmitter release from excitatory and inhibitory synapses was severely reduced, primarily probably because voltage-dependent Ca 2ϩ channels were impaired. It remained unclear, however, which neurexin variants actually influence exocytosis and Ca 2ϩ channels, which domain of neurexins is required for this function, and which Ca 2ϩ -channel subtypes are regulated. Here, we show by electrophysiological recordings that transgenic neurexin 1␣ rescues the release and Ca 2ϩ -current phenotypes, whereas transgenic neurexin 1 has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1␣ rescued the knock-out phenotype independent of the ␣-neurexin gene deleted, these data are consistent with a redundant function among different ␣-neurexins. In both knock-out and transgenically rescued mice, ␣-neurexins selectively affected the component of neurotransmitter release that depended on activation of N-and P/Q-type Ca 2ϩ channels, but left L-type Ca 2ϩ channels unscathed. Our findings indicate that ␣-neurexins represent organizer molecules in neurotransmission that regulate N-and P/Q-type Ca 2ϩ channels, constituting an essential role at synapses that critically involves the extracellular domains of neurexins.
Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.
␣-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three ␣-neurexin genes show a severe reduction of synaptic release. Although analyses of ␣-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca 2ϩ channels, it remained unclear whether ␣-neurexins have a general role in Ca 2ϩ -dependent exocytosis and how they may affect Ca 2ϩ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by Ͼ50% in adult ␣-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca 2ϩ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that ␣-neurexins do not affect the activation or inactivation properties of Ca 2ϩ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for ␣-neurexins in Ca 2ϩ -triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.
Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs), occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX) and a highly conserved co-receptor protein (Orco). The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM) action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs) expressing Or22a inside the fly's antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native neurons are required to study the presently unknown additional mechanisms involved in OSN sensitization.
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