Whether contact of an axon with a dendrite is a necessary inductive signal for the assembly of functional presynaptic machinery is controversial. Combining FM1-43 imaging with retrospective immunocytochemistry, we observe many functional synaptic vesicle (SV) release sites lacking postsynaptic specializations in cultured hippocampal neurons. These "orphan" release sites share the same exocytic machinery and mechanisms of endocytic recycling as mature synaptic sites. Moreover, quantitative analysis of FM1-43 destaining at these orphan release sites reveals similar kinetics with slightly lower release probabilities. Time-lapse imaging of FM1-43 reveals that orphans are generated by complete or partial mobilization of synaptic release sites that retain their functionality in transit. Orphan clusters fuse with existing synaptic release sites or form novel release sites onto dendrites. Mobilization and stabilization of orphan boutons to new sites of dendritic contact may represent a necessary presynaptic counterpart to postsynaptic changes observed during development and plasticity in the CNS.

The likelihood with which an action potential elicits neurotransmitter release, the release probability (p r ), is an important component of synaptic strength. Regulatory mechanisms controlling several steps of synaptic vesicle (SV) exocytosis may affect p r , yet their relative importance in determining p r and eliciting temporal changes in neurotransmitter release at individual synapses is largely unknown. We have investigated whether the size of the active zone cytomatrix is a major determinant of p r and whether changes in its size lead to corresponding alterations in neurotransmitter release. We have used a fluorescent sensor of SV exocytosis, synaptophysin-pHluorin, to measure p r at individual synapses with high accuracy and employed a fluorescently labeled cytomatrix protein, Bassoon, to quantify the amount of active zone cytomatrix present at these synapses. We find that, for synapses made by a visually identified presynaptic neuron, p r is indeed strongly correlated with the amount of active zone cytomatrix present at the presynaptic specialization. Intriguingly, active zone cytomatrices are frequently subject to synapse-specific changes in size on a time scale of minutes. These spontaneous alterations in active zone size are associated with corresponding changes in neurotransmitter release. Our results suggest that the size of the active zone cytomatrix has a large influence on the reliability of synaptic transmission. Furthermore, they implicate mechanisms leading to rapid structural alterations at active zones in synapse-specific forms of plasticity. release probability | synaptic vesicle docking | active zone cytomatrix | plasticity | pHluorin S ynaptic transmission between most neurons is unreliable as a presynaptic action potential elicits neurotransmitter (NT) release at any individual synapse with a release probability (p r ) of less than one. This probability can vary considerably among synapses of the same axon, often in a target-specific manner (1-3). Similarly, synapses onto a single neuron often display widely varying release probabilities (4-7). Moreover, p r can undergo sustained activity-dependent changes over time (8)(9)(10)(11). These observations suggest that p r is an important determinant of synaptic strength that is tightly controlled by the presynaptic neuron and/or, indirectly, by its postsynaptic target.Although numerous studies have highlighted specific regulatory mechanisms affecting p r of synapses at rest, a concise picture of how p r at individual synapses is determined is only beginning to emerge. Synaptic vesicle (SV) exocytosis is a multistep process during which SVs have to dock at the active zone cytomatrix and undergo a priming step before they are "readily releasable," i.e., immediately available for fusion with the plasma membrane in response to calcium influx through voltage-gated calcium channels (12). Evidence that the size of a synapse's readily releasable pool (RRP) of SVs is closely correlated with its p r (7, 13) suggests that p r is mainly determined by pro...

The acid-sensitive ion channel ASIC1 is a proton-gated ion channel from the mammalian nervous system. Its expression in sensory neurons and activation by low extracellular pH suggest that ASIC is involved in transmitting nociceptive impulses produced by the acidification caused by injury or inflammation. However, ASIC1 expression is not restricted to sensory neurons. To understand the functional role of ASIC1 in the CNS we investigated its expression and subcellular distribution therein. In particular, we examined the presence of ASIC1 in domains where the local pH may drop sufficiently to activate ASIC1 under physiological conditions. Immunostaining with specific antibodies revealed broad expression of ASIC1 in many areas of the adult rat brain including the cerebral cortex, hippocampus and cerebellum. Within cells, ASIC1 was found predominantly throughout the soma and along the branches of axons and dendrites. ASIC1 was not enriched in the microdomains where pH may reach low values, such as in synaptic vesicles or synaptic membranes. Pre-or postsynaptic ASIC1 was not gated by synaptic activity in cultured hippocampal neurons. Blockage or desensitization of ASIC1 with amiloride or pH 6.7, respectively, did not modify postsynaptic currents. Finally, the ontogeny of ASIC1 in mouse brain revealed constant levels of expression of ASIC1 protein from embryonic day 12 to the postnatal period, indicating an early and almost constant level of expression of ASIC1 during brain development.

No abstract

Wigerius et al. identify the polarity protein AMOT-130 as vital for dendritic spine morphogenesis. They show that reduced Lats1 kinase activity in the neonatal brain is required for the recruitment of AMOT-130 to postsynaptic compartments to stabilize dendritic spines.

Neurexins are a diverse family of cell adhesion molecules that localize to presynaptic specializations of CNS neurons. Heterologous expression of neurexins in non-neuronal cells leads to the recruitment of postsynaptic proteins in contacting dendrites of co-cultured neurons, implicating neurexins in synapse formation. However, isoform-specific knockouts of either all α- or all β-neurexins show defects in synaptic transmission but an unaltered density of glutamatergic synapses, a finding that argues against an essential function of neurexins in synaptogenesis. To address the role of neurexin in synapse formation and function, we disrupted the function of all α- and β-neurexins in cultured hippocampal neurons by shRNA knockdown or by overexpressing a neurexin mutant that is unable to bind to postsynaptic neurexin ligands. We show that neurexin perturbation results in an attenuation of neurotransmitter release that is in large part due to a reduction in the number of readily releasable synaptic vesicles. We also find that neurexin perturbation fails to alter the ability of neurons to form synapses, but rather leads to more frequent synapse elimination. These experiments suggest that neurexins are dispensable for the formation of initial synaptic contacts, but play an essential role in the stabilization and functional maturation of synapses.

Neuronal activity is thought to drive the remodeling of circuits in the mammalian cerebral cortex. However, its precise function in the underlying formation and elimination of glutamatergic synapses has remained controversial. To clarify the role of activity in synapse turnover, we have assessed the effects of inhibition of glutamate release from a sparse subset of cultured hippocampal neurons on synapse turnover. Sustained chemogenetic attenuation of release through presynaptic expression of a designer receptor exclusively activated by designer drugs (DREADD) had no effect on the formation or elimination of glutamatergic synapses. Sparse expression of tetanus neurotoxin light chain (TeNT-LC), a synaptobrevin-cleaving protease that completely abolishes neurotransmitter release, likewise did not lead to changes in the rate of synapse elimination, although it reduced the rate of synapse formation. The stability of active and silenced synapses correlated with measures of synapse size. While not excluding a modulatory role in synapse elimination, our findings show that synaptic activity is neither required for the removal nor the maintenance of glutamatergic synapses between hippocampal neurons. Our results also demonstrate that the stability of glutamatergic synapses scales with their size irrespective of their activity.