Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse.

Ribbon-type synapses in inner hair cells of the mammalian cochlea encode the complexity of auditory signals by fast and tonic release through fusion of neurotransmitter-containing vesicles. At any instant, only about 100 vesicles are tethered to the synaptic ribbon, and about 14 of these are docked to the plasma membrane, constituting the readily releasable pool. Although this pool contains about the same number of vesicles as that of conventional synapses, ribbon release sites operate at rates of about two orders of magnitude higher and with submillisecond precision. How these sites replenish their vesicles so efficiently remains unclear. We show here, using two-photon imaging of single release sites in the intact cochlea, that preformed vesicles derived from cytoplasmic vesicle-generating compartments participate in fast release and replenishment. Vesicles were released at a maximal initial rate of 3 per millisecond during a depolarizing pulse, and were replenished at a rate of 1.9 per millisecond. We propose that such rapid resupply of vesicles enables temporally precise and sustained release rates. This may explain how the first auditory synapse can encode with indefatigable precision without having to rely on the slow, local endocytic vesicle cycle.

Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity.

Despite their wide use, the physiological relevance of organotypic slices remains controversial. Such cultures are prepared at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphology might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch-clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepared at postnatal days (P) 14, 17 and 21 were found to be developmentally equivalent to organotypic slices cultured for 1, 2 and 3 weeks, respectively, in terms of development of synaptic transmission and dendritic morphology. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine density and proportions of different spine types were also similar in both preparations,at these corresponding stages. The most notable difference between organotypic and acute slices was a four- to five-fold increase in the absolute frequency of glutamatergic (but not GABAergic)miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal analysis, resulting in an increased total synapse number. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed constant in both preparations over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both preparations at are markably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.

Fm1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea.

Neural transmission of complex sounds demands fast and sustained rates of synaptic release from the primary cochlear receptors, the inner hair cells (IHCs). The cells therefore require efficient membrane recycling. Using two-photon imaging of the membrane marker FM1-43 in the intact sensory epithelium within the cochlear bone of the adult guinea pig, we show that IHCs possess fast calcium-dependent membrane uptake at their apical pole. FM1-43 did not permeate through the stereocilial mechanotransducer channel because uptake kinetics were neither changed by the blockers dihydrostreptomycin and d-tubocurarine nor by treatment of the apical membrane with BAPTA, known to disrupt mechanotransduction. Moreover, the fluid phase marker Lucifer Yellow produced a similar labeling pattern to FM1-43, consistent with FM1-43 uptake via endocytosis. We estimate the membrane retrieval rate at approximately 0.5% of the surface area of the cell per second. Labeled membrane was rapidly transported to the base of IHCs by kinesin-dependent trafficking and accumulated in structures that resembled synaptic release sites. Using confocal imaging of FM1-43 in excised strips of the organ of Corti, we show that the time constants of fluorescence decay at the basolateral pole of IHCs and apical endocytosis were increased after depolarization of IHCs with 40 mm potassium, a stimulus that triggers calcium influx and increases synaptic release. Blocking calcium channels with either cadmium or nimodipine during depolarization abolished the rate increase of apical endocytosis. We suggest that IHCs use fast calcium-dependent apical endocytosis for activity-associated replenishment of synaptic membrane.