Munc13-1 is a presynaptic protein with an essential role in synaptic vesicle priming. It contains a diacylglycerol (DAG)/beta phorbol ester binding C(1) domain and is a potential target of the DAG second messenger pathway that may act in parallel with PKCs. Using genetically modified mice that express a DAG/beta phorbol ester binding-deficient Munc13-1(H567K) variant instead of the wild-type protein, we determined the relative contribution of PKCs and Munc13-1 to DAG/beta phorbol ester-dependent regulation of neurotransmitter release. We show that Munc13s are the main presynaptic DAG/beta phorbol ester receptors in hippocampal neurons. Modulation of Munc13-1 activity by second messengers via the DAG/beta phorbol ester binding C(1) domain is essential for use-dependent alterations of synaptic efficacy and survival.

It has been well established that the volume of secretory vesicles can be modulated. However, we present the first data demonstrating that the amount of transmitter in a vesicle can regulate its volume. Amperometry and transmission electron microscopy have been used to determine that L-3,4-dihydroxyphenylalanine and reserpine increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles as well as their catecholamine content. Because changes in vesicular catecholamine content are tracked by changes in vesicle volume, our results indicate that when quantal size is altered via the vesicular monoamine transporter the concentration of catecholamines within the vesicles remains relatively constant. This previously unidentified cellular response provides new insight into how catecholamines can be packaged in and released from secretory vesicles.

Membrane fusion plays a central role in the synaptic vesicle cycle. While many of the pre- and postfusion events have been investigated at room temperature, few researchers have investigated these processes at more physiologically relevant temperatures. We have used autaptic cultures of hippocampal neurons to investigate changes in the size and refilling rate of the readily releasable pool (RRP) of synaptic vesicles brought about by an increase in temperature from 25 to 35 degrees C. We have also examined temperature-dependent changes in spontaneous and action potential (AP)-evoked release as well as the fraction of the RRP that is released during an AP. Although we found a threefold increase in the refilling rate of the RRP at the higher temperature, there was no apparent change in the size of the RRP with increased temperature. Moreover, we observed a slight but significant decrease in the quanta released during an AP. This increased refilling rate and decreased release probability resulted in a reduction of both the degree and time course of synaptic depression during high frequency stimulation at the higher temperature. This reduction in synaptic depression was accompanied by an increased maintenance of the synchronous component of release during high frequency stimulation. These findings indicate that the dynamics of vesicular supply and release in hippocampal neurons at room temperature are significantly different at near physiological temperatures and could affect our present understanding of the way in which individual neurons and networks of neurons process information.

Processing of sound in the cochlea involves both afferent and efferent innervation. The Na,K-ATPase (NKA) is essential for cells that maintain hyperpolarized membrane potentials and sodium and potassium concentration gradients. Heterogeneity of NKA subunit expression is one mechanism that tailors physiology to particular cellular demands. Therefore, to provide insight into molecular differences that distinguish the various innervation pathways in the cochlea, we performed a variety of double labeling experiments with antibodies against three of the α isoforms of the NKA (NKAα1-3) and markers identifying particular subsets of neurons or supporting cells in whole mount preparations of the organ of Corti and spiral ganglion. We found that the NKAα3 is abundantly expressed within the membranes of the spiral ganglion somata, the type I afferent terminals contacting the inner hair cells, and the medial efferent terminals contacting the outer hair cells. We also found expression of the NKAα1 in the supporting cells that neighbor the inner hair cells and express the glutamate transporter GLAST. These findings suggest that both the NKAα1 and NKAα3 are poised to play an essential role in the regulation of the type I afferent synapses, the medial efferent synapses, and also glutamate transport from the afferent-inner hair cell synapse.

In the vestibular periphery a unique postsynaptic terminal, the calyx, completely covers the basolateral walls of type I hair cells and receives input from multiple ribbon synapses. To date, the functional role of this specialized synapse remains elusive. There is limited data supporting glutamatergic transmission, K ϩ or H ϩ accumulation in the synaptic cleft as mechanisms of transmission. Here the role of glutamatergic transmission at the calyx synapse is investigated. Whole-cell patch-clamp recordings from calyx endings were performed in an in vitro whole-tissue preparation of the rat vestibular crista, the sensory organ of the semicircular canals that sense head rotation. AMPA-mediated EPSCs showed an unusually wide range of decay time constants, from Ͻ5 to Ͼ500 ms. Decay time constants of EPSCs increased (or decreased) in the presence of a glutamate transporter blocker (or a competitive glutamate receptor blocker), suggesting a role for glutamate accumulation and spillover in synaptic transmission. Glutamate accumulation caused slow depolarizations of the postsynaptic membrane potentials, and thereby substantially increased calyx firing rates. Finally, antibody labelings showed that a high percentage of presynaptic ribbon release sites and postsynaptic glutamate receptors were not juxtaposed, favoring a role for spillover. These findings suggest a prominent role for glutamate spillover in integration of inputs and synaptic transmission in the vestibular periphery. We propose that similar to other brain areas, such as the cerebellum and hippocampus, glutamate spillover may play a role in gain control of calyx afferents and contribute to their high-pass properties.