Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.

KEY POINTS: Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. ABSTRACT: Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) - a haem-containing plant enzyme - or antibodies against synaptotagmin-1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2 O2 ) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization-induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP-tagged syt1 construct and subsequent H2 O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss-of-function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins.

The introduction of pHluorin, a pH-sensitive GFP, by Miesenbock and colleagues provided a versatile tool to studies of vesicle trafficking, in particular synaptic vesicle exocytosis and endocytosis. By tagging pHluorin to the luminal region of the synaptic vesicular protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) or other synaptic vesicle-specific proteins such as the vesicular glutamate transporter-1, we are able to directly track synaptic vesicle endocytosis in response to stimuli in a molecularly specific manner. Here, we describe the process of imaging synaptic vesicle endocytosis in response to extracellular stimulation in dissociated neuronal cultures of hippocampal neurons obtained from rats-also applicable to mice-using pHluorin-tagged vesicular glutamate transporter-1 as a reporter.

One month in the life of a neuron: longitudinal single-unit electrophysiology in the monkey visual system.

Conventional recording methods generally preclude following the activity of the same neurons in awake animals across days. This limits our ability to systematically investigate the principles of neuronal specialization, or to study phenomena that evolve over multiple days such as experience-dependent plasticity. To redress this shortcoming, we developed a drivable, chronically implanted microwire recording preparation that allowed us to follow visual responses in inferotemporal (IT) cortex in awake behaving monkeys across multiple days, and in many cases across months. The microwire bundle and other implanted components were MRI compatible and thus permitted in the same animals both functional imaging and long-term recording from multiple neurons in deep structures within a region the approximate size of one voxel (<1 mm). The distinct patterns of stimulus selectivity observed in IT neurons, together with stable features in spike waveforms and interspike interval distributions, allowed us to track individual neurons across weeks and sometimes months. The long-term consistency of visual responses shown here permits large-scale mappings of neuronal properties using massive image libraries presented over the course of days. We demonstrate this possibility by screening the visual responses of single neurons to a set of 10,000 stimuli.