Tuhin Virmani

An Isolated Pool of Vesicles Recycles at Rest and Drives Spontaneous Neurotransmission

Spontaneous synaptic vesicle fusion is a common property of all synapses. To trace the origin of spontaneously fused vesicles in hippocampal synapses, we tagged vesicles with fluorescent styryl dyes, antibodies against synaptotagmin-1, or horseradish peroxidase. We could show that synaptic vesicles recycle at rest, and after spontaneous exo-endocytosis, they populate a reluctantly releasable pool of limited size. Interestingly, vesicles in this spontaneously labeled pool were more likely to re-fuse spontaneously compared to vesicles labeled with activity. We found that blocking vesicle refilling at rest selectively depleted neurotransmitter from spontaneously fusing vesicles without significantly altering evoked transmission. Furthermore, in the absence of the vesicle SNARE protein synaptobrevin (VAMP), activity-dependent and spontaneously recycling vesicles could mix, suggesting a role for synaptobrevin in the separation of the two pools. Taken together these results suggest that spontaneously recycling vesicles and activity-dependent recycling vesicles originate from distinct pools with limited cross-talk with each other.

TrkB Has a Cell-Autonomous Role in the Establishment of Hippocampal Schaffer Collateral Synapses

Neurotrophin signaling has been implicated in the processes of synapse formation and plasticity. To gain additional insight into the mechanism of BDNF and TrkB influence on synapse formation and synaptic plasticity, we generated a conditional knock-out for TrkB using the cre/loxp system. Using three different cre-expressing transgenic mice, three unique spatial and temporal configurations of TrkB deletion were obtained with regard to the hippocampal Schaffer collateral synapse. We compare synapse formation in mutants in which TrkB is ablated either in presynaptic or in both presynaptic and postsynaptic cells at early developmental or postdevelopmental time points. Our results indicate a requirement for TrkB at both the presynaptic and postsynaptic sites during development. In the absence of TrkB, synapse numbers were significantly reduced. In vivo ablation of TrkB after synapse formation did not affect synapse numbers. In primary hippocampal cultures, deletion of TrkB in only the postsynaptic cell, before synapse formation, also resulted in deficits of synapse formation. We conclude that TrkB signaling has a cell-autonomous role required for normal development of both presynaptic and postsynaptic components of the Schaffer collateral synapse.

Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling

The speed of synaptic vesicle recycling determines the efficacy of neurotransmission during repetitive stimulation. Synaptotagmins are synaptic C2‐domain proteins that are involved in exocytosis, but have also been linked to endocytosis. We now demonstrate that upon expression in transfected neurons, a short splice variant of synaptotagmin 7 that lacks C2‐domains accelerates endocytic recycling of synaptic vesicles, whereas a longer splice variant that contains C2‐domains decelerates recycling. These results suggest that alternative splicing of synaptotagmin 7 acts as a molecular switch, which targets vesicles to fast and slow recycling pathways.

Progressively reduced synaptic vesicle pool size in cultured neurons derived from neuronal ceroid lipofuscinosis-1 knockout mice

The neuronal ceroid lipofuscinoses are a newly-recognized group of lysosomal storage disorders in which neurodegeneration predominates. The pathophysiological basis for this is unknown. In the current paper, we sought to determine whether neurons that lack the enzyme responsible for the infantile form of neuronal ceroid lipofuscinosis (INCL) display abnormalities in culture that could be related to the clinical disorder. Electrophysiological and fluorescent dye studies were performed using cortical neuronal cultures established from postnatal day 2 palmitoyl-protein thioesterase-1 (Ppt1) knockout mice. We found a 30% reduction in synaptic vesicle number per bouton that was progressive with time in culture as well as an elevation in lysosomal pH, whereas a number of passive and active membrane properties of the neurons were normal. The reduction in vesicle pool size was also reflected in a decrease in the frequency of miniature synaptic currents. The progressive and gradual decline in vesicle numbers and miniature event frequency we observed here may be an early indicator of synapse degeneration, in keeping with observations during competitive stimulation at the neuromuscular junction or age-related synapse elimination recently reported by others. PPT1 did not colocalize with synaptic vesicle or synapse markers, suggesting that lysosomal dysfunction leads indirectly to the synaptic abnormalities. We conclude that from an early age, neurons deficient in PPT1 enzyme activity display intrinsically abnormal properties that could potentially explain key features of the clinical disease, such as myoclonus and seizures.

Light Scattering and Phase Behavior of Lysozyme-Poly(Ethylene Glycol) Mixtures

Measurements of liquid-liquid phase transition temperatures (cloud points) of mixtures of a protein (lysozyme) and a polymer, poly(ethylene glycol) (PEG) show that the addition of low molecular weight PEG stabilizes the mixture whereas high molecular weight PEG was destabilizing. We demonstrate that this behavior is inconsistent with an entropic lysozyme-PEG depletion interaction and suggest that an energetic lysozyme-PEG attraction is responsible. In order to independently characterize the lysozyme-PEG interactions, light scattering experiments on the same mixtures were performed to measure second and third virial coefficients. These measurements indicate that PEG induces repulsion between lysozyme molecules, contrary to the depletion prediction. Furthermore, it is shown that third virial terms must be included in the mixtures free energy in order to qualitatively capture our data.

Fast synaptic vesicle reuse slows the rate of synaptic depression in the CA1 region of hippocampus

During short-term synaptic depression, neurotransmission rapidly decreases in response to repetitive action potential firing. Here, by blocking the vacuolar ATPase, alkalinizing the extracellular pH, or exposing hippocampal slices to pH buffers, we impaired neurotransmitter refilling, and electrophysiologically tested the role of vesicle reuse in synaptic depression. Under all conditions, synapses onto hippocampal CA1 pyramidal cells showed faster depression with increasing stimulation frequencies. At 20 Hz, compromising neurotransmitter refilling increased depression within 300 ms reaching completion within 2 s, suggesting a minimal contribution of reserve vesicles to neurotransmission. In contrast, at 1 Hz, depression emerged gradually and became significant within 100 s. Moreover, the depression induced by pH buffers was reversible with a similar frequency dependence, suggesting that the frequency-dependent increase in depression was caused by impairment of rapid synaptic vesicle reuse. These results indicate that synaptic vesicle trafficking impacts the kinetics of short-term synaptic plasticity at an extremely rapid time scale.

Phorbol Esters Target the Activity-Dependent Recycling Pool and Spare Spontaneous Vesicle Recycling

Using electrophysiology and styryl dye imaging, we studied the effect of phorbol 12-myristate 13-acetate (PMA) on activity-dependent and spontaneous vesicle recycling. In electrophysiological experiments, we found that the PMA effect depended on the maturational state of the synapses. Spontaneous neurotransmitter release from nascent synapses without a functional readily releasable pool (RRP) was unresponsive to PMA application. In contrast, mature synapses responded robustly to PMA application, consistent with previous studies. Using styryl dye imaging, we found that there was a PMA-dependent increase in the size of the RRP when PMA was present before, during, or after activity-dependent dye uptake, suggesting that this effect involves an increase in the population of the RRP by vesicles recruited from the reserve pool. Additionally, we found that when PMA was present during spontaneous dye uptake, there was an increase in dye labeling, and these additional dye-loaded vesicles showed rapid destaining in response to strong stimulation and were also releasable by hypertonic sucrose. In contrast, these observations were not reproducible when PMA treatment was performed after spontaneous dye uptake and extracellular dye washout. Together, these findings suggest that the increased spontaneous neurotransmission in the presence of PMA was attributable to release of vesicles from the RRP rather than an effect of PMA on the spontaneously recycling pool. Thus, the phorbol esters selectively regulate the activity-dependent pool of vesicles, indicating that priming mechanisms that prepare vesicles for fusion, which are targeted by phorbol esters, are different for the spontaneous and evoked forms of fusion.

Synaptic Vesicle Recycling Adapts to Chronic Changes in Activity

Synaptic vesicle recycling is essential for maintaining neurotransmission during rhythmic activity. To test whether the demands imposed by ambient activity influences synaptic vesicle trafficking, we compared the kinetics of synaptic depression in hippocampal versus neocortical cultures, which have high and low levels of intrinsic activity, respectively. In response to moderate 10 Hz stimulation, hippocampal synapses depressed less compared with neocortical synapses, although they reused vesicles more slowly. Therefore, during stimulation, hippocampal synapses used more vesicles from the reserve pool, whereas neocortical synapses relied on vesicle reuse. In hippocampal cultures, chronic block of network activity increased synaptic depression by decreasing the rate of vesicle mobilization, with little effect on the rate of vesicle reuse. In contrast, in neocortical cultures, an increase in the normally low network activity reduced synaptic depression by robustly increasing vesicle reuse with no effect on vesicle mobilization. These results suggest that synaptic vesicle trafficking and the resulting synaptic dynamics adapt to meet the changing demands on neurotransmitter release. Furthermore, during these functional modifications, synapses use alternate strategies to adjust to changes in activity.

Clinicopathological characteristics of freezing of gait in autopsy-confirmed Parkinson's disease.

Twenty‐five percent to sixty percent of Parkinsons disease (PD) patients reportedly have freezing of gait, leading to impaired mobility, falls, and decreased quality of life. Several factors have been associated with gait freezing in PD patients. We analyze for these factors in autopsy‐proven PD patients.

Familial Orthostatic Tremor: An Additional Report in Siblings

Orthostatic tremor (OT) was a term first used to describe tremor that occurred in the legs while patients were standing, and which was relieved while patients were walking, seated, or supine.1 The disorder can be very disabling, and the treatment, which is largely unsuccessful, remains empiric.2,3 Although there is some evidence of a dopaminergic deficit,4 the exact circuit responsible for the high frequency, 13–18 Hz tremor that is pathognomonic for this disorder remains unclear. OT is thought to be sporadic rather than familial, which makes the search for a pathophysiologic mechanism particularly challenging. There are only 2 prior case reports of OT in siblings.5,6 Here we report a third sibling pair, each of whom had clinical and electrophysiologic evidence of OT.