Faculty Bibliography

Synaptic vesicles release neurotransmitter at chemical synapses, thus initiating the flow of information in neural networks. To achieve this, vesicles undergo a dynamic cycle of fusion and retrieval to maintain the structural and functional integrity of the presynaptic terminals in which they reside. Moreover, compelling evidence indicates these vesicles differ in their availability for release and mobilization in response to stimuli, prompting classification into at least three different functional pools. Ongoing studies of the molecular and cellular bases for this heterogeneity attempt to link structure to physiology and clarify how regulation of vesicle pools influences synaptic strength and presynaptic plasticity. We discuss prevailing perspectives on vesicle pools, the role they play in shaping synaptic transmission, and the open questions that challenge current understanding.

The usefulness of genetically encoded probes for optical monitoring of neuronal activity and brain circuits would be greatly advanced by the generation of multiple indicators with non-overlapping color spectra. Most existing indicators are derived from or spectrally convergent on GFP. We generated a bright, red, pH-sensitive fluorescent protein, pHTomato, that can be used in parallel with green probes to monitor neuronal activity. SypHTomato, made by fusing pHTomato to the vesicular membrane protein synaptophysin, reported activity-dependent exocytosis as efficiently as green reporters. When expressed with the GFP-based indicator GCaMP3 in the same neuron, sypHTomato enabled concomitant imaging of transmitter release and presynaptic Ca(2+) transients at single nerve terminals. Expressing sypHTomato and GCaMP3 in separate cells enabled the simultaneous determination of presynaptic vesicular turnover and postsynaptic sub- and supra-threshold responses from a connected pair of neurons. With these new tools, we observed a close size matching between pre- and postsynaptic compartments, as well as interesting target cell-dependent regulation of presynaptic vesicle pools. Lastly, by coupling expression of pHTomato- and GFP-based probes with distinct variants of channelrhodopsin, we provided proof-of-principle for an all-optical approach to multiplex control and tracking of distinct circuit pathways.

Activity-dependent gene expression triggered by Ca(2+) entry into neurons is critical for learning and memory, but whether specific sources of Ca(2+) act distinctly or merely supply Ca(2+) to a common pool remains uncertain. Here, we report that both signaling modes coexist and pertain to Ca(V)1 and Ca(V)2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. Ca(V)1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca(2+) to drive local CaMKII aggregation and trigger communication with the nucleus. In contrast, Ca(V)2 channels must elevate [Ca(2+)](i) microns away and promote CaMKII aggregation at Ca(V)1 channels. Consequently, Ca(V)2 channels are approximately 10-fold less effective in signaling to the nucleus than are Ca(V)1 channels for the same bulk [Ca(2+)](i) increase. Furthermore, Ca(V)2-mediated Ca(2+) rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca(2+), further attenuating Ca(V)2-mediated gene expression.

Neurotransmission depends on movements of transmitter-laden synaptic vesicles, but accurate, nanometer-scale monitoring of vesicle dynamics in presynaptic terminals has remained elusive. Here, we report three-dimensional, real-time tracking of quantum dot-loaded single synaptic vesicles with an accuracy of 20 to 30 nanometers, less than a vesicle diameter. Determination of the time, position, and mode of fusion, aided by trypan blue quenching of Qdot fluorescence, revealed that vesicles starting close to their ultimate fusion sites tended to fuse earlier than those positioned farther away. The mode of fusion depended on the prior motion of vesicles, with long-dwelling vesicles preferring kiss-and-run rather than full-collapse fusion. Kiss-and-run fusion events were concentrated near the center of the synapse, whereas full-collapse fusion events were broadly spread.

Homeostatic scaling of synaptic strengths is essential for maintenance of network "gain", but also poses a risk of losing the distinctions among relative synaptic weights, which are possibly cellular correlates of memory storage. Multiplicative scaling of all synapses has been proposed as a mechanism that would preserve the relative weights among them, because they would all be proportionately adjusted. It is crucial for this hypothesis that all synapses be affected identically, but whether or not this actually occurs is difficult to determine directly. Mathematical tests for multiplicative synaptic scaling are presently carried out on distributions of miniature synaptic current amplitudes, but the accuracy of the test procedure has not been fully validated. We now show that the existence of an amplitude threshold for empirical detection of miniature synaptic currents limits the use of the most common method for detecting multiplicative changes. Our new method circumvents the problem by discarding the potentially distorting subthreshold values after computational scaling. This new method should be useful in assessing the underlying neurophysiological nature of a homeostatic synaptic scaling transformation, and therefore in evaluating its functional significance.

Recurrent excitatory circuits face extreme challenges in balancing efficacy and stability. We recorded from CA3 pyramidal neuron pairs in rat hippocampal slice cultures to characterize synaptic and circuit-level changes in recurrent synapses resulting from long-term inactivity. Chronic tetrodotoxin treatment greatly reduced the percentage of connected CA3-CA3 neurons, but enhanced the strength of the remaining connections; presynaptic release probability sharply increased, whereas quantal size was unaltered. Connectivity was decreased in activity-deprived circuits by functional silencing of synapses, whereas three-dimensional anatomical analysis revealed no change in spine or bouton density or aggregate dendrite length. The silencing arose from enhanced Cdk5 activity and could be reverted by acute Cdk5 inhibition with roscovitine. Our results suggest that recurrent circuits adapt to chronic inactivity by reallocating presynaptic weights heterogeneously, strengthening certain connections while silencing others. This restricts synaptic output and input, preserving signaling efficacy among a subset of neuronal ensembles while protecting network stability.

Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the Ca(V)1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying Ca(V)1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD

Down syndrome (DS) is the most prevalent form of mental retardation caused by genetic abnormalities in humans. This has been successfully modeled in mice to generate the Ts65Dn mouse, a genetic model of DS. This transgenic mouse model shares a number of physical and functional abnormalities with people with DS, including changes in the structure and function of neuronal circuits. Significant abnormalities in noradrenergic (NE-ergic) afferents from the locus coeruleus to the hippocampus, as well as deficits in NE-ergic neurotransmission are detected in these animals. In the current study we characterized in detail the behavioral phenotype of Ts65Dn mice, in addition to using pharmacological tools for identification of target receptors mediating the learning and memory deficits observed in this model of DS. We undertook a comprehensive approach to mouse phenotyping using a battery of standard and novel tests encompassing: (i) locomotion (Activity Chamber, PhenoTyper, and CatWalk), (ii) learning and memory (spontaneous alternation, delayed matching-to-place water maze, fear conditioning, and Intellicage), and (iii) social behavior. Ts65Dn mice showed increased locomotor activity in novel and home cage environments. There were significant and reproducible deficits in learning and memory tests including spontaneous alternation, delayed matching-to-place water maze, Intellicage place avoidance and contextual fear conditioning. Although Ts65Dn mice showed no deficit in sociability in the 3-chamber test, a marked impairment in social memory was detected. Xamoterol, a beta1-adrenergic receptor (beta1-ADR) agonist, effectively restored the memory deficit in contextual fear conditioning, spontaneous alternation and novel object recognition. These behavioral improvements were reversed by betaxolol, a selective beta1-ADR antagonist. In conclusion, our results demonstrate that this mouse model of Down syndrome displays cognitive deficits which are mediated by an imbalance in the noradrenergic system. In this experimental model of Down syndrome a selective activation of beta1-ADR does restore some of these behavioral deficits. Further mechanistic studies will be needed to investigate the failure of noradrenergic system and the role of beta1-ADR in cognitive deficit and pathogenesis of DS in people. Restoring NE neurotransmission or a selective activation of beta1)-ADR needs to be further investigated for the development of any potential therapeutic strategy for symptomatic relief of memory deficit in DS. Furthermore, due to the significant involvement of noradrenergic system in the cardiovascular function further safety and translational studies will be needed to ensure the safety and efficacy of this approach

Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination

In excitable cells, membrane depolarization and activation of voltage-gated Ca(2)+ (Ca(V)) channels trigger numerous cellular responses, including muscle contraction, secretion, and gene expression. Yet, while the mechanisms underlying excitation-contraction and excitation-secretion coupling have been extensively characterized, how neuronal activity is coupled to gene expression has remained more elusive. In this article, we will discuss recent progress toward understanding the relationship between patterns of channel activity driven by membrane depolarization and activation of the nuclear transcription factor CREB. We show that signaling strength is steeply dependent on membrane depolarization and is more sensitive to the open probability of Ca(V) channels than the Ca(2)+ entry itself. Furthermore, our data indicate that by decoding Ca(V) channel activity, CaMKII (a Ca(2)+/calmodulin-dependent protein kinase) links membrane excitation to activation of CREB in the nucleus. Together, these results revealed some interesting and unexpected similarities between excitation-transcription coupling and other forms of excitation-response coupling