Faculty Bibliography

Synaptic homeostasis, induced by chronic changes in neuronal activity, is well studied in cultured neurons, but not in more physiological networks where distinct synaptic circuits are preserved. We characterized inactivity-induced adaptations at three sets of excitatory synapses in tetrodotoxin-treated organotypic hippocampal cultures. The adaptation to inactivity was strikingly synapse specific. Hippocampal throughput synapses (dentate-to-CA3 and CA3-to-CA1) were upregulated, conforming to homeostatic gain control in order to avoid extreme limits of neuronal firing. However, chronic inactivity decreased mEPSC frequency at CA3-to-CA3 synapses, which were isolated pharmacologically or surgically. This downregulation of recurrent synapses was opposite to that expected for conventional homeostasis, in apparent conflict with typical gain control. However, such changes contributed to an inactivity-induced shortening of reverberatory bursts generated by feedback excitation among CA3 pyramids, safeguarding the network from possible runaway excitation. Thus, synapse-specific adaptations of synaptic weight not only contributed to homeostatic gain control, but also dampened epileptogenic network activity

Voltage-gated Ca(2+) channels (VGCCs) are membrane proteins that determine the activity and survival of neurons, and mutations in the P/Q-type VGCCs are known to cause cerebellar ataxia. VGCC dysfunction may also underlie acquired peripheral and central nervous system diseases associated with small-cell lung cancer, including Lambert-Eaton myasthenic syndrome (LEMS) and paraneoplastic cerebellar ataxia (PCA). The pathogenic role of anti-VGCC antibody in LEMS is well established. Although anti-VGCC antibody is also found in a significant fraction of PCA patients, its contribution to PCA is unclear. Using a polyclonal peptide antibody against a major immunogenic region in P/Q-type VGCCs (the extracellular Domain-III S5-S6 loop), we demonstrated that such antibody was sufficient to inhibit VGCC function in neuronal and recombinant VGCCs, alter cerebellar synaptic transmission, and confer the phenotype of cerebellar ataxia. Our data support the hypothesis that anti-VGCC antibody may play a significant role in the pathogenesis of cerebellar dysfunction in PCA

Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca2+ channels. After membrane depolarization, Ca2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include autism or autism spectrum disorder along with severe cardiac arrhythmia and developmental abnormalities. Most cases of TS arise from a sporadic single nucleotide change that generates a mutation (G406R) in the pore-forming subunit of the L-type Ca2+ channel Ca(V)1.2. We found that the TS mutation powerfully and selectively slows VDI while sparing or possibly speeding the kinetics of CDI. The deceleration of VDI was observed when the L-type channels were expressed with beta1 subunits prominent in brain, as well as beta2 subunits of importance for the heart. Dissociation of VDI and CDI was further substantiated by measurements of Ca2+ channel gating currents and by analysis of another channel mutation (I1624A) that hastens VDI, acting upstream of the step involving Gly406. As highlighted by the TS mutation, CDI does not proceed to completeness but levels off at approximately 50%, consistent with a change in gating modes and not an absorbing inactivation process. Thus, the TS mutation offers a unique perspective on mechanisms of inactivation as well as a promising starting point for exploring the underlying pathophysiology of autism

Synaptic vesicles are responsible for releasing neurotransmitters and are thus essential to brain function. The classical mode of vesicle recycling includes full collapse of the vesicle into the plasma membrane and clathrin-mediated regeneration of a new vesicle. In contrast, a nonclassical mode known as 'kiss-and-run' features fusion by a transient fusion pore without complete loss of vesicle identity and offers possible advantages for increasing the throughput of neurotransmission. Studies of vesicular traffic have benefited greatly from fluorescent probes like FM dyes and synaptopHluorin. However, intrinsic properties of these probes limit their ability to provide a simple and precise distinction between classical and nonclassical modes. Here we report a novel optical probe specific to full collapse fusion, capitalizing on the size and superior photo-properties of photoluminescent quantum dots (Qdots). Qdots with exposed carboxyl groups were readily taken up by synaptic vesicles in an activity-, Ca(2+)-, and clathrin-dependent manner. Electron microscopy showed that Qdots were harbored within individual vesicles in a 1:1 ratio. The release of Qdots was activity- and Ca(2+)-dependent, similar to FM dyes. As artificial cargo, approximately 15 nm in diameter, Qdots will not escape vesicles during kiss-and-run but only with full collapse fusion. Strikingly, Qdots unloaded with kinetics substantially slower than destaining of FM dye, indicating that full-collapse fusion contributed only a fraction of all fusion events. As a full-collapse-fusion-responsive reporter, Qdots will likely promote better understanding of vesicle recycling at small CNS nerve terminals

The properties of synaptic transmission were first elucidated at the neuromuscular junction. More recent work has examined transmission at synapses within the brain. Here we review the remarkable progress in understanding the biophysical and molecular basis of the sequential steps in this process. These steps include the elevation of Ca2+ in microdomains of the presynaptic terminal, the diffusion of transmitter through the fusion pore into the synaptic cleft and the activation of postsynaptic receptors. The results give insight into the factors that control the precision of quantal transmission and provide a framework for understanding synaptic plasticity

Intracellular Ca(2+) regulates numerous proteins and cellular functions and can vary substantially over submicron and submillisecond scales, so precisely localized fast detection is desirable. We have created a approximately 1-kDa biarsenical Ca(2+) indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca(2+)] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca(2+), with a K(d) of approximately 100 microM, <1-ms kinetics and good Mg(2+) rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca(2+) waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled alpha(1C) L-type calcium channels shows fast-rising depolarization-evoked Ca(2+) transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca(2+) buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca(2+) from a tiny minority of clustered open channels. With high Ca(2+) buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca(2+) domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca(2+)] dynamics

The sympathetic nervous system regulates cardiac function through the activation of adrenergic receptors (ARs). beta(1) and beta(2)ARs are the primary sympathetic receptors in the heart and play different roles in regulating cardiac contractile function and remodeling in response to injury. In this study, we examine the targeting and trafficking of beta(1) and beta(2)ARs at cardiac sympathetic synapses in vitro. Sympathetic neurons form functional synapses with neonatal cardiac myocytes in culture. The myocyte membrane develops into specialized zones that surround contacting axons and contain accumulations of the scaffold proteins SAP97 and AKAP79/150 but are deficient in caveolin-3. The beta(1)ARs are enriched within these zones, whereas beta(2)ARs are excluded from them after stimulation of neuronal activity. The results indicate that specialized signaling domains are organized in cardiac myocytes at sites of contact with sympathetic neurons and that these domains are likely to play a role in the subtype-specific regulation of cardiac function by beta(1) and beta(2)ARs in vivo

LTP and other rapidly induced forms of synaptic modification tune individual synaptic weights, whereas slower forms of plasticity such as adaptation to inactivity are thought to keep neurons within their firing limits and preserve their capability for information processing. Here we describe progress in understanding the relationship between LTP and adaptation to inactivity. A prevailing view is that adaptation to inactivity is purely postsynaptic, scales synaptic strength uniformly across all synapses, and thus preserves relative synaptic weights without interfering with signatures of prior LTP or the relative capacity for future LTP. However, recent evidence in hippocampal neurons indicates that, like LTP, adaptation to AMPA receptor blockade can draw upon a repertoire of synaptic expression mechanisms including enhancement of presynaptic vesicular turnover and increased quantal amplitude mediated by recruitment of homomeric GluR1 AMPA receptors. These pre- and postsynaptic changes appeared coordinated and preferentially expressed at subset of synapses, thereby increasing the variability of miniature EPSCs. In contrast to the NMDA receptor-, Ca2+ entry-dependent induction of LTP, adaptation to inactivity may be mediated by attenuation of voltage-sensitive L-type Ca2+ channel function. The associated intracellular signaling involves elevation of betaCaMKII, which in turn downregulates alphaCaMKII, a key player in LTP. Thus, adaptation to inactivity and LTP are not strictly independent with regard to mechanisms of signaling and expression. Indeed, we and others have found that responses to LTP-inducing stimuli can be sharply altered by prior inactivity, suggesting that the slow adaptation changes the rules of plasticity-an interesting example of 'metaplasticity'

The number and diversity of plasticity mechanisms in the brain raises a central question: does a neural circuit store all memories by stereotyped application of the available plasticity mechanisms, or can subsets of these mechanisms be selectively engaged for specific memories? The uniform architecture of the cerebellum has inspired the idea that plasticity mechanisms like cerebellar long-term depression (LTD) contribute universally to memory storage. To test this idea, we investigated a set of closely related, cerebellum-dependent motor memories. In mutant mice lacking Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), the maintenance of cerebellar LTD is abolished. Although memory for an increase in the gain of the vestibulo-ocular reflex (VOR) induced with high-frequency stimuli was impaired in these mice, memories for decreases in VOR gain and increases in gain induced with low-frequency stimuli were intact. Thus, a particular plasticity mechanism need not support all cerebellum-dependent memories, but can be engaged selectively according to the parameters of training