David A. Richards

Ben Burns was a pioneer of operations research and of the statistical analysis of neuronal activity. During the war, Ben served in Solly Zuckermann's operations research unit, which included a period of active service in the Mediterranean. After the war he worked with G. L. Brown at the National Institute for Medical Research (NIMR), where he investigated the effects of agents that affected neuromuscular transmission. In 1950 he moved to the Physiology Department of McGill University in Montreal, where he explored the properties of neural networks in neurologically isolated slabs of cerebral cortex and established the mechanisms responsible for maintaining rhythmic periods of excitation in isolated nerve networks. He subsequently provided evidence that self-re-exciting neural networks were implicated in establishing the respiratory rhythm. While at McGill, Ben initiated a number of highly original cross-disciplinary studies concerning the physiological bases of learning, memory and attention. He returned to NIMR in 1966 to head the Division of Physiology and Pharmacology, where he continued his investigations of visual perception. Ben was an ingenious experimenter and devised a number of mechanical and electronic devices for the statistical analysis of nerve cell activity at a time when digital computers were largely unavailable for biological work. In his 1968 book, The uncertain nervous system , he expressed his view that the interdisciplinary nature of central neurophysiology required of those who studied it a knowledge of classical physiology, experimental psychology, applied mathematics and electronic engineering. His broad view of the subject inspired a generation of students.

The Munc13 family of exocytosis regulators has multiple Ca2+-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates in vitro membrane fusion and platelet exocytosis. We show that Munc13-4 enhances in vitro soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome fusion in a Ca2+- and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4–SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca2+. However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca2+. Interestingly, Ca2+-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca2+-liganding aspartate residues of the C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrine-stained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13dJinx) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca2+. In summary, we show that Munc13-4 conveys Ca2+ sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.

Summary: PIP2 and PIP3 are implicated in a wide variety of cellular signaling pathways at the plasma membrane. We have used STORM imaging to localize clusters of PIP2 and PIP3 to distinct nanoscale regions within the plasma membrane of PC12 cells. With anti-phospholipid antibodies directly conjugated with AlexaFluor 647, we found that PIP2 clusters in membrane domains of 64.5±27.558 nm, while PIP3 clusters had a size of 125.6±22.408 nm. With two color direct STORM imaging we show that >99% of phospholipid clusters have only one or other phospholipid present. These results indicate that lipid nano-domains can be readily identified using super-resolution imaging techniques, and that the lipid composition and size of clusters is tightly regulated.

Numerous studies indicate a role for the actin cytoskeleton in secretion. Here, we have used evanescent wave and widefield fluorescence microscopy to study the involvement of the actin cytoskeleton in secretion from PC12 cells. Secretion was assayed as loss of ANF-EmGFP in widefield mode. Under control conditions, depolarization induced secretion showed two phases: an initial rapid rate of loss of vesicular cargo (tau = 1.4 s), followed by a slower, sustained drop in fluorescence (tau = 34.1 s). Pretreatment with Latrunculin A changed the kinetics to a single exponential, slightly faster than the fast component of control cells (1.2 s). Evanescent wave microscopy allowed us to examine this at the level of individual events, and revealed equivalent changes in the rates of vesicular arrival at the plasma membrane immediately following and during the sustained phase of release. Co-transfection of mCherry labeled β-actin and ANF-EmGFP demonstrated that sites of exocytosis had an inverse relationship with sites of actin enrichment. Disruption of visualized actin at the membrane resulted in the loss of specificity of exocytic site.

Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell≫CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP‐expressing mice and immunolabeling for the zinc transporter ZnT‐3 to reveal the pre‐ and postsynaptic components of granule cell≫CA3 pyramidal cell synapses following pilocarpine‐epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity 1 month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and 3 months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT‐3 labeling of presynaptic giant boutons apposed to GFP‐expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus.

Release of neurotransmitters from synaptic vesicles is a central event in synaptic transmission. Recent evidence suggests that synaptic vesicles fuse with the plasma membrane by multiple routes during exocytosis, but the regulation and physiological implications of this choice are unclear. At hippocampal synapses in culture, two modes of synaptic vesicle exocytosis can be distinguished by virtue of the rate and extent of loss of a fluorescent lipid marker (FM1-43). Here we investigate these two modes of exocytosis using fluorescence imaging of FM1-43, combined with quantitative Ca(2+) imaging using Oregon green BAPTA-1 (OGB1), to examine how the balance of exocytic mode changes during a stimulus train. Our findings are twofold: that the full fusion mode becomes progressively favoured through the course of a 5 or 10 Hz stimulus train, and that this occurs in parallel with presynaptic accumulation of calcium. Blockade of calcium accumulation with AM-EGTA also prevents the conversion of exocytic mode. This conversion of exocytic mode may provide insight as to the mechanisms underpinning short term plasticity.

Release of neurotransmitters from synaptic vesicles is a central event in synaptic transmission. Recent evidence suggests that synaptic vesicles fuse with the plasma membrane by multiple routes during exocytosis, but the regulation and physiological implications of this choice are unclear. At hippocampal synapses, two modes of synaptic vesicle exocytosis can be distinguished by virtue of the rate and extent of loss of a fluorescent lipid marker (FM1‐43). Here these two modes of exocytosis were investigated with a combination of electrophysiological recording and fluorescence imaging. It is shown that these exocytic modes result in distinct postsynaptic consequences, such that so‐called ‘kiss‐and‐run’ exocytosis results in negligible activation of AMPA receptors, compared to the robust postsynaptic responses elicited by apparent full fusion. In contrast NMDA receptors are robustly activated by this form of glutamate delivery. Addition of cyclothiazide, which blocks AMPA receptor desensitization, reveals that the relatively slow rate of release of glutamate during kiss‐and‐run exocytosis shifts the population of AMPA receptors into a desensitized state, rather than simply being insufficient for receptor activation. These findings provide further support for the existence of a fusion pore mediated mode of exocytosis, and demonstrate that these two exocytic modes directly affect the throughput of synaptic transmission.

Synaptotagmin (syt) I is thought to act as a Ca²⁺ sensor that regulates neuronal exocytosis. Fifteen additional isoforms of syt have been identified, but their functions are less well understood. Here, we used PC12 cells to test the idea that different isoforms of syt impart cells with distinct metal (i.e., Ca²⁺, Ba²⁺, and Sr²⁺) requirements for secretion. These cells express syt's I and IX (syt IX sometimes referred to as syt V), which have low apparent metal affinities, at much higher levels than syt VII, which we show has a relatively high apparent affinity for metals. We found that syt I and VII partially colocalize on large dense core vesicles and that upregulation of syt VII produces a concomitant increase in the divalent cation sensitivity of catecholamine release from PC12 cells. Furthermore, RNA interference-mediated knockdown of endogenous syt VII reduced the metal sensitivity of release. These data support the hypothesis that the complement of syt's expressed by a cell, in conjunction with their metal affinity, determines the divalent cation sensitivity of exocytosis.