Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.

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Neurotransmitter release is a highly efficient secretory process exhibiting resistance to fatigue and plasticity attributable to the existence of distinct pools of synaptic vesicles (SVs), namely a readily releasable pool and a reserve pool from which vesicles can be recruited after activity. Synaptic vesicles in the reserve pool are thought to be reversibly tethered to the actin-based cytoskeleton by the synapsins, a family of synaptic vesicle-associated phosphoproteins that have been shown to play a role in the formation, maintenance, and regulation of the reserve pool of synaptic vesicles and to operate during the post-docking step of the release process. In this paper, we have investigated the physiological effects of manipulating synapsin levels in identified cholinergic synapses of Aplysia californica. When endogenous synapsin was neutralized by the injection of specific anti-synapsin antibodies, the amount of neurotransmitter released per impulse was unaffected, but marked changes in the secretory response to high-frequency stimulation were observed, including the disappearance of post-tetanic potentiation (PTP) that was substituted by post-tetanic depression (PTD), and increased rate and extent of synaptic depression. Opposite changes on post-tetanic potentiation were observed when synapsin levels were increased by injecting exogenous synapsin I. Our data demonstrate that the presence of synapsin-dependent reserve vesicles allows the nerve terminal to release neurotransmitter at rates exceeding the synaptic vesicle recycling capacity and to dynamically change the efficiency of release in response to conditioning stimuli (e.g., post-tetanic potentiation). Moreover, synapsin-dependent regulation of the fusion competence of synaptic vesicles appears to be crucial for sustaining neurotransmitter release during short periods at rates faster than the replenishment kinetics and maintaining synchronization of quanta in evoked release.

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca 2؉ -dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynapticreleasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.

Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.motor control | olivo-cerebellar loop | complex spikes T he cerebellar cortex is involved in a wealth of functions, from the control of posture to higher cognitive processes (1-3). Purkinje cells (PCs) are key processing units of the cerebellar cortex (4): each PC receives more than 175,000 parallel fiber synaptic inputs carrying information about the ongoing sensorymotor context. It also receives a single inferior olive afferent, the climbing fiber, which triggers a complex spike (CS), modulates PC firing (5), controls synaptic input plasticity, and has been proposed to carry error and clock signals to the cerebellum (2, 4-8). PCs are grouped in multiple parasagittal microzones, each receiving projections from separate areas of the inferior olive and projecting to subregions of the cerebellar nuclei (CN) (9-12). In the CN, PCs make inhibitory contacts on excitatory neurons that project to various premotor areas and propagate cerebellar computations to the motor system. Anatomical evidence indicates that PC terminals also contact CN inhibitory neurons that target inferior olive cells (13,14). This nucleoolivary pathway is topographically organized in multiple parallel projections to the inferior olive subnuclei (15), suggesting the existence of closed olivary-cortico-nuclear loops. Therefore, the discharge of a population of PCs in a microzone might not only shape the output of the cerebellum but also control its afferent climbing-fiber signal. Previous studies have shown that stimulation of the nucleo-olivary pathway significantly reduces olivary cell firing (16-18) and that pharmacological and genetic manipulations of PCs or olivary cell activity induce reciprocal modulations of the firing rate of PCs and climbing fibers (19, 20).These results indicate that PCs may tonically modulate the nucleo-olivary pathway. However, whether...

The mossy fiber (MF)-granule cell (GC) pathway conveys multiple modalities of information to the cerebellar cortex, converging on Purkinje cells (PC), the sole output of the cerebellar cortex. Recent in vivo experiments have shown that activity in GCs varies from tonic firing at a few hertz to phasic bursts Ͼ500 Hz. However, the responses of parallel fiber (PF)-PC synapses to this wide range of input frequencies are unknown, and there is controversy regarding several frequency-related parameters of transmission at this synapse. We performed recordings of unitary synapses and combined variance-mean analysis with a carefully adapted extracellular stimulation method in young and adult rats. We show that, although the probability of release at individual sites is low at physiological calcium concentration, PF-PC synapses release one or more vesicles with a probability of 0.44 at 1.5 mM [Ca 2ϩ ] e . Paired-pulse facilitation was observed over a wide range of frequencies; it renders burst inputs particularly effective and reproducible. These properties are primarily independent of synaptic weight and age. Furthermore, we show that the PF-PC synapse is able to sustain transmission at very high frequencies for tens of stimuli, as a result of accelerated vesicle replenishment and an apparent recruitment of release site vesicles, which appears to be a central mechanism of paired-pulse facilitation at this synapse. These properties ensure that PF-PC synapses possess a dynamic range enabling the temporal code of MF inputs to be transmitted reliably to the PC.