The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca2+ sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (sytnull) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C2 domains, C2A and C2B. Five, highly conserved, acidic residues in both the C2A and C2B domains of synaptotagmin coordinate the binding of Ca2+ ions, and biochemical studies have characterized several in vitro Ca2+-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca2+-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca2+-binding aspartate residues in the C2B domain (D416, 418N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca2+ affinity of evoked transmitter release. These studies show that the Ca2+-binding motif of the C2B domain of synaptotagmin is essential for synaptic transmission

Temperature dependence of soleus H-reflex and M wave in young and older women

The purpose of this study was to investigate the effect of altered local temperature on soleus H-reflex and compound muscle action potential (M wave) in young and older women. H-reflex and M wave responses were elicited in 10 young (22.3±3.3 years) and 10 older (72.5±3.2 years) women at three muscle temperatures: control (34.2±0.3°C), cold (31.3±0.5°C) and warm (37.1±0.2°C). H-reflex output, expressed as the ratio between maximal H-reflex and maximal M wave (Hmax/Mmax), was lower in the older, compared with the younger, group, regardless of temperature. In control temperature conditions, for example, the Hmax/Mmax ratio was 36.8±24% in the young and 25.4±20% in the older (P<0.05). Warming had no effect on the H-reflex output in either group, whilst cooling increased H-reflex output only in the younger group (+28%). In both groups, cooling increased (+5.3%), and warming decreased (-5.5%) the H-reflex latency. This study confirms that older individuals experience a reduced ability to modulate the reflex output in response to a perturbation. In a cold environment, for example, the lack of facilitation in the reflex output, along with a delayed reflex response could be critical to an older individual in responding to postural perturbations thus potentially compromising both static and dynamic balance

Genetic analysis of synaptotagmin 2 in spontaneous and Ca(2+)-triggered neurotransmitter release

Synaptotagmin 2 resembles synaptotagmin 1, the Ca(2+) sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca(2+)-induced but not in hypertonic sucrose-induced release, suggesting that synaptotagmin 2 mediates Ca(2+) triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca(2+) triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling ‘release slots' for synaptic vesicles at the active zone