Using biochemical and morphologic techniques, our research focuses on synaptic function in primary neuronal cell cultures. Studies utilize the clostridial neurotoxins (tetanus and seven serotypes of botulinum) whose catalytic domains are zinc endopeptidases that block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion with the presynaptic membrane. Consequently, the toxins have become valuable tools for understanding neurotransmitter release, membrane trafficking, and protein sorting, transport, and targeting. The identity of toxin receptor(s), organelles involved in toxin uptake, the mechanism of toxin translocation into the neuronal cytoplasm, intracellular trafficking of the toxins, and toxin effect on synaptic vesicle trafficking remain subjects of active research. Intoxication in vivo with botulinum neurotoxin causes a fatal flaccid paralysis of voluntary muscle. Given their paralytic effect, the toxins are important therapeutic agents for a number of neurologic disorders characterized by excessive muscle contraction, including cerebral palsy. Botulinum poisoning remains a public health problem, and the toxin is considered a potential agent of bioterrorism; a more complete understanding of its intracellular functioning will aid in designing an appropriate therapy.

Synaptic Vesicle Recycling
Parfitt, Behar
We reported previously that the action of botulinum neurotoxin (BoNT) serotype A on cultured spinal cord neurons was unique among the C. botulinum toxin serotypes in that it appeared to block synaptic vesicle exocytosis while allowing some endocytosis to continue (Neale et al., J Cell Biol. 1999;147:1249). In an effort to examine this phenomenon with improved temporal resolution and sensitivity, we have monitored synaptic vesicle endocytosis and exocytosis by the quantitative kinetic measurement of synaptic staining and destaining with fluorescent FM dyes. Both BoNT A–blocked and untreated cultures are stained with FM2-10 during five minutes of potassium depolarization in the presence of dye. In control cultures, essentially all the FM dye is lost on subsequent depolarization. The initial fast phase of destaining fits a single exponential; a second, slower process sometimes manifests after one to two minutes. BoNT A–treated cultures, which take up about 50 percent less FM dye than controls, lose only about 50 percent on destaining and show markedly different destaining kinetics. The rate of the initial fast phase is reduced compared with the controls and lasts for a shorter time. The rate of the second process is greatly reduced. In control cultures in the presence of a very low extracellular calcium concentration, destaining kinetics are analogous to those seen with normal calcium in BoNT A–treated cultures. The pool of synaptic vesicles is believed to comprise three subgroups: a reserve pool that does not normally participate in synaptic transmission; a recycling pool (RP) that is fully stainable after prolonged exposure to FM dyes; and a readily releasable pool (RRP) containing vesicles that are rapidly exocytosed at the start of high-frequency stimulation and that have been identified morphologically (by others) as those vesicles docked at the active zone.

Based on our current findings, we propose that part of the lethal effect of BoNT A intoxication may be attributable to the severe retardation of the rate of replenishment of vesicles in the RRP rather than solely to a direct inhibition of vesicle exocytosis. Similarly, low calcium may allow fusion of readily releasable vesicles but not support movement of vesicles from the recycling to the readily releasable pool. We will test our hypothesis by examining the distribution of labeled vesicles in the electron microscope after brief periods of depolarization.

Uptake and Translocation of Botulinum Neurotoxin
Keller, Behar
BoNT serotypes A and E cleave the same synaptic protein, although BoNT A is more potent and remains active for longer than BoNT E (Keller et al., FEBS Lett. 1999;456:137; Keller et al., 2001). Potassium stimulation greatly enhances the uptake of BoNT into spinal cord neurons in cell culture, implicating recycling synaptic vesicles as the uptake compartment. Short exposure to toxin in depolarizing medium produces a saturable uptake. Movement of toxin across the vesicle membrane into the neuronal cytosol requires low intravesicular pH. Bafilomycin A1 inhibits the vacuolar ATPase, preventing vesicle acidification. The drug was used to trap toxin within vesicles in order to study the synchronized translocation of toxin across the vesicle membrane. Toxin translocation occurs about 25 minutes after uptake and is complete within 90 minutes. Bafilomycin is less effective at blocking the translocation of BoNT E than of BoNT A, indicating that BoNT E is able to cross the membrane in the presence of a more shallow pH gradient. This difference may reflect a fundamental difference in intracellular toxin trafficking, perhaps relevant to the long duration of action of BoNT A. The data indicate that the potency of BoNT A is related to an aspect of toxin action other than the efficiency of translocation.