No abstract

We have determined the parameters necessary to fabricate reproducible neuronal patterns which we are using to begin studying fundamental issues in developmental neurobiology. The addition of a beam homogenizer, as well as a new surface preparation, has enabled the routine production of reproducible, high-resolution (2−20 μm) organosilane patterns. The effects of surface preparation and beam dosage were monitored using X-ray photoelectron spectroscopy (XPS) and proof of patterning is provided by high-resolution imaging XPS. We also report the guidance of neuronal adhesion and neurite outgrowth and the creation of reproducibly defined circuits of embryonic (E18−19) rat hippocampal neurons using these patterned surfaces in vitro. We have achieved a >50% rate of pattern formation, and at times the rate approaches 90%. We are using these patterns to address the issue of how geometric pattern cues might be used to affect cell-to-cell communication and we report the preliminary results on the synaptic development of the hippocampal neurons using dual patch-clamp electrophysiology. We monitored neurite outgrowth and the emergence of both spontaneous and evoked synaptic activity for both patterned and unpatterned (control) hippocampal cultures. The results indicate the intriguing possibility that geometry itself may be a modulating or trophic factor for cell development.

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.