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Substantia nigra neurons release dopamine from their somatodendritic regions. A long-unresolved question is whether this release occurs by exocytosis or by a nonvesicular mechanism. We used carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events in approximately 90% of cells in resting conditions. These events had charge integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had effects similar to those of glutamate. The frequency of resting and stimulated amperometric events was much lower at 21-22 degreesC than at 32-35 degreesC. The addition of Cd2+ (50 microM), a blocker of voltage-dependent Ca2+ channels, to the bath solution blocked the stimulatory effects of glutamate. These results suggest that dopamine is released from the somata of substantia nigra neurons by exocytosis and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion in brain slices.

Needle-type voltammetric ultramicroelectrodes show exceptional sensitivity for the detection of redox-active substances, rapid response times, and total tip diameters in the lower micrometer range. These characteristics make them ideal for analyzing the chemical environment and the activity of isolated living cells, which in their various forms are the microscopic building blocks of human, animal, and other life forms. Prerequisites for successful local electrochemical measurements in the vicinity of the tiny biological objects are gentle, stress-free, and accurate placement of the tip at the cell, exact knowledge of the tip-to-cell distance, and appropriate selectivity of the ultramicroelectrode tip for species that may change in concentration as a result of cellular actions such as growth, respiration, or transmitter and metabolite uptake or release. The concepts of single-cell microelectrochemistry are considered and an overview is given of recent results on the fundamental mechanisms of cell functions.

A non-optical shear-force-based detection scheme for accurately controlling the tip-to-sample distance in scanning electrochemical microscopy (SECM) is presented. With this approach, the detection of the shear force is accomplished by mechanically attaching a set of two piezoelectric plates to the scanning probe. One of the plates is used to excite the SECM tip causing it to resonate, and the other acts as a piezoelectric detector of the amplitude of the tip oscillation. Increasing shear forces in close proximity to the sample surface lead to a damping of the vibration amplitude and a phase shift, effects that are registered by connecting the detecting piezoelectric plate to a dual-phase analogue lock-in amplifier. The shear force and hence distance-dependent signal of the lock-in amplifier is used to establish an efficient, computer-controlled closed feedback loop enabling SECM imaging in a constant-distance mode of operation. The details of the SECM setup with an integrated piezoelectric shear-force distance control are described, and approach curves are shown. The performance of the constant-distance mode SECM with a non-optical detection of shear forces is illustrated by imaging simultaneously the topography and conductivity of an array of Pt-band microelectrodes.

BackgroundChitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH3.Principal FindingsThis study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides.Conclusion/SignificanceWe provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production.