RESUMEN

Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Single neurons of Aplysia californica were cultured on control and conditioned medium-treated cover slips (CM, derived from central ganglia of Aplysia) and fed with L-15 medium plus 20% hemolymph. After 5 h 36.9 +/- 3.3% of all neurons plated on CM-treated cover slips sprouted, whereas only 22.9 +/- 2.7% sprouted in the control group. After 24 h the average neurite length in the presence of putative conditioning factor(s) was 621 +/- 42 microns versus 411 +/- 20 microns in control, but the number of primary processes was the same (4.3 +/- 0.5 versus 4.2 +/- 0.9).

Asunto(s)

RESUMEN

1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl2) on acetylcholine-, carbachol-, and glutamate-activated currents on Aplysia neurons. Hg2+ was applied with microperfusion. 2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl- dependent and reversed at about -60 mV, while the slower component was K+ dependent and reversed at greater than -80 mV. 3. Hg2+ (0.1-10 microM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl- current in medial neurons. With only a 1-min preapplication of Hg2+, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 microM Hg2+. 4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1-10 microM Hg2+ caused a complete and irreversible blockade of K(+)-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl2 that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 microM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl2 as well. 5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg2+ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg2+ toxicity.

Asunto(s)

RESUMEN

1. The effect of mercuric(II) chloride on kinetic parameters of carbachol-activated single chloride channels were studied in cultured neurons of the marine mollusk, Aplysia californica. 2. Single neurons of Aplysia were cultured in L-15 medium containing 1 mM beta-D-xyloside, which improved the success rate for gigaseal formation by 46%. Carbachol-activated single chloride channels were recorded in the cell-attached patch clamp configuration. Recordings with control solution (1 microM carbachol) and with test solution (1 microM carbachol + 1 microM HgCl2) were performed successively on the same neuron. 3. In both the control and the test solution the open and closed time distributions were fitted with a double-exponential function. However, kinetic analysis revealed that Hg2+ caused a significant reduction of the mean closed time (10.37 +/- 1.08 vs. 3.32 +/- 0.02 msec) and of the second time constant tau 2 of the closed time distribution (2.09 +/- 0.05 vs. 0.66 +/- 0.5 msec). The reduction of tau 2, i.e., fewer events in the longer closed state under the action of Hg2+, may be the physical cause for the reduction of the mean closed time and thus underlies the increased open probability p0 (0.13 +/- 0.01 vs. 0.29 +/- 0.01 msec) of carbachol-activated chloride channels. 4. Inorganic Hg2+ affects the acetylcholine receptor at lower concentrations than previously reported.

Asunto(s)

RESUMEN

Carbachol and acetylcholine (ACh)-activated whole-cell chloride currents were recorded in identified neurons of Aplysia californica. Application of 0.1-10 microM HgCl2 potentiated the chloride response and reduced the rate of desensitization. To investigate the underlying mechanism we recorded single chloride channels in a cell-attached patch configuration. Recordings were performed successively on the same neuron with 1 microM carbachol and with 1 microM carbachol + 1 microM HgCl2, respectively. The slope conductance did not change significantly, but there was a manyfold increase in the open probability, Po. This may underlie the Hg(2+)-induced enhancement of carbachol- and ACh-evoked chloride currents in Aplysia neurons and may serve as a model for the neurotoxic effect of Hg2+ on the mammalian ACh receptor.

Asunto(s)

Asunto(s)

RESUMEN

Electrophysiologic parameters such as input resistance and response to microperfusion of neurotransmitters vary under circumstances where neurons from isolated ganglia of Aplysia californica are subjected to either long-term (several hours) blockade of active sodium transport or to hypo- or hyperosmotic solutions. Since one of multiple possible events under these circumstances is neuronal volume changes, we have developed a system using cultured Aplysia neurons and confocal scanning laser microscopy to directly monitor cell volume when the osmolarity of the perfusing solution is altered and when sodium transport is blocked. Volume changes of greater than 30% were observed, accompanied by changes in surface area of greater than 15%. The volume increase secondary to sodium pump inhibition and hypotonic solutions and the volume decrease secondary to hypertonic solutions were reversible. Our results demonstrate that neuronal volume may change dramatically and raise the possibility that dynamic changes in neuronal cell volume may have physiological importance.

Asunto(s)