Faculty Bibliography

Aequorin, a protein that emits light in the presence of calcium, was injected in the presynaptic terminal of the squid giant synapse. This injection was preceded by intracellular tetraethylammonium administration, which prolonged the duration of the presynaptic action potential. After this procedure light emission was evoked by single presynaptic spikes capable of releasing synaptic transmitter. In a second set of experiments, presynaptic tetraethylammonium injection was followed by the administration of tetrodotoxin extracellularly, which abolished the presynaptic action potential. Under these conditions artificial depolarization of the presynaptic terminal triggered the release of synaptic transmitter, in a graded manner. However, as previously reported by other authors, membrane potential steps to an internal positive value of approximately plus 90 mV (the suppression potential) produced a blockage of transmitter release for the duration of the imposed potential. Synaptic transmission recurred, nevertheless, as the current injection was terminated. A similar set of experiments, performed after the intracellular injection of aequorin in the presynaptic fiber, demonstrated that the aequorin light response was evoked by membrane potential steps capable of releasing synaptic transmitter. If the membrane potential was made positive to the 'suppression' level, no light response was evoked but the light emission appeared, as did transmitter release, at the end of the current pulse. These experiments demonstrate that release of transmitter is directly correlated with intracellular calcium concentration and that the suppression potential is compatible with the existence of a calcium equilibrium potential at the presynaptic terminal.

The theoretical basis of current source-density (CSD) analysis in the central nervous system is described. Equations relating CSD, the current flow vector, and the extracellular field potential are given. It is shown that the CSD provides superior resolution of neuronal events when compared to conventional field-potential analysis. Expressions for the CSD in rectangular Cartesian coordinates are derived, including the general case of anisotropic, inhomogeneous conductive tissue, and a coordinate system rotated with respect to the principal axes (APPENDIX). The minimum number of spatial dimensions for accurate CSD analysis is discussed. The conductivity tensor was experimentally measured in frog and toad cerebella. All three principal components of the tensor were evaluated and their spatial gradients determined to be negligible. It was also shown that the conductivity was independent of potential. Thus the anuran cerebellum is anisotropic, homogeneous, and ohmic. On the basis of these results the appropriate mathematical expression for the CSD was selected

This paper represents a systematic, semirigorous attempt to optimize the technique of current source-density (CSD) analysis experimentally. We compared different spatial differentiation formulas in terms of accuracy, aliasing, and smoothing, and provide experimental and theoretical rationale for their use. Sources of error have also been investigated. Expressions were derived to enable one to estimate the relative magnitude of errors due to electrical noise, uncertainty in tip position of recording electrodes, and error in the conductivity tensor. Corresponding experiments illlustrating the validity of such estimates are also presented. Methods to determine the optimum interelectrode spacing are given, based on computations of spatial energy-density spectra in the anuran cerebellum. The application of the technique of CSD analysis developed in this, and the accompanying paper, to the vestibulocerebellar input in the toad cerebellum provided significantly better temporal and spatial resolution of neuronal events than conventional field-potential analysis. Considerations germane to the optimum application of this technique to other neural structures are also discussed