Showing papers by "Harold Gainer published in 1986"

Action potentials and frequency-dependent secretion in the mouse neurohypophysis.

Abstract: The frequency-dependence of secretion of arginine vasopressin (AVP) from the mouse neural lobe in vitro was studied and found to be comparable to that reported for the rat neural lobe in vitro. For a stimulus train of 600 pulses, the secretion of AVP per pulse (i.e., facilitation) increased to a maximum at 20 Hz. Compound intracellular action potentials were recorded from the mouse neural lobe using optical recording methods and potentiometric dyes. These extrinsic optical signals reflect the true time courses of transmembrane potential changes (e.g., action potentials), and the action potentials recorded from mouse neural lobes had a duration of 5 ms; at half-maximum peak height. Optical recordings during repetitive stimulation showed that significant spike broadening occurred in each subsequent spike at 10 and 16 Hz stimulation. These data are consistent with a spike broadening hypothesis of frequency-dependent facilitation in the neural lobe. However, 4-aminopyridine, a drug which causes spike broadening in neural tissues by blocking potassium channels, did not produce an increase in secretion of AVP per stimulus from the mouse neural lobe.

Calcium-activated proteolysis of neurofilament proteins in the squid giant neuron.

Abstract: The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium-activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [gamma-32P]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220-kilodalton (NF-220) and a high-molecular-weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium-activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 110K protein is the putative side arm of the NF-220. In contrast, neither the HMW or the NF-220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium-activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium-activated proteolysis generated characteristic fragments that could be labeled with the anti-intermediate filament antibody.

Optical Studies of Excitation and Secretion in Vertebrate Nerve Terminals

Abstract: Optical methods that employ potential sensitive molecular probes are shown to monitor rapid changes in membrane potential from populations of nerve terminals in the neurohypophyses of amphibia and mammals. Ca++ and Na+ contributions to the action potential are demonstrated and, in the mouse, variations in light scattering are detected following membrane potential changes known to trigger the release of peptide hormones. Calcium antagonists such as Cd++, Mn++, Co++, and Ni++ block an after-hyperpolarization in the frog that we attribute to the effect of a calcium mediated potassium conductance. Block of sodium and potassium currents with TTX and TEA reveals a pure calcium action potential which is eliminated by 0.5 mN Cd++. In the mouse, the magnitude of the light scattering signal depends upon the frequency of stimulation and the extracellular concentration of Ca++, as well as on the presence of secretagogues such as 4-aminopyridine. A part of this intrinsic optical signal that seems to be correlated with neuropeptide release is blocked by inorganic calcium antagonists, while a faster component related to the action potential itself remains. The weak wavelength dependence of the light scattering signal, contrasted with the strong wavelength dependence of the extrinsic absorption signal provided by linear potentiometric probes (e.g., merocyanines), may permit one to monitor simultaneoulsy, in a stained preparation, the voltage changes in the nerve terminals and the release of secretory products. The inherently fast responses of the two optical measurements may then improve our ability to resolve early events in the coupling of excitation to secretion. Supported in part by USPHS grant NS 16824.

Ultrastructural Studies of Peptide Coexistence in Corticotropin-Releasing Factor- and Arginine-Vasopressin­ Containing Neurons

Abstract: It has become clear over the last several years that the coexistence of more than one transmitter in the same neuron is a widespread phenomenon in the nervous system, yet the physiological implications of this coexistence are only beginning to be understood (Hokfelt et al., 1984). A complete description of the roles of coexisting neurotransmitters should include (1) knowledge of the locations, actions, and dose-response curves of the receptors for the multiple transmitters, (2) analysis of the quantity and kinetics of release of the transmitters, and (3) determination of whether the release of multiple transmitters in the same cell can be independently regulated. An important determinant of the regulation of release of transmitters is their intracellular packaging. If different transmitters are localized in different populations of secretory vesicles in the same nerve ending, then the possibility of separate regulation of their release exists. However, if different transmitters are packaged in the same secretory vesicles, then independent regulation of their release by the nerve ending is unlikely.

Optical Studies on Ionic Channels in Intact Vertebrate Nerve Terminals

Abstract: Optical techniques for the detection of transmembrane electrical events have found a variety of applications over the past decade because they offer certain advantages over more conventional measurements. Because the membranes of interest are not mechanically violated, the methods may be relatively noninvasive. Spatial resolution is limited only by microscope optics and noise considerations; it is possible to measure changes in membrane potential from regions of a cell having linear dimensions on the order of 1 μm. Temporal resolution is limited by the response time of the probes and by the bandwidth imposed on the measurement, again by noise considerations, and response times faster than any known membrane time constant may be achieved. Because mechanical access is not required, unusual latitude is possible in the choice of preparation, and voltage changes may be monitored in membranes that are otherwise inaccessible. Finally, since no recording electrodes are employed, and the measurement is actually made at a distance from the preparation—in the image plane of an optical apparatus—it is possible to record changes in potential simultaneously from a large number of discrete sites.