Ian M. Cooke - US grants

This research exploits the cardiac ganglion of crabs and lobsters as a model in which to seek basic knowledge of endogenous neuronal properties, their biophysical bases, the mechanisms by which they are modified and controlled by synaptic interactions, both electronically and chemically mediated, mechanisms of integration, and the mode of action on an integrating neural system of a peptide neurohormone. The ganglion exhibits spontaneity, stable rhythmicity and burst impulse firing. With 9 neurons, it is the smallest neural system that can be isolated functionally intact which exhibits major features of central nervous system functioning. Electrophysiological techniques employing several intracellular and extracellular electrodes simultaneously will be used. They permit monitoring of all impulse traffic, evaluation of synaptic interactions and recording of endogenously generated potential changes such as pacemaker (PMP) and burst forming or 'driver' potentials (DP). The characterization of DPs, recently found to persist in the absence of other responses in tetrodotoxin, will be refined and extended. Functional isolation and biophysical characterization of DP's with voltage clamping techniques will be undertaken. Functional and electronmicroscopical (EM) localization of the sites of chemical and electronic synaptic interaction will be studied. The cardioactive peptide hormone(s) of crab pericardial organs will be separated and their mode of action in enhancing burst rate, duration and coordination analyzed. Knowledge gained from study of this model system may be expected to contribute and have general relevance to basic understanding of CNS functioning.

Research will be continued on the in vitro crab sinus gland neurosecretory system. The biophysical bases of electrical responses recorded intracellularly from the peptide neurohormone secreting terminals will be studied, and changes of electrical responses resulting from all other manipulations to be employed monitored. Fine-structural changes in terminals will also be monitored in parallel electron-microscope studies. Stimulus-secretion coupling is to be studied with improved time resolution to correlate intracellular changes during repetitive stimulation with 'facilitation' of erythrophore concentrating hormone (ECH) release. The rapid, semi-quantitative crab leg segment assay, sensitive to 5 times 10 to the minus 12th power molar synthetic ECH, will be used to determine ECH released into the perfusate. Involvement of metabolically dependent processes in acute release and in subsequent replenishment of hormone in terminals will be examined. Organ culture of the sinus gland system will be undertaken for studies employing isotopically labelled ECH precursor amino acids. Selective application of amino acids or drugs to somata or terminals is possible and will be exploited to localize processes involved in replenishment of hormone released from the terminals. The localization and time course of isotope distribution will be followed, not only by counting of perfusate collected during stimulation, but also by autoradiography of whole mounts, and of light and electron microscope sections. Experiments will test the effect on replenishment processes of secretory activity and seek the mechanism by which stimulation of these processes occurs. Electron-microscope examination of the terminals marked with Procion rubine injected through the intracellular recording electrode will permit correlation of electrophysiological characteristics of individual terminals with the type of neurosecretory granules each contains. Identification of the terminal type releasing ECH will be attempted.

Information is sought to answer questions about the relationship between the differing electrical characteristics of neurons that secrete different peptide hormones and their biochemical and morphological characteristics. The neurons will be studied intact within an acutely isolated preparation and isolated in cell culture. In culture, the neurons synthesizing different hormones, as indicated by their immunoreactivity to specific antisera, assume distinct morphologies under defined conditions. The cultured neurons having different form show distinct differences in their electrical characteristics. The material used for these studies is the X-organ - sinus gland of the tropical land crab, Cardisoma carnifex. Earlier work has demonstrated that this crustacean neurosecretory system is directly analogous to the major vertebrate neurosecretory system, the hypothalamic-neurohypophyseal system, and that at every level of analysis yet undertaken the physiological and cell biological mechanisms involved are directly comparable. Many of the observations obtainable with the crab material, however, would not be possible with vertebrate material. The crab neurosecretory system is easily isolated and consists exclusively of peptidergic neurons and supporting tissue. The terminals are unusually large (up to 30um), permitting recording with intracellular or patch electrodes. Antisera to several peptide- hormonal activities permit typing of neurons and terminals. Isolated X-organ neurons immediately commence regeneration of processes in unconditioned dishes and defined media. Conventional microelectrode techniques and dye injection will be used to characterize neurons in acutely isolated systems. Both conventional and patch-clamping techniques will be applied to the neurons in culture. The regional distribution in the membrane of channels giving rise to different ionic currents will be explored by testing the effects of axotomy and by localized recording. Regional sensitivity to putative neurotransmitter agents will be explored. As feasible, changes with time in culture or with changed morphology induced by altered culturing conditions will be detailed.

Peptide secretion probably occurs from most neurons. It modulates CNS function as well as serving neuroendocrine regulation. Knowledge of peptide secretory mechanisms is limited to a very few experimentally tractable systems. This work proposes rigorous experiments on a uniquely suitable invertebrate preparation to understand how secretion of neuropeptides is related to physiological stimuli, the electrical responses and changes of cytoplasmic [Ca++] and [Na+] that these produce in the nerve terminals, and the possible modulation of these by hormones. The X-organ - sinus gland (XOSG) of the crab (Cardisoma carnifex) will be used. Analogous to the vertebrate hypothalamic-neurohypophyseal system in its role and physiology, it is purely peptidergic with 3 major hormonal activities that can be immunocytologically localized and quantified in perfusates by bioassay or ELISA. The XOSG is easily isolated; terminals and preterminal swellings form a neurohemal organ (the SG), and many are large enough for intracellular recording. Terminals can be dissociated for patch-clamping. Somata dissociated from the XO grow in defined culture. For each of the 3 hormonal activities, crustacean hyperglycemic hormone, red-pigment concentrating hormone and molt-inhibiting hormone, the following questions are to be addressed: Is the rate of basal secretion governed by [Ca++]i or [Na+]i, and what controls these? Is the response to stimulation accounted for by entry of Ca++, or is release from internal stores involved? Does [Na+]i influence evoked secretion? Does release occur only at contacts with blood spaces, or at other sites as well? Is secretion modulated by hormones, and at what sites and by what mechanisms? What limits the amount of releasable hormone, and is there peripheral restocking? In isolated XOSGs, for the 3 hormonal activities, basal secretion and secretion evoked by raised [K+]o and axonal stimulation will be followed by analysis of perfusate aliquots. The effects of ionic composition, pharmacological agents and temperature that alter these will then also be tested for effects on electrical responses. "Whole terminal" patch-clamp will characterize ionic currents of isolated terminals which will then be identified immunocytologically. Fura-2 and SBFI will be used to optically assess [Ca]i and [Na]i in terminals and cultured neurons. Capacitance measurements and computer-aided, video-enhanced microscopy will be used to track secretion from terminals and growth cones. Immunocytologically identified terminals will be characterized at the EM level together with changes resulting from secretory activity.