Ian M. Cooke

Reliable, Responsive Pacemaking and Pattern Generation With Minimal Cell Numbers: the Crustacean Cardiac Ganglion

Investigations of the electrophysiology of crustacean cardiac ganglia over the last half-century are reviewed for their contributions to elucidating the cellular mechanisms and interactions by which a small (as few as nine cells) neuronal network accomplishes extremely reliable, rhythmical, patterned activation of muscular activity—in this case, beating of the neurogenic heart. This ganglion is thus a model for pacemaking and central pattern generation. Favorable anatomy has permitted voltage- and space-clamp analyses of voltage-dependent ionic currents that endow each neuron with the intrinsic ability to respond with rhythmical, patterned impulse activity to nonpatterned stimulation. The crustacean soma and initial axon segment do not support impulse generation but integrate input from stretch-sensitive dendrites and electrotonic and chemically mediated synapses on axonal processes in neuropils. The soma and initial axon produce a depolarization-activated, calcium-mediated, sustained potential, the “driver potential,” so-called because it drives a train of impulses at the “trigger zone” of the axon. Extreme reliability results from redundancy and the electrotonic coupling and synaptic interaction among all the neurons. Complex modulation by central nervous system inputs and by neurohormones to adjust heart pumping to physiological demands has long been demonstrated, but much remains to be learned about the cellular and molecular mechanisms of action. The continuing relevance of the crustacean cardiac ganglion as a relatively simple model for pacemaking and central pattern generation is confirmed by the rapidly widening documentation of intrinsic potentials such as plateau potentials in neurons of all major animal groups. The suite of ionic currents (a slowly inactivating calcium current and various potassium currents, with variations) observed for the crustacean cardiac ganglion have been implicated in or proven to underlie a majority of the intrinsic potentials of neurons involved in pattern generation.

Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jellyfish (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and purification procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by D-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20mM EDTA buffer; it was optimal in the presence of 10mM Ca2+ or Mg2+. Two chromatographic purification methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42kDa by SDS-PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG.

Electrical activity and release of neurosecretory material in crab pericardial organs

Abstract 1. 1. One of the pair of pericardial organs (POs) of Libinia emarginata was removed to a chamber and arranged for simultaneous electrical stimulation and recording. Fluid bathing the PO was assayed for excitatory effect on the isolated, perfused crab heart. 2. 2. A low level of spontaneous release of excitatory material and spontaneous electrical activity, interpreted as action potentials in one or a few axons, were observed in many POs. 3. 3. A high rate of release of active material was observed only when stimulation resulted in a compound action potential showing a large component representing fibers of 1 4 to 1 2 m/sec conduction velocity. 4. 4. This electrical component and release of excitatory material showed the same threshold and optimal values of stimulus voltage, and the same maximum, 10/sec, for response to repetitive stimulation. 5. 5. The excitatory effect of the fluid was proportional to the number of stimuli given. 6. 6. It is concluded that action potentials propagated in axons of neurosecretory cells result in release of neurosecretory material from the terminals.

Somatostatin and altered medium osmotic pressure elicit rapid changes in prolactin release from the rostral pars distalis of the tilapia, Oreochromis mossambicus, in vitro

Prolactin (PRL) cells in the rostral pars distalis of the tilapia Oreochromis mossambicus respond to somatostatin (SRIF) and reduced medium osmotic pressure within 10-20 min of exposure during perifusion incubation. Pieces of rostral pars distalis tissue were removed from freshwater-adapted tilapia and were preincubated in [3H]leucine in static culture (355 m phi smolal) for 48 hr. Following preincubation, they were placed in the perifusion apparatus and baseline release was established for 3 hr in hyperosmotic medium (355 m phi smolal). Exposure to hyposmotic medium (280 m phi smolal) resulted in a rapid and steep rise in the release of [3H]PRL, which remained elevated for more than 2 hr. When SRIF was added simultaneously with hyposmotic medium, the rise in PRL release normally initiated by reduced osmotic pressure was prevented. Somatostatin also quickly reduced release that had been previously elevated by exposure to hyposmotic medium. The time course of these changes suggests that SRIF and altered osmotic pressure act on PRL secretion in at least partial independence of effects which they may have on PRL synthesis in the tilapia pituitary.

Electrical Activity of Neurosecretory Terminals and Control of Peptide Hormone Release

Nerve cells specialized for the release of peptide hormones to the circulation, i.e., classic neurosecretory cells, retain their full complement of neuronal properties. Their activity is under control of the CNS through excitatory and inhibitory synaptic mediation. They integrate these influences with their own capabilities for endogenous activity to ultimately generate action potentials propagated to the secretory terminals. They represent the “final neuroendocrine pathway” (Knowles, 1974; E. Scharrer, 1965). In this chapter, I wish to examine the relationship between those action potentials that are propagated to the secretory terminals and release of peptide hormones from them. How much of the extensive, detailed knowledge of the mechanisms governing the release of transmitters at synapses (for reviews, see, for example, Katz, 1969; Gerschenfeld, 1973) is applicable to release of peptides from neurosecretory terminals? Are there modifications of the electrical activity of neurosecretory cells, particularly their terminals, that are related to peptide secretion? For example, is the longer duration of action potentials, well documented for the neuron somata, also a feature of neurosecretory axons and terminals, and what is its significance? What is the significance for hormone release of the “spontaneous” activity often recorded from neurosecretory cells, and of firing in bursts or patterned activity?

Separation of neuronal sites of driver potential and impulse generation by ligaturing in the cardiac ganglion of the lobster,Homarus americanus

SummaryThis study documents the ability of individual neurons of theHomarus americanus cardiac ganglion to produce driver potentials (regenerative, 20 mV, 200 ms depolarizing responses to depolarization).Partial block of impulse traffic, achieved by ligaturing, indicates that chemically mediated synaptic transmission is not essential to burst formation, initiation or coordination, but increases burst duration (Figs. 1, 9); electrotonic coupling suffices.Ligatures placed within 1.2 mm of the soma of Cells 1 or 2 separate impulse supporting axon from a soma-proximal neurite region which does not support impulses, but generates a driver potential in response to depolarizing current (Figs. 2, 6, 10). Driver potentials persist with ligatures as close as 200 μm. Such a ligature excludes neuronal interaction via axon collaterals observable with lucifer yellow (Fig. 7), thus establishing that driver potentials are endogenous. Driver potentials are unaffected by TTX (Fig. 2, Table 1).Driver potentials ofHomarus neurons isolated by ligaturing were compared with those ofPortunus isolated by TTX. The measured parameters are very similar (Table 1), as are effects of altered membrane holding potential (Figs. 3, 4) and drugs (Fig. 5). Driver potentials are reversibly blocked by Mn (4 mmol/l) or Cd (0.5 mmol/l). Tetraethylammonium chloride (5–50 mmol/l) reversibly augments the amplitude and duration of driver potentials; hyperpolarizing afterpotentials remain.Driver potential responses during repetitive stimulation indicate a relatively refractory period and capability for graded responsiveness (Fig. 8).Anterior neurons (Cells 1 or 2) isolated by ligaturing rarely exhibited spontaneity (Figs. 10, 11). Cell 3, isolated by 3 ligatures, consistently showed rhythmic burst generation (Fig. 11) arising from pacemaker depolarization.The capability of driver potential generation in response to non-specific depolarization endows individual neurons with their pattern-forming ability.

Postsynaptic membrane response predicted from presynaptic input pattern in lobster cardiac ganglion.

Given the pattern of impulses impinging on a neuron, it should be possible to predict its output firing pattern if enough is known about pre- and postsynaptic properties. A quantitative reproduction of the first part of this input-output conversion is reported, namely the translation of input pattern into a sequence of postsynaptic membrane potential variations.

Topographical localization of function in the cardiac ganglion of the crab,Portunus sanguinolentus

SummaryThe location and role in burst formation of electrotonic and chemically mediated interaction among cardiac ganglion neurons ofPortunus sainguinolentus was studied with intra- and extracellular recording and stimulation, and by dye injection (Fig. 1).The soma-proximal neurite region generates TTX-resistant, 200 ms, 20 mV ‘driver potentials’, but not impulses; the axon initiates TTX-sensitive impulses, but not driver potentials (Fig. 3).A pressure block by ligaturing (Figs. 4–7) shows that chemically mediated synaptic potentials (epsps) are not essential to synchronous bursting, but burst duration is reduced in their absence. Electrotonic conduction of slow and driver potentials is adequate to maintain rhythmic, synchronous bursting.The anterior three cells remain electrotonically- and dye-coupled when isolated by a ligature 250 μm posterior of their somata (Figs. 1, 12). They exhibit spontaneous, synchronous, rhythmic bursting.Epsps and impulse initiation of the two posterior large cells occur in the anterior half of the ganglion (Figs. 5, 7, 8, 12). Electrotonic and dye coupling remain following ligaturing 750 μm anterior of their somata (Fig. 11).Driver potentials and impulses of small cells can be recorded by electrotonic conduction in posterior large cells after ligaturing the trunk. Small cell activity is influenced by current passed into a large cell (Figs. 8, 9, 13).Each neuron has endogenous capability for producing a driver potential. Initiation of these is synchronized by electrotonic spread of slow potentials and small cell-initiated epsps. Proximity of the sites of these interactions and those of large cell impulse initiation ensure synchronous impulse bursts of the large cell axons.

Effect of calcium omission on neurosecretion and electrical activity of crab pericardial organs

Abstract Fluid bathing an isolated crab pericardial organ was assayed on the isolated, perfused crab heart in order to follow release of heart excitatory neurosecretory material in response to electrical stimulation. The propagated, compound action potential produced by stimulation was monitored extracellularly from the neurohemal structure. When calcium is omitted from the bathing saline, release of neurosecretory material is reduced to the unstimulated level. However, the compound potential is not depressed.

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release

Electrophysiological measurements of cell capacitance (Cm) and biochemical assays of [3H] serotonin ([3H]5-hydroxytryptamine or [3H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced Cm changes (ΔCm), indicative of exocytosis, with the same voltage and Ca2+ dependency as the inward Ca2+ currents (ICa). Ba2+ was able to substitute for Ca2+ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca2+-versus Ba2+-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [3H]5-HT to study hormone release from cell populations. [3H]5-HT was released in a depolarization-, Ca2+- and time-dependent manner. Ba2+ also substituted for Ca2+ in depolarization-induced [3H]5-HT release. Thapsigargin, used to deplete Ca2+ stores, had no effects on Ca2+-triggered Cm increases, but Ca2+-triggered [3H]5-HT release was abolished. Ba2+-triggered [3H]5-HT release, however, was only slightly affected by Ca2+ store depletion. Ba2+ was found to act directly as a secretagogue of [3H]5-HT in intact cells, but not in Cm measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action.