Mahlon E. Kriebel

PACEMAKER PROPERTIES OF TUNICATE HEART CELLS

1. The primary pacemakers in the tunicate heart are located near the cardiovascular junctions close to the raphe that connects the V-shaped heart to the pericardium. However, small isolated pieces of the ring of myocardial cells at the ends of the heart were found to have nearly equal pacemaker capabilities. Arrhythmia in one primary pacemaker region was found to result from activity of two or more centers.2. Following isolation of a small piece of tissue in sea water, 20-50% of the cells were observed to pulsate and usually within a few minutes all cells contracted synchronously. Reversals in the direction of conduction in strips of myocardium without any primary pacemaker region were observed.3. The C center located in the middle of the heart was found to be dormant during normal heart activity but in opened animals it was activated by increasing the blood pressure.

Porocytosis: a new approach to synaptic function

We propose a new approach to address the question of how a single quantum of neurotransmitter is secreted from a presynaptic terminal whose clustered secretory vesicles are locally bathed in high levels of calcium ions [Proceedings of the Symposium on Bioelectrogenesis (1961) 297-309; The Physiology of Synapses (1964) Chapters 1, 4, 5, 6; How the Self Controls its Brain (1994) Chapters 1, 4, 5, 6; Science 256 (1992) 677-679]. This hypothesis, which we term porocytosis, posits that the post-synaptic quantal response results from transmitter secreted through an array of docked vesicle/secretory pore complexes. The transient increase in calcium ions, which results from the voltage activated calcium channels, stimulates the array of secretory pores to simultaneously flicker open to pulse transmitter. Porocytosis is consistent with the quantal nature of presynaptic secretion and transmission, and with available biochemical, morphological and physiological evidence. It explains the frequency dependency of quantal size as a function of the secretion process. It permits a signature amount of transmitter release for different frequencies allowing a given synapse to be employed in different behavioral responses. The porocytosis hypothesis permits fidelity of secretion and the seemingly apposed characteristic of synaptic plasticity. The dynamics inherent in an array insure a constant quantal size as a function of the number of units within the array. In this hypothesis, plasticity is a consequence of concurrent pre- and post-synaptic changes due to a change in array size. Changes in the number of docked vesicle-secretory pore complexes composing the array can explain facilitation, depletion, graded excitation-secretion and long term plasticity.

Detached, Purified Nerve Terminals From Skate Electric Organ for Biochemical and Physiological Studies

Electric organs of skate (Raja species) dissociate to form populations of individual electrocytes when incubated in saline solutions containing collagenase. The rate of dissociation was highly temperature dependent, with an apparent Q10 of > 6 in the range of 6 degrees-26 degrees C. The number of electrocytes per organ was relatively constant and independent of electric organ size, whereas mean cell diameters increased with organ size. The activities of two cholinergic marker enzymes, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in extracts of whole fresh organs were much less than those reported for the electric ray Torpedo, suggesting a lower volume of terminals in the organ. Electrocytes prepared from collagenase-treated organs had good resting potentials and generated postsynaptic evoked potentials. Spontaneous and electrode pressure-evoked miniature endplate potentials (MEPPs) were readily recorded from isolated electrocytes. Incubation periods of more than 4 days in collagenase at 6 degrees C produced electrocytes with good resting potentials and very low MEPP frequencies, indicating denervation. Detachment of terminals and decreased MEPP frequencies were concurrent. The time course of denervation was followed with the appearance of ChAT and AChE activities in a small particulate fraction derived from washed electrocytes. Peak activities of both enzymes were seen at 4 days of incubation at 16 degrees C, but after 20 h at 16 degrees C. Electrocytes from 4-day, 6 degrees C incubations showed detached, mitochondria-rich nerve terminals and dissociated Schwann cells. In unfixed preparations examined with Nomarski optics, isolated nerve terminals were recognized and distinguished from nucleated Schwann cells. Electron micrographs show that isolated terminals were similar to attached terminals just before they dissociated. The MEPP frequencies and evoked potentials were normal at terminals just before dissociation. We conclude that the transmitter release process was normal in detached terminals and in terminals free of Schwann cells.

Porocytosis: Fusion Pore Array Secretion of Neurotransmitter

We believe that there is sufficient experimental evidence to support the premise that transmitter is secreted by the simultaneous activation of arrays of fusion pores at docked vesicles. This process is initiated by the action potential that activates calcium channels to increase the number of cytoplasmic calcium ions. Calcium ions trigger fusion pores to flicker open causing transmitter to diffuse from vesicular stores. We define the term porocytosis to identify this process and use the term synaptomere to indicate the anatomical and physiological functional unit of the synapse or junction. Our model shows that the simultaneous flicker of fusion pores in an array can generate unitary-end plate potentials (u-EPPs) and miniature end plate potentials (MEPPs) and that activation of all fusion pores produces EPPs. U-EPPs and EPPs generated with the model show mean values and coefficients of variation similar to experimental observations. The model is robust in that the number of docked vesicles can vary and these can be full to empty depending on nerve frequencies and vesicular traffic. The model shows that the overall process of excitation-secretion coupling is highly deterministic. At the neuromuscular junction, secretion from arrays of fusion pores ensures that a muscle fiber action potential is always produced over a range of frequencies because all transmitter release sites are activated. Our model shows that transmission at the synaptomere guarantees fidelity of information transfer at different frequencies. This characteristic shows a dynamic relationship of the secretory process to memory and learning.

CHOLINOCEPTIVE AND ADRENOCEPTIVE PROPERTIES OF THE TUNICATE HEART PACEMAKER

1. Many previous investigators have reported that acetylcholine has little or no effect on the beat frequency of intact but isolated hearts. However, it was found that when hearts were split open, Ach at low concentrations (10-8 g./ml.) stopped the heart beat for up to a minute. Atropine blocked Ach.2. D-tubocurarine at a concentration of 3 x 10-7 g./ml. stopped the beat of opened hearts. Its effect was additive to that of Ach.3. Adrenaline (10-5 g./ml.) could stop pacemakers. However, a doubling in beat frequency was frequently observed with higher concentrations. Doubling in frequency resulted from an ectopic center which alternated with the primary center, thus driving the heart at twice its original frequency. In hearts which showed arrhythmia resulting from activity of more than one pacemaker center, adrenaline at a low concentration (10-5 g./ml.) usually stopped one or the other of the centers, decreasing the beat frequency by half.4. Hearts recovered in low concentrations of either Ach or adrenalin...

The synaptic bouton acts like a salt shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.

KCl STIMULATION AND ULTRASTRUCTURAL RESPONSES OF TANNIC ACID‐STAINED CHOLINERGIC SYNAPTIC TERMINALS

The quantal‐vesicular hypothesis equates miniature end‐plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense‐core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid—elasmobranch saline, KCl stimulation produced MEPPs at 40/s/μm2of terminal surface for several minutes with gradual reduction to spontaneous levels by 25–30min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800MEPPs/μm3of terminals having densities of 567 vesicles/μm3. No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.