Long-term growth in vitro of isolated, fully differentiated neurones from the central nervous system of an adult insect

A method is described for the isolation and growth in vitro of fully differentiated neurones from the thoracic ganglia of adult cockroaches. The presence of insect blood in the culture system is shown to promote growth. The morphology of the growing neurones and the plasticity of the branching processes are described and growth rates are measured. Using a fluorescent Ca2+ indicator dye, changes of intracellular calcium levels in the growing neurones in response to K+ depolarization have been measured. The results, indicating the presence of voltage-dependent Ca2+ channels on neuronal processes in vitro, show that neurones can be maintained in a functional state for several weeks by this technique. Such preparations could prove useful for studying a variety of physiological and pharmacological properties of neurones, including the mechanisms controlling growth, synapse formation and neuronal interactions with other cell types. <br/

The role of venous pressure in regulation of output from the heart of the Octopus, Eledone Cirrhosa (Lam.)

1. The influence of perfusion pressure on stroke volume and heart rate was examined in the isolated ventricle. Input pressure, within the physiological range (10–20 cm of water), had a direct effect upon stroke volume and heart rate. Output back pressure had an inverse effect upon stroke volume and no effect upon heart rate. 2. Sites that could vary input pressure were investigated by selective denervation in the whole animal. The results indicated that the efferent branchial vessel and auricle may be involved, as well as the branchial hearts and lateral venae cavae. 3. It is proposed that the pressure of venous blood has a limited effect upon ventricular output in vivo. <br/

Cardiac performance in response to loading pressures in Busycon Canauculatum (Gastropoda) and Mercenaria Mercenaria (Bivalvia)

The performance of the isolated hearts of a gastropod, Busycon canaliculatum (L.), and a bivalve, Mercenaria mercenaria(L.), were examined at different perfusion levels around the expected physiological ranges. Both hearts followed the Frank-Starling relationship with regard to stroke volume versus preload, but the heart-rate response was species-dependent. The argument is developed that the molluscs might functionally apply Starling's Law of the heart to accommodate increased output during exercise. At the expected in vivo filling pressures the power output of the two hearts was the same (15–30 × 10?6Wg?1 ventricular tissue), but the Mercenaria filling levels were considerably lower. This clearly indicates that the cardiac muscle of each of the two species has evolved to operate at specific pressure ranges. Electrical recordings from the surface of the myocardium in the perfused Busycon hearts confirm that the shape changes reported in the literature, dealing with stretched myocardium, also occur for changes in the whole heart at realistic loading pressures. These results support previous conclusions that the cardiac output is controlled by the duration of the action potential plateau. <br/

The contribution of the branchial heart to the accessory branchial pump in the Octopoda

Experimental and anatomical observations upon the Octopoda suggest that the branchial hearts are not the sole contributors to the increase in venous blood pressure between the anterior vena cava and the afferent branchial vessel. The lateral venae cavae are proposed as an additional source of pressure generation, thus contributing to the octopod accessory branchial pump. <br/

The ventilation cycle in Octopus

Pressure measurements made at various points inside the mantle show that the ventilatory stream of the resting animal is driven by very small (often less than 0.5 cm H2O) pressure differences. Inspiration occupies less than one third of the total cycle time, while flow across the gills is evidently continuous, since there is always a pressure differential between the prebranchial and postbranchial parts of the mantle cavity. The fact that branchial heartbeats do not correlate with ventilatory movements is further evidence that water flow through to the gills is both steady and continuous. <br/

Mitochondrial metabolism reveals a functional architecture in intact islets of Langerhans from normal and diabetic Psammomys obesus

The cells within the intact islet of Langerhans function as a metabolic syncytium, secreting insulin in a coordinated and oscillatory manner in response to external fuel. With increased glucose, the oscillatory amplitude is enhanced, leading to the hypothesis that cells within the islet are secreting with greater synchronization. Consequently, non-insulin-dependent diabetes mellitus (NIDDM; type 2 diabetes)-induced irregularities in insulin secretion oscillations may be attributed to decreased intercellular coordination. The purpose of the present study was to determine whether the degree of metabolic coordination within the intact islet was enhanced by increased glucose and compromised by NIDDM. Experiments were performed with isolated islets from normal and diabetic Psammomys obesus. Using confocal microscopy and the mitochondrial potentiometric dye rhodamine 123, we measured mitochondrial membrane potential oscillations in individual cells within intact islets. When mitochondrial membrane potential was averaged from all the cells in a single islet, the resultant waveform demonstrated clear sinusoidal oscillations. Cells within islets were heterogeneous in terms of cellular synchronicity (similarity in phase and period), sinusoidal regularity, and frequency of oscillation. Cells within normal islets oscillated with greater synchronicity compared with cells within diabetic islets. The range of oscillatory frequencies was unchanged by glucose or diabetes. Cells within diabetic (but not normal) islets increased oscillatory regularity in response to glucose. These data support the hypothesis that glucose enhances metabolic coupling in normal islets and that the dampening of oscillatory insulin secretion in NIDDM may result from disrupted metabolic coupling. <br/

Cardiac performance in response to loading pressures and perfusion with 5-hydroxytryptamine in the isolated heart of Busycon Canaliculatum (Gastropoda, Prosobranchia)

In this study the effects of a molluscan neurotransmitter, 5-hydroxytryptamine (5-HT), were examined on the isolated and pumping heart of the gastropod mollusc Busycon canaliculatum. Unlike in previous studies, the response was measured in such a way as to equate it with cardiac output. In addition, the effects on the myogram form and the manner of perfusate ejection were also examined. As would be expected from previous studies, 5-HT affects heart rate, showing a positive chronotropic response at a threshold of around 10?9 moll?1. Stroke volume shows little evidence of being regulated by 5-HT concentration. This observation was unexpected as 5-HT is commonly reported to regulate the ‘force’ of contraction in a molluscan heart and, at constant perfusion conditions, this might have been expected to find expression as an increase in stroke volume. 5-HT does, however, have a very clear dose-dependent effect on the aortic pressure pulse amplitude and duration. Amplitude increases markedly (250%) over the concentrations used (10?10-10?6moll?1) with a threshold around 10?8 moll?1 The effect on the duration has the same threshold but the opposite result, with a reduction to approximately 50% of the original value. The same amount of perfusate is therefore being ejected at a higher pressure and flow rate. It is suggested that this might have important implications for a soft-bodied animal with a hydrostatic skeleton. The electrical activity of the heart was also examined and showed that 5-HT increased the amplitude of both the spike and plateau phase of the action potential. The duration of the latter was reduced. This is discussed with reference to other studies. <br/

Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra grevilleana

We have adapted the self-referencing microelectrode technique to allow sensitive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a selective microelectrode through the gradient next to the cell wall or membrane. The electrode is moved at a known frequency and between known points. The differential electrode output values are converted into a directional measurement of flux by the Fick equation. By coupling the newly developed oxygen-selective self-referencing electrochemical microelectrode (SREM-O2) system with self-referencing ionselective proton measurements (SRIS-H+) we have characterized oxygen and proton fluxes from a single cell of the filamentous green algaSpirogyra gre illeana (Hass.). Oxygen showed a net efflux and protons showed a net influx when the cell was illuminated. These photosynthesis-dependent fluxes were found to be spatially associated with the chloroplasts and were sensitive to treatment with dichlorophenyldimethylurea. In the dark the directions of oxygen and proton fluxes were reversed. This oxygen influx was associated with mitochondrial respiration and was reduced by 78% when the cells was treated with 0.5 mM KCN. The residual cyanide-resistant respiration was inhibited by the application of 5 mM salicylhydroxamic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O2 system to measure and characterize metabolic fluxes at a level of sensitivity that allows for subcellular resolution. These measurements, in conjunction with SERIS-H+ measurements, have led to new insights in our understanding of basic cellular physiology in plant cells.<br/