Catecholamine-containing neurons in the peripheral nervous system of Aplysia

Numerous green-fluorescent neurons have been revealed by means of the glyoxylic acid histochemical method in cryostat sections of several organs of two Adriatic aplysiid gastropods, Aplysia depilans and A. fasciata. Catecholamine-containing perikarya and their processes have been found to be especially abundant in the vaginal portion of the large hermaphrodite duct, in the penis and its sheath, and in the gill. In the reproductive organs, two subpopulations of catecholamine-containing neurons could be distinguished according to their size and location. Axons of larger neurons formed bundles which seemed to project at the CNS.     

Defensive behaviour in the pond snail, Lymnaea stagnalis: the whole body withdrawal associated with exsanguination

The freshwater snail L. stagnalis is known to be able to respond to a strong, noxious stimulus with a full retraction of the foot and head into the shell accompanied with expelling the blood through the hemal pore. We have found that this behavioural response, besides graded local retractions, can be triggered by mild tactile stimulation provided that the stimulus is repeated several times. Only a complete exsanguination could be obtained, indicating that it is an all-or-none defensive behaviour. In an electrophysiological investigation of isolated brain, a number of similarities were found between this all-or-none behaviour and the so-called input 3 to central neurons, as described by Benjamin and Winlow. These include ability to be selectively activated by high calcium solutions, and blocked by keeping the snails in a spoiled water. Injection of snails with naloxone (0.5-2.0/micrograms/g) or ergotamine (0.4/microgram/g) blocked selectively the whole body withdrawal induced by tactile stimulation, but not that induced by injection of a high calcium saline or acetylcholine solution, indicating that enkephalinergic and/or dopaminergic mechanosensory neurons might be involved. The consideration of available data has led to a working hypothesis that the activity of input 3 might be the neurophysiological correlate of the high threshold all-or-none whole body withdrawal associated with exsanguination.     

Silver staining of the nucleoli of pig embryonic kidney cells during the hyperactivation and inhibition of the synthesis of nucleolar RNA by UV microirradiation

Silver staining of the nucleoli in pig embryo kidney cells (PK) was studied during the cell cycle and also upon mature nucleoli modifications induced by UV microirradiation. During anaphase only four silver-stained granules were revealed in each daughter set of chromosomes in the four nucleolus-organizing regions (NORs). In the following 1-2 hours, the number of granules in the NORs rapidly increased up to 25-30 per nucleus. During the next 20-25 hours of the cell cycle, the number of silver-stained granules was slowly doubling as the nucleoli grew in size. UV microirradiation of one nucleolus in the nucleus with two nucleoli induced a profound degradation of the injured nucleolus and a compensatory hypertrophy of the intact one. Such nucleolar modifications were accompanied by redistribution of the silver-stained granules between the injured and non-injured nucleoli and by alterations in the levels of nucleolar RNA synthesis in the NORs. These data support a hypothesis that silver-stained proteins may be involved in the regulation of the nucleolar activity.     

Permeability and damage of the erythrocyte membrane at -1 degree C to -9 degrees C shown by the NMR relaxation method

Amount of inter--and extracellular unfrozen water in the intact human erythrocytes as well as tau a, "a life time" of water molecules in these cells, have been determined. The progressive compression of the erythrocytes during freezing them as deep as -5 degrees C has been found to submit to the ideal osmometer law. The membrane impermeability to Mn2+ ions has been disturbed when the temperature falls to -7 degrees C and below, the erythrocytes reaching "the minimal volume".     

Effect of serotonin and acetylcholine on neurons in the central nervous system of snails

Differences in the distribution of neurons distinguished by their responses to serotonin and acetylcholine were found in the central nervous system ofHelix pomatia. When applied to the body of the neuron acetylcholine hyperpolarizes the cell more often than it depolarizes it, but depolarization predominates in some regions, e.g., on the dorsal surface of the visceral ganglion. In most cases serotonin stimulates activity and induces depolarization or the appearance of pacemaker oscillations of membrane potential. The oscillogenic effect of serotonin is characteristic, in particular, of white (peptidergic) neurons and the depolarization effect is characteristic of other neurons, including the paired giant metacerebral neurons which contain serotonin in their cytoplasm. Both effects failed to appear in sodium-free solution. A group of neurons in which hyperpolarization was observed in response to serotonin application was found in the visceral ganglion of hibernating snails. The same cells in active snails were stimulated by serotonin. A giant neuron with two variously located cholinergic structures is present on the ventral surface of the ganglion among this group of cells: acetylcholine hyperpolarized it when applied to the cell body but depolarized it when applied to the axon.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.