Reduced H+ channel activity disrupts pH homeostasis and calcification in coccolithophores at low ocean pH.

Coccolithophores are major producers of ocean biogenic calcite, but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H+ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H+ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H+ channels. Heavily calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H+ load and are likely to be most vulnerable to future decreases in ocean pH.

Effects of the microbial secondary metabolites pyrrolnitrin, phenazine and patulin on INS-1 rat pancreatic ß-cells.

The effects on pancreatic ß-cell viability and function of three microbial secondary metabolites pyrrolnitrin, phenazine and patulin were investigated, using the rat clonal pancreatic ß-cell line, INS-1. Cells were exposed to 10-fold serial dilutions (range 0-10 µg mL(-1)) of the purified compounds for 2, 24 and 72 h. After 2 h exposure, only patulin (10 µg mL(-1)) was cytotoxic. All compounds showed significant cytotoxicity after 24 h. None of the compounds altered insulin secretion with 2 and 20 mM glucose after 2 h. However, after 24 h treatment, phenazine and pyrrolnitrin (10 and 100 ng mL(-1)) potentiated insulin production and glucose-stimulated insulin secretion, whereas patulin had no effect. Exposure (24 h) to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 ng mL(-1)) caused similar increases in the Ca(2+) content of INS-1 cells. The outward membrane current was inhibited after 24 h exposure to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 or 100 ng mL(-1)). This study presents novel data suggesting that high concentrations of pyrrolnitrin and phenazine are cytotoxic to pancreatic ß-cells and thus possibly diabetogenic, whereas at lower concentrations these agents are nontoxic and may be insulinotropic. The possible role of such agents in the development of cystic fibrosis-related diabetes is discussed.

Whole-cell recording of light-evoked photoreceptor responses in a slice preparation of the cuttlefish retina.

A new tissue slice preparation of the cuttlefish eye is described that permits patch-clamp recordings to be acquired from intact photoreceptors during stimulation of the retina with controlled light flashes. Whole-cell recordings using this preparation, from the retinas of very young Sepia officinalis demonstrated that the magnitude, latency, and kinetics of the flash-induced photocurrent are closely dependent on the magnitude of the flash intensity. Depolarizing steps to voltages more positive than -40 mV, from a membrane holding potential of -60 mV, induced a transient inward current followed by a larger, more sustained outward current in these early-stage photoreceptors. The latter current resembled the delayed rectifier (I(K)) already identified in many other nerve cells, including photoreceptors. This current was activated at -30 mV from a holding potential of -60 mV, had a sustained time course, and was blocked in a dose-dependent manner by tetraethylammonium chloride (TEA). The smaller, transient, inward current appeared at potentials more positive than -50 mV, reached peak amplitude at -30 mV and decreased with further depolarization. This current was characterized as the sodium current (I(Na)) on the basis that it was inactivated at holding potentials above -40 mV, was blocked by tetrodotoxin (TTX) and was insensitive to cobalt. Intracellular perfusion of the photoreceptors, via the patch pipette, demonstrated that U-73122 and heparin blocked the evoked photocurrent in a dose-dependent manner, suggesting the involvement of the phospholipase C (PLC) and inositol 1,4,5-triphosphate (InsP(3)), respectively, in the phototransduction cascade. Perfusion with cyclic GMP increased significantly the evoked photocurrent, while the inclusion of phorbol-12,13-dibutyrate reduced significantly the evoked photocurrent, supporting the involvement of cGMP and the diacylglycerol (DAG) pathways, respectively, in the cuttlefish transduction process.

Dopamine modulates synaptic activity in the optic lobes of cuttlefish, Sepia officinalis.

The effects of dopamine on spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) in three different classes of neurones within the optic lobe of cuttlefish were investigated using whole-cell voltage clamp techniques in a slice preparation. The neuronal types were centrifugal and amacrine neurones, located in the inner granular cell layer, and medullar interneurones, located within the central medulla of the optic lobes. The results demonstrate that bath application of dopamine (50 microM) reversibly reduced both the frequency and amplitude of sEPSCs and of sIPSCs in these optic lobe neurones. The inhibitory effects of DA were dose-dependent and neither D1- nor D2-like receptors appear to be implicated, but probably D4-like receptors are involved in these actions. By pre-applying tetrodotoxin (TTX, 0.5 microM), to block action potential-dependent EPSCs and IPSCs, it is shown that dopamine has no effect on the amplitude, frequency or decay time constant of the mEPSCs or mIPSCs. The results are the first to identify a specific physiological action of dopamine on cephalopod brain activity, they indicate that this effect is probably presynaptic to the specific classes of cells recorded from, and they provide information on the pharmacological profile of the receptors involved. The widespread inhibitory effect of dopamine on the activity of cuttlefish optic lobe neurones is discussed in the context of comparable data from vertebrate preparations and the actions of other neuromodulators in the cuttlefish brain.

Cholinergic and glutamatergic spontaneous and evoked excitatory postsynaptic currents in optic lobe neurons of cuttlefish, Sepia officinalis.

Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from two different classes of neurons in the optic lobes of the cuttlefish brain and their synaptic activities analyzed and compared. The cell types were as follows: efferent centrifugal neurons, with cell bodies in the inner granule layer and axons projecting to the retina, and interneurons local to the medulla. For both neuronal groups, the sEPSCs reversal potentials were around 0 mV and there were no significant differences in their mean amplitude and rise times. However, the sEPSCs from the centrifugal neurons had a significantly higher frequency and faster decay time constant than those recorded from the medulla. Tetrodotoxin (TTX) reduced the mean frequency of the sEPSCs from both the medulla and centrifugal neurons by 69.66 +/- 4.05% and 57.80 +/- 3.87%, respectively, implying that more than half of these excitatory synaptic inputs were due to action potential-mediated release of neurotransmitter. Pharmacological examination revealed that the centrifugal neurons were driven by spontaneous synaptic inputs mediated by glutamatergic and cholinergic receptors, because co-application of the glutamate antagonist kynurenic acid (KYNA) and the nicotinic antagonist mecamylamine hydrochloride (MCM) resulted in complete blockade of these excitatory inputs. For the medulla neurons, the synaptic inputs were driven by glutamate and other transmitters yet to be identified. Evoked EPSCs (eEPSCs) were recorded from both types of neurons by stimulating the appropriate optic nerve bundles; in centrifugal neurons, the eEPSCs were blocked by co-application of KYNA and MCM, whereas in the medulla neurons, KYNA alone either totally or partially blocked the eEPSCs.

Cholinergic modulation of L-type calcium current in isolated sensory hair cells of the statocyst of octopus, Eledone cirrhosa.

Whole-cell voltage-clamp recordings from dissociated hair cells of the statocyst of octopus, Eledone cirrhosa, demonstrated that application of ACh, carbachol or muscarine (10 microM) reversibly decreased the amplitude of L-type calcium current (I(Ca,L)), while nicotine (10-100 microM) did not have any effect. Furthermore, atropine blocked the effect of ACh and agonists suggesting that ACh reduces I(Ca,L) through activation of muscarinic receptors. Internal dialysis of these cells with guanosine 5'-O-3-thiotriphosphate (GTPgammaS), a non-hydrolysable GTP analogue, mimicked the ACh-induced inhibition of I(Ca,L) and occluded any further ACh-induced inhibition. Internal dialysis of these hair cells with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) reduced the ACh-induced inhibition I(Ca,L). The inhibitory effects of ACh were abolished by pre-incubation of these cells with pretussis toxin (PTX) suggesting that ACh-induced inhibition of I(Ca,L) involves a PTX-sensitive G protein pathway.

Cephalopod neural networks.

Cephalopods have arguably the largest and most complex nervous systems amongst the invertebrates; but despite the squid giant axon being one of the best studied nerve cells in neuroscience, and the availability of superb information on the morphology of some cephalopod brains, there is surprisingly little known about the operation of the neural networks that underlie the sophisticated range of behaviour these animals display. This review focuses on a few of the best studied neural networks: the giant fiber system, the chromatophore system, the statocyst system, the visual system and the learning and memory system, with a view to summarizing our current knowledge and stimulating new studies, particularly on the activities of identified central neurons, to provide a more complete understanding of networks within the cephalopod nervous system.

Modulation of spontaneous and evoked EPSCs and IPSCs in optic lobe neurons of cuttlefish Sepia officinalis by the neuropeptide FMRF-amide.

The effects of the neuropeptide FMRFa on spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs), as well as on evoked EPSCs and IPSCs, in two types of neurons within the central optic lobe of cuttlefish were examined using the whole-cell voltage-clamp technique. FMRFa (1-10 micro m) did not affect cell membrane resting potentials, but reversibly reduced both the frequency and amplitude of sEPSCs in neurons within the medulla region of the optic lobe while increasing the frequency and amplitude of their sIPSCs. For centrifugal neurons in the inner granule cell layer of the optic lobe, FMRFa (1-10 micro m) decreased both the frequency and amplitude of sEPSCs. In the presence of tetrodotoxin (0.5 micro m), neither the interevent interval, nor amplitude distributions of the miniature EPSCs or the miniature IPSCs, were affected by FMRFa, implying a presynaptic action of FMRFa on the optic lobe neurons. Bath application of the neuropeptide also abolished or reduced in amplitude the evoked EPSCs and increased the amplitude of evoked IPSCs in optic lobe neurons, showing that FMRFa induced similar effects on evoked as on spontaneous postsynaptic currents. These results demonstrate the complex range of modulatory effects FMRFa can have within central nervous system circuits.