Synaptic physiology and mitochondrial function in crayfish tonic and phasic motor neurons

Phasic and tonic motor neurons of crustaceans differ strikingly in their junctional synaptic physiology. Tonic neurons generally produce small excitatory postsynaptic potentials (EPSPs) that facilitate strongly as stimulation frequency is increased, and normally show no synaptic depression. In contrast, phasic neurons produce relatively large EPSPs with weak frequency facilitation and pronounced depression. We addressed the hypothesis that mitochondrial function is an important determinant of the features of synaptic transmission in these neurons. Mitochondrial fluorescence was measured with confocal microscopy in phasic and tonic axons and terminals of abdominal and leg muscles after exposure to supravital mitochondrial fluorochromes, rhodamine-123 (Rh123) and 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). Mitochondria of tonic axons and neuromuscular junctions had significantly higher mean Rh123 and 4-Di-2-Asp fluorescence than in phasic neurons, indicating more accumulation of the fluorochromes. Mitochondrial membrane potential, which is responsible for Rh123 uptake and is related to mitochondrial oxidative activity (the production of ATP by oxidation of metabolic substrates), is likely higher in tonic axons. Electron microscopy showed that tonic axons contain approximately fivefold more mitochondria per microm2 cross-sectional area than phasic axons. Neuromuscular junctions of tonic axons also have a much higher mitochondrial content than those of phasic axons. We tested the hypothesis that synaptic fatigue resistance is dependent on mitochondrial function in crayfish motor axons. Impairment of mitochondrial function by uncouplers of oxidative phosphorylation, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone, or by the electron transport inhibitor sodium azide, led to marked synaptic depression of a tonic axon and accelerated depression of a phasic axon during maintained stimulation. Iodoacetate, an inhibitor of glycolysis, and chloramphenicol, a mitochondrial protein synthesis inhibitor, had no significant effects on either mitochondrial fluorescence or synaptic depression in tonic or phasic axons. Collectively, the results provide evidence that mitochondrial oxidative metabolism is important for sustaining synaptic transmission during maintained stimulation of tonic and phasic motor neurons. Tonic neurons have a higher mitochondrial content and greater oxidative activity; these features are correlated with their greater resistance to synaptic depression. Conversely, phasic neurons have a lower mitochondrial content, less oxidative activity, and greater synaptic fatigability.     

PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.     

A comparative study of the interaction of phosphoric acid dichlorovinyl esters with a neurotoxic esterase from the brain of hens and rats

In order to validate a rodent biochemical model of delayed neurotoxicity of organophosphates (OP) inhibition of rat and hen brain neurotoxic esterase (NTE) by some dichlorovinyl phosphates and phosphonates was studied in vitro and in vivo. It was shown that compounds investigated exhibited the similar inhibitory potency to NTE from both species in vitro, in addition rat and hen NTE showed the same sensitivity to variation of the structure of OP inhibitors. A good correlation was found between pI50 estimated with enzymes from rat and hen trains: r2 = 0.951, n = 18, p < or = 0.05. NTE activities were also measured in rat and hen brains after acute administration of various dosages of potent axonopathic compound dipropyldichlorovinyl phosphate. The results obtained indicate that difference in species susceptibility to neurotoxic action of OP, in particular the absence of ataxia in rats, is not caused by difference in target enzyme sensitivity to axonopathic organophosphates.     

Choline esters and biogenic amines in the hypobranchial gland of 55 molluscan species of the neogastropod Muricoidea superfamily

As many as 55 neogastropod molluscs, all belonging to the Muricoidea superfamily, have been investigated for occurrence and contents, in their hypobranchial gland (HG), of choline esters and, subordinately, biogenic amines. Very high amounts of esters, strictly localized in the median area of the HG, were found in all dye-secreting molluscs. The choline esters were represented by murexine, dihydromurexine and senecioylcholine. A fourth ester, acryloylcholine, occurred in the HG of a single, non dye-secreting mollusc. All the compounds displayed potent neuromuscular blocking actions in all examined vertebrate and invertebrate species, as well as potent nicotinic actions. Muscarinic effects were either lacking or unimportant. In addition to choline esters the HG occasionally contained known and hitherto unknown biogenic amines: tyramine, octopamine, 5-hydroxytryptamine, histamine, urocanylhistamine and imidazole-propionylhistamine. The interest of extending the search of bioactive compounds to carnivorous, predatory molluscs other than those described in this paper and, more, extensively, to any molluscan species provided with 'venomous' glands or apparatuses, is emphasized.     

5 GHz, 12 mW NMOS frequency divider

Reports on the development of an NMOS 2:1 frequency divider circuit that operates to over 5 GHz. It is powered from a 2.5 V supply and dissipates only 0.012 W. These results indicate that the use of silicon MOS technology may be extended to very-high-speed low-power applications     

Effects of amperozide, 8-OH-DPAT, and FG 5974 on operant responding for ethanol

Ventilatory acclimatization to sustained hypoxia (VASH) is the time-dependent increase in ventilation that occurs during prolonged exposure to hypoxia. We tested the hypothesis that carotid body (CB) dopaminergic mechanisms are down-regulated during VASH, which would allow CB afferent discharge and ventilation to increase beyond the initial response to hypoxia. Domperidone (DOM; 1.0 mg.kg-1) was administered intravenously to block CB dopamine (DA) receptors after VASH was complete in awake goats. DOM caused a significant augmentation of the ventilatory response to hypoxia in acclimatized goats, failing to support the hypothesis. We conclude that inhibitory CB dopaminergic function is not significantly reduced following prolonged hypoxia, and that down-regulation of CB dopaminergic mechanisms may not be involved in VASH in the goat.