Coral snake venoms: mode of action and pathophysiology of experimental envenomation

Coral snakes, the New World Elapidae, are included in the genera Micniroides and Micrurus. The genus Mlcrurus comprises nearly all coral snake species and those which are responsible for human snake-bite accidents. The following generalizations concerning the effects induced by their venoms, and their venom-properties can be made. Coral snake venoms are neurotoxic, producing loss of muscle strenght and death by respiratory paralysis. Local edema and necrosis are not induced nor blood coagulation or hemorrhages. Proteolysis activity is absent or of very low grade. They display phospholipase A2 activity. Nephrotoxic effects are not evoked. The main toxins from elapid venoms are postsynaptic and presynaptic neurotoxins and cardiotoxins. Phospholipases A2 endowed with myonecrotic or cardiotoxin-like properties are important toxic components from some elapid venoms. The mode of action of Micrurus frontalis, M. lemniscatus, M. corallinus and M. fulvius venoms has been investigated in isolated muscle preparations and is here discussed. It is shown that while M. frontalis and M. lemniscatus venoms must contain only neurotoxins that act at the cholinergic end-plate receptor (postsynaptic neurotoxins), M. corallinus venom also inhibits evoked acetylcholine release by the motor nerve endings (presynaptic neurotoxin-like effect) and M. fulvius induces muscle fiber membrane depolarization (cardiotoxin-like effect). The effects produced by M. corallinus and M. fulvius venoms in vivo in dogs and M. frontalis venom in dogs and monkeys are also reported.

Localization of NMDA receptors in the cerebral cortex: a schematic overview

The fundamental role of N-methyl-D-aspartate (NMDA) receptors in many cortical functions has been firmly defined, as has its involvement in a number of neurological and psychiatric diseases. However, until recently very little was known about the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has provided specific tools which have greatly expanded our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have shown that NMDA receptors are preferentially localized on dendritic spines, have disclosed an unknown fraction of presynaptic NMDA receptors on both excitatory and inhibitory axon terminals, and demonstrated that cortical astrocytes do express NMDA receptors. These studies suggest that the effects induced by the activation of NMDA receptors are not due solely to the opening of NMDA channels on neuronal postsynaptic membranes, as previously assumed, but that the activation of presynaptic and glial NMDA receptors may mediate part of these effects

Reversal by methylene blue of tetanic fade induced in cats by nitric oxide

Previous data from our laboratory have indicated that nitric oxide (NO) acting at the presynaptic level increases the amplitude of muscular contraction (AMC) of the phrenic-diaphragm preparations isolated from indirectly stimulated rats, but, by acting at the postsynaptic level, it reduces the AMC when the preparations are directly stimulated. In the present study we investigated the effects induced by NO when tetanic frequencies of stimulation were applied to in vivo preparations (sciatic nerve-anterior tibial muscle of the cat). Intra-arterial injection of NO (0.75-1.5 mg/kg) induced a dose-dependent increase in the Wedensky inhibition produced by high frequencies of stimulation applied to the motor nerve. Intra-arterial administration of 7.2 µg/kg methylene blue did not produce any change in AMC at low frequencies of nerve stimulation (0.2 Hz), but antagonized the NO-induced Wedensky inhibition. The experimental data suggest that NO-induced Wedensky inhibition may be mediated by the guanylate cyclase-cGMP pathway

Identification of the main generator source of longitudinal muscle contraction in the earthworm ventral nerve cord

The main generator source of a longitudinal muscle contraction was identified as an M (mechanical-stimulus-sensitive) circuit composed of a presynaptic M-1 neuron and a postsynaptic M-2 neuron in the ventral nerve cord of the earthworm, Amynthas hawayanus, by simultaneous intracellular response recording and Lucifer Yellow-CH injection with two microelectrodes. Five-peaked responses were evoked in both neurons by a mechanical, but not by an electrical, stimulus to the mechanoreceptor in the shaft of a seta at the opposite side of an epidermis-muscle-nerve-cord preparation. This response was correlated to 84% of the amplitude, 73% of the rising rate and 81% of the duration of a longitudinal muscle contraction recorded by a mechano-electrical transducer after eliminating the other possible generator sources by partitioning the epidermis-muscle piece of this preparation. The pre- and postsynaptic relationship between these two neurons was determined by alternately stimulating and recording with two microelectrodes. Images of the Lucifer Yellow-CH-filled M-1 and M-2 neurons showed that both of them are composed of bundles of longitudinal processes situated on the side of the nerve cord opposite to stimulation. The M-1 neuron has an afferent process (A1) in the first nerve at the stimulated side of this preparation and the M-2 neuron has two efferent processes (E1 and E3) in the first and third nerves at the recording side where their effector muscle cell was identified by a third microelectrode.

Persistent attenuation and enhancement of the earthworm main muscle contraction generator response induced by repeated stimulation of a peripheral neuron

Responses evoked in the earthworm, Amynthas hawayanus, main muscle contraction generator M-2 (postsynaptic mechanical-stimulus-sensitive) neuron by threshold mechanical stimuli in 2-s intertrial intervals (ITI) were used as the control or unconditioned responses (UR). Their attenuation induced by decreasing these intervals in non-associative conditioning and their enhancement induced by associating the unconditioned stimuli (US) to a train of short (0.1 s) hyperpolarizing electrical substitutive conditioning stimuli (SCS) in the Peri-Kästchen (PK) neuron were measured in four parameters, i.e., peak numbers (N) and amplitude ()averaged from 120 responses, sum of these amplitudes (SAMP) and the highest peak amplitude (V) over a period of 4 min. Persistent attenuation similar to habituation was induced by decreasing the control ITI to 0.5 s and 2.0 s in non-associative conditioning within less than 4 min. Dishabituation was induced by randomly pairing one of these habituated US to an electrical stimulus in the PK neuron. All four parameters of the UR were enhanced by forward (SCS-US), but not backward (US-SCS), association of the US with 25, 100 and 250-Hz trains of SCS with 40-ms interstimulus intervals (ISI) for 4 min and persisted for another 4 min after turning off the SCS. The enhancement of these parameters was proportional to the SCS frequencies in the train. No UR was evoked by the SCS when the US was turned off after 4 min of classical conditioning.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.

Differentiation of autonomic reflex control begins with cellular mechanisms at the first synapse within the nucleus tractus solitarius

Visceral afferents send information via cranial nerves to the nucleus tractus solitarius (NTS). The NTS is the initial step of information processing that culminates in homeostatic reflex responses. Recent evidence suggests that strong afferent synaptic responses in the NTS are most often modulated by depression and this forms a basic principle of central integration of these autonomic pathways. The visceral afferent synapse is uncommonly powerful at the NTS with large unitary response amplitudes and depression rather than facilitation at moderate to high frequencies of activation. Substantial signal depression occurs through multiple mechanisms at this very first brainstem synapse onto second order NTS neurons. This review highlights new approaches to the study of these basic processes featuring patch clamp recordings in NTS brain slices and optical techniques with fluorescent tracers. The vanilloid receptor agonist, capsaicin, distinguishes two classes of second order neurons (capsaicin sensitive or capsaicin resistant) that appear to reflect unmyelinated and myelinated afferent pathways. The differences in cellular properties of these two classes of NTS neurons indicate clear functional differentiation at both the pre- and postsynaptic portions of these first synapses. By virtue of their position at the earliest stage of these pathways, such mechanistic differences probably impart important differentiation in the performance over the entire reflex pathways.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

Nitric oxide (NO) is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS) in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC) superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

Reduced hippocampal GABAergic function in Wistar audiogenic rats

Epilepsy is a neurological disorder associated with excitatory and inhibitory imbalance within the underlying neural network. This study evaluated inhibitory γ-amino-butyric acid (GABA)ergic modulation in the CA1 region of the hippocampus of male Wistar rats and Wistar audiogenic rats (aged 90 ± 3 days), a strain of inbred animals susceptible to audiogenic seizures. Field excitatory postsynaptic potentials and population spike complexes in response to Schaffer collateral fiber stimulation were recorded in hippocampal slices before and during application of picrotoxin (50 µM, 60 min), a GABA A antagonist, and the size of the population spike was quantified by measuring its amplitude and slope. In control audiogenic-resistant Wistar rats (N = 9), picrotoxin significantly increased both the amplitude of the population spike by 51 ± 19% and its maximum slope by 73 ± 21%. In contrast, in slices from Wistar audiogenic rats (N = 6), picrotoxin caused no statistically significant change in population spike amplitude (33 ± 46%) or slope (11 ± 29%). Data are reported as means ± SEM. This result indicates a functional reduction of GABAergic neurotransmission in hippocampal slices from Wistar audiogenic rats.

Morphometric analysis of feedforward pathways from the primary somatosensory area (S1) of rats

We used biotinylated dextran amine (BDA) to anterogradely label individual axons projecting from primary somatosensory cortex (S1) to four different cortical areas in rats. A major goal was to determine whether axon terminals in these target areas shared morphometric similarities based on the shape of individual terminal arbors and the density of two bouton types: en passant (Bp) and terminaux (Bt). Evidence from tridimensional reconstructions of isolated axon terminal fragments (n=111) did support a degree of morphological heterogeneity establishing two broad groups of axon terminals. Morphological parameters associated with the complexity of terminal arbors and the proportion of beaded Bp vs stalked Bt were found to differ significantly in these two groups following a discriminant function statistical analysis across axon fragments. Interestingly, both groups occurred in all four target areas, possibly consistent with a commonality of presynaptic processing of tactile information. These findings lay the ground for additional work aiming to investigate synaptic function at the single bouton level and see how this might be associated with emerging properties in postsynaptic targets.