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Neurofilament proteins (NFs) are made by co-polymerization of three intermediate filament proteins, NF-L, NF-M and NF-H and constitute the most abundant cytoskeletal element in large myelinated axons. NFs have a well-established role as intrinsic determinants of axon caliber with all the functional implications, but the role of each individual NF subunit is much less clear. The aim of our study was to examine functional properties of large myelinated axons with altered morphology from mice bearing a targeted disruption of each NF genes (NF-L -/-, NF-M-/- and NF-H -/- mice). Membrane properties, action potentials and single axon refractory period were measured in isolated sciatic nerves in vitro, using intra-axonal microelectrode recording in conjunction with current-clamp technique. Some results were obtained from whole nerves by sucrose-gap recording. The NF-knockout mice showed several deficits in physiological properties of low-threshold fibers. In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L -/- and NF-M -/- transgenic animals (control, 39.9+/-1.8 m/s, NF-M -/-; 23.5+/-1. 4 m/s, and NF-L-/-; 12.0+/-0.7 m/s, mean+/-S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22-26 degrees C). However, in spite of their preserved caliber, large myelinated axons in NF-H -/- mice also showed a significant decrease in conduction velocity (22.8+/-1.0 m/s, mean+/-S.E.M.). Although action potential amplitudes, duration and shape did not differ between control axons and transgenic animals, the refractory period was prolonged in NF-H -/- and NF-M -/- animals. Intracellular injections of 200 ms depolarizing and hyperpolarizing currents revealed outward and inward rectification in all animal groups. In comparison to control animals, NF-H -/- mice expressed a significant decrease in outward rectification. Potassium channel blockers (4AP and TEA) and cesium ions were able to block outward and inward rectification in all myelinated axons in qualitatively the same manner. These results suggest that NF-H may have a specific role in modulating ion channel functions in large myelinated fibers.

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Neurofilaments (NFs; made by copolymerization of three intermediate filament proteins NF-L, NF-M, and NF-H, for light, medium, and heavy) constitute the most abundant cytoskeletal structure in large myelinated axons. The presence of aberrant NF accumulation has been associated with neurodegenerative diseases (such as ALS). The possible causal role of NF in neurodegeneration has been supported by studies on recently available transgenic mice in which expression of human NF-H (hNF-H +/+) leads to overt neuropathy. We have examined electrophysiological properties of myelinated axons in hNF-H +/+ mice using intraaxonal microelectrode recording from isolated sciatic and tibial nerves. Transgenic mice showed several deficits in physiological properties of low threshold myelinated fibers: conduction velocity and resting membrane potential were significantly decreased (20 +/- 1.6 vs 40 +/- 2 m/s; -71.3 +/- 0.9 vs -75.5 +/- 0.5 m/s; mean +/- SE; n = 25; 22 degrees C). While the amplitude of action potentials was of comparable size (82 +/- 5 vs 86 +/- 3 mV) duration of action potential (at half-amplitude, AP/2) in hNF-H +/+ was significantly prolonged (0.82 +/- 0.02 vs 0.65 +/- 0.02 ms). Voltage-current properties of axonal membrane indicate a significant decrease in inward and outward rectification. Occasionally, impaled axons of hNF-H +/+ showed membrane oscillations and repetitive activity (reminiscent of fasciculations) never observed in normal animals. These results are compatible with an imbalance between ion conductances in axons from transgenic animals (an increase in Na(+) and a decrease in K(+) conductances), in agreement with recent suggestion based on clinical studies on ALS patients (H. Bostock et al., 1995, Brain 118, 217-225). One may hypothesize that these changes could contribute to neurodegenerative processes (i.e., via an increase in [Na(+)](i)), as well as clinical symptoms (fasciculations) observed in patients with degenerative motor neuron diseases.

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We studied conduction velocity in peripheral nerves and the block of synaptic transmission produced by lack of glucose in hippocampal slices from 4- and 12-month-old streptozotocin-induced diabetic rats and their age-matched controls. In sural nerves of young and old diabetic rats, the conduction velocity was reduced by 30-35%. In slices from young diabetics, CA1 synaptic transmission was more sensitive to aglycemia than in control slices. However, all slices from older rats showed comparable increases in CA1 synaptic sensitivity to aglycemia. We conclude that the cerebral adaptation to diabetic hyperglycemia apparent in the hippocampus of young rats is masked in older rats by an age-dependent increase in sensitivity to lack of glucose.

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After blockade of the voltage-dependent potassium conductances by intracellular application of 4-aminopyridine and tetraethylammonium in frog myelinated axons, a set of brief (0.1 ms) intracellular depolarizing pulses or a long (200 ms) depolarizing pulse evoked a train of action potentials. Under both experimental conditions a hyperpolarizing afterpotential appeared (duration 367 ms +/- 34, mean +/- S.E., n = 15). The purpose of this study was to investigate the properties of this hyperpolarizing afterpotential. It was found that the hyperpolarizing afterpotential increases in amplitude with: (1) the number of sodium-dependent action potentials; (2) action potential broadening (following potassium channels blockade); and (3) the level of depolarization during a current step. Application of tetrodotoxin prevented the activation of the hyperpolarizing afterpotential by any of the above stimuli. The hyperpolarizing afterpotential was unaffected by: (1) 8-acetyl-strophanthidin, an agent that poisons the electrogenic pumping in the axon; (2) blocking calcium influx with extracellular 10 mM magnesium or 2 mM manganese; and (3) buffering of the intracellular calcium, using EGTA in the recording microelectrode. Extracellular application of tetraethylammonium, but not 4-aminopyridine, reduced the hyperpolarizing afterpotential. The hyperpolarizing afterpotential reversed at >> -92 mV. Increasing the external potassium concentration from 2 to 10 mM shifted the reversal potential +14.5 mV, indicating that the hyperpolarizing afterpotential is a potassium mediated conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

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Intracellular microelectrode recordings were used to examine the effects of the potassium channel blockers: 4-aminopyridine, a selective blocker of fast potassium conductances gKf1 and gKf2,13 and tetraethylammonium, a blocker of gKf1, gKf2 and the slow conductance gKs,13 on the repetitive activity of large myelinated axons of frog. The blockers were applied intracellularly by diffusional leak of the agents from the recording microelectrode containing either 4-aminopyridine or a mixture of 4-aminopyridine and tetraethylammonium. A decrease in outward rectification, a measure of the block of the potassium conductances, was evident within 5 min of axon impalement. Within 30 min 80% of maximal blockade was observed during prolonged recording sessions (> 1 h). Parallel with the resistance increase, the action potential duration increased (up to 5 ms). This was attributed to the block of gKf2. The excitability regularly increased, manifested as a train of action potentials (a decrease in accommodation) for a maximum of 200 ms (54 +/- 8 vs 111 +/- 22, 4-aminopyridine vs 4-aminopyridine-tetraethylammonium, respectively, n = 8 and 6, P < 0.006). The presence of 4-aminopyridine-tetraethylammonium in the microelectrodes decreased the spike frequency adaptation (the instantaneous action potential frequency per spike interval number) observed in fibres treated with 4-aminopyridine alone (32 +/- 9 vs 7 +/- 1 Hz; 4-aminopyridine vs 4-aminopyridine-tetraethylammonium, n = 8 and 6, P < 0.04). This effect was attributed to block of gKs by the tetraethylammonium.(ABSTRACT TRUNCATED AT 250 WORDS)

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Cerebral electrical stimulation (CES), born from research on electroanesthesia in the seventies, consists of the application of a pulsating current of small intensity (usually less than 1 mA, and below the threshold of perception) through the skull, e.g., in daily 30-min sessions. Claims of biological effectiveness (neurochemical, hormonal and EEG changes, naloxone-reversible analgesia in rats, etc.) and of clinical effectiveness (anxiety, depression, cognitive functions in alcoholics) have often relied on poorly controlled data. A recent controlled study in the treatment of opiate withdrawal has been positive. The present double-blind controlled study compares active CES with sham stimulation in 64 alcohol-dependent males. Over 4 weeks, both treatment groups improved significantly in most aspects. In the active treatment group additional significant improvement was observed in week-end alcohol consumption, and in two psychological measures: depression and stress symptoms index, but not in general drinking behavior.

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Characteristics of voltage-dependent currents in cultured frog Schwann cells were investigated by the whole-cell clamp technique. An inward current was detectable at a membrane potential level more positive than -50 mV and reached a maximum value at about -10 mV, while no rectifying channel was present. The inward current was carried by Na+ ions, because the extrapolated reversal potential of the current agreed with the calculated ENa, and the current was sensitive to tetrodotoxin. The membrane potential for half-maximal inactivation was -82 mV. The inactivation curve indicated that more than 90% of the Na+ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (-10 mV, 13 degrees C). The electrophysiological characteristics of the Na+ current in the cultured frog Schwann cells were compared with those in other tissues. This Na+ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half-maximal inactivation potential and in the slower inactivation time course.

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1. Intracellular microelectrode current-clamp technique was used to study the steady-state membrane properties of single intact large primary afferent axons (conduction velocity > 10 m/s) attached to isolated hemisected frog spinal cord. 2. Hyperpolarizing electrotonic potentials (ETPs) had a slow complex multiphasic charging. This complex charging could be approximated by two time constants: one in the range of 70-210 ms, the other of < 20 ms. 3. Two regions of outward and inward rectification hyperpolarized to the resting membrane potential were observed, in addition to the previously characterized outward rectification active at potentials depolarized to resting membrane potential. The peak and steady-state input resistance of these axons in tetrodotoxin Ringer solution was on average 65.6 +/- 21.1 and 31.1 +/- 10.8 M omega, mean +/- SE, respectively. 4. Application of external tetraethylammonium (10-20 mM) significantly depolarized the axon and decreased the outward rectification just hyperpolarized to the resting membrane potential. This outward rectification could also be blocked by external barium ions (2-10 mM). 5. Activation of an inward or anomalous rectification in these axons was observed 300-600 ms after the start of a current pulse. In addition, a depolarizing afterpotential (DAP) (1-3 mV in amplitude, 500 ms-10 s in duration) was evident after a current pulse in which inward rectification had been activated. This DAP most likely reflected the slow inactivation of the inwardly rectifying conductance. 6. Inward rectification was blocked by external application of cesium ions (1-3 mM) but it was insensitive to external application of barium ions (2-10 mM). The blockade of the voltage attenuation was accompanied by a disappearance of the DAP and an increase in the charging time constant of the axon. This blockade resulted in a single linear voltage-current (V-I) relationship. Axons now had, on average, an input resistance of 114 +/- 19.1 M omega. 7. Reducing the concentration of external potassium ions increased both the peak and steady-state slope resistance. Reducing the external sodium concentration altered the ETPs and the V-I relationship little but it consistently reduced the magnitude and length of the DAP. These results are compatible with the hypothesis that anomalous rectification is a mixed ionic conductance dependent on potassium and sodium ions in the external media. 8. Overall, the V-I relationship of these intact axons had both linear and nonlinear regions reflecting the activity of numerous slowly activating and inactivating conductances. (ABSTRACT TRUNCATED AT 400 WORDS)

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The Intensive Care Unit is the area in patient care where the amount of patient data from a variety of sources is particularly large. The problem for clinicians lies in the ability to gather, and use these data in the decision making process. A well designed computer based patient data management system, incorporating a variety of data analysis tools, would have a dramatic impact in patient care in an environment such as this. The PDB System has been in continuous use at the Montreal General Hospital's Surgical and Trauma Intensive Care Unit since Jan. 88. Its initial implementation in two beds in our SICU has allowed the complete replacement of the conventional patient paper record. It is used by all ICU staff, including nurses, physicians, and ward clerks for the recording/viewing of all patient vital data, laboratory data, medications, and optionally chart notes. In addition, medical staff has the option to use the entered data to perform a variety of data analysis procedures.

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Baclofen has been shown to be a selective agonist for a subclass of GABA receptors (GABAB) in many regions of the vertebrate nervous system. On the intraspinal terminals of dorsal roots (DRT), it evokes a pure hyperpolarizing response. We have previously shown that the response of DRT to GABA and some of its analogs (e.g. kojic amine) in isolated frog spinal cord is dual in nature, consisting of a bicuculline-sensitive depolarizing component and a bicuculline-resistant hyperpolarizing component. Under the working hypothesis that the hyperpolarizing component of the GABA-evoked response is mediated by the activation of GABAB receptors, we have examined, using the sucrose gap technique, some characteristics of the response of DRT to baclofen. We have found that this response is stereospecific (L-baclofen being about 100 times more potent than D-baclofen), dependent on [K]o (response amplitude inversely related to [K]o), blocked by barium (0.5 mM causing a reduction of the response amplitude to 37% of control), and is not significantly affected by 4-aminopyridine, nor by inorganic calcium channel blockers (manganese, cobalt, cadmium). Some proposed GABAB antagonists (delta-aminovaleric acid, delta-aminolaevulinic acid, phaclofen) are also rather ineffective at blocking it. These results are therefore consistent with the notion that the baclofen-evoked response of DRT is mediated by an increase in conductance to potassium ions.

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1. Intracellular microelectrode recordings from large sensory and motor myelinated axons in spinal roots of Rana pipiens were used to study the effects of dendrotoxin (DTX), a specific blocker of a fast activating potassium current (GKf1). 2. Dendrotoxin reduced the ability of myelinated sensory and motor axons to accommodate to a constant stimulus. A depolarizing current step, which normally evoked only one action potential, after dendrotoxin treatment (200-500 nM) produced a train of action potentials. These spike trains lasted 29 +/- 2.8 (SE) ms on average in sensory fibers (n = 18) and 40.2 +/- 4.5 ms in motor fibers (n = 9). 3. After dendrotoxin treatment, in addition to a reduction in the ability to accommodate to a constant stimulus, a slowing in the rate of action potential generation was evident (spike frequency adaptation). 4. Dendrotoxin had no effect on the rising phase of conducted action potentials evoked by peripheral stimulation. Together with a lack of effect on the absolute refractory period, these results indicate that dendrotoxin does not affect sodium channel activity. 5. The steady-state voltage/current relationship was unchanged in response to hyperpolarizing current pulses; however, there was a significant increase in cord resistance in response to depolarizing current steps, demonstrating that DTX decreases outward rectification. 6. A computer model based on Hodgkin and Huxley equations was developed, which included the three voltage-dependent potassium conductances described by Dubois. The model reproduced major experimental results: removal of the conductance, termed GKf1, reduced the accommodation in the early phase of a continuous stimulus, indicating that this current could be responsible for the early accommodation. The hypothesis that the slow potassium conductance GKs regulates late accommodation and action potential frequency adaptation is also supported by the computer model. 7. In summary, these results suggest that in amphibian myelinated sensory and motor axons, the activity of potassium conductances can account for accommodation and adaptation without involvement of sodium conductance activity.

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It has been reported that 6-aminomethyl-3-methyl-4H,1,2,4-benzothiadiazine-1, 1-dioxide (AMBD, TAG) is a specific blocker of taurine and beta-alanine responses in the central nervous system. We have re-examined the effect of AMBD on amino acid and synaptically evoked responses recorded from isolated hemisected frog spinal cords by means of the sucrose gap technique. When indirect responses were blocked by adding tetrodotoxin (0.2 microM) or manganese chloride (2 mM) to the normal Ringer solution, AMBD (0.01-0.5 mM) selectively antagonized taurine, beta-alanine, hypotaurine and kojic amine evoked depolarizations of primary afferents at their intramedullary part (dorsal root terminals, DRT) and on dorsal root ganglia (DRG), without significantly affecting responses to glutamate (on DRT), glycine (on DRT) or GABA (on DRT and DRG). Depolarizing responses to taurine and beta-alanine (1 mM) were depressed by up to 50% with 0.1 mM AMBD and often completely antagonized with 0.25 mM AMBD. In normal Ringer solution, AMBD selectively antagonized the dorsal root potential evoked by ventral root stimulation (VR-DRP, threshold at 0.02 mM AMBD, 90% block with 0.25 mM); other synaptic potentials increased in duration and/or amplitude, demonstrating a strong convulsant effect of AMBD. Thus, the depolarizing responses of taurine, beta-alanine and hypotaurine on primary afferents are pharmacologically indistinguishable from the VR-DRP. These results are in agreement with the proposal that taurine or a taurine-like substance (possibly beta-alanine or hypotaurine) is the mediator of VR-DRP in amphibian spinal cord.

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The mechanism of primary afferent depolarization (PAD) was studied in the isolated frog spinal cord using intrafibre recording (microelectrodes filled with 0.6 M potassium sulfate) from large myelinated axons of dorsal roots. Standard current-clamp technique was used to obtain voltage-current (V-I) relationship. It was found that: (i) PAD is voltage dependent: its amplitude and rate of rise are increased with hyperpolarization; (ii) the slope of the linear part of the V-I curve obtained during PAD is decreased compared with the V-I curve at rest; (iii) the PAD equilibrium potential, estimated by extrapolation, ranged from -66 to -40 mV. These results suggest that PAD is associated with an increase in conductance of primary afferent terminals and thus seem to provide the first experimental evidence for the hypothesis that shunting of primary afferent membrane is the mechanism of presynaptic inhibition in the vertebrate nervous system.

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Drug responses of isolated hemisected frog spinal cords were examined by means of the sucrose-gap technique. The glutamate-induced depolarizations (glu-d) of motoneurones (recorded from ventral roots), and primary afferents (recorded from dorsal roots), were followed by an afterhyperpolarization (glu-a.h.). The depolarization induced by DL-homocysteic acid (DLH) was only occasionally followed by an afterhyperpolarization (DLH-a.h.). The glu-a.h. on both roots persisted in the presence of tetrodotoxin (TTX, 0.1-1 microM), or Ringer solution containing 10 mM-Mg2+; 0.1 mM-Ca2+ or 2 mM-Mn2+; 0.2 mM-Ca2+. This indicated that the response was neither due to the release of endogenous neurally active substances nor to the activation of a Ca2+-sensitive K+ conductance. The glu-a.h. was reduced or blocked by K+-free Ringer solution, 3-acetylstrophanthin (3-Ac-Str; 1 microM) or Li+ ions, and was therefore attributed to the activity of the electrogenic Na+ pump. The duration of depolarization induced by glu or DLH was increased in the presence of K+-free Ringer solution, 1 microM 3-Ac-Str or Li ions. It is therefore suggested that the electrogenic Na+ pump may play a role in limiting the duration of depolarization induced by the action of excitatory amino acids. The re-admission of K+ ions to preparations which had been incubated in K+-free Ringer solution produced a transient hyperpolarization (K-a.h.) of the membrane potential of ventral roots which is also attributable to the activation of the electrogenic Na+ pump. Both the K-a.h. and the glu-a.h. were enhanced in Ca2+-free Ringer solution. It is therefore suggested that the Ca2+ ions may modulate the activity of the electrogenic pump in central nervous tissue.

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In rats under urethane anesthesia, a twin set of multibarreled micropipettes was used to record simultaneously in situ the somatic population spike, the positive field and the dendritic negative field (EPSP) evoked by commissural stimulation, and to apply microiontophoretically GABAergic and cholinergic agents at each level. Relying on the typical somatic responses evoked by local application of GABA and acetylcholine (ACh), an electrode can be positioned in the pyramidal layer with good accuracy (in the range of 50 microns) and the pharmacology of the excitatory drive impinging upon the apical dendrite concomitantly investigated.

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Explant cultures from adult bullfrog sympathetic ganglia can be maintained in vitro for several months and are suitable for electrophysiological recording. The cultured neurons display morphological, electrophysiological and pharmacological characteristics similar, in most respects, to those reported for acutely isolated sympathetic ganglia. Individual cells were visualized by Nomarski optics and impaled with a glass micropipette, which was used for voltage recording and current injection. The average specific membrane properties, calculated from cell dimensions and responses to current injection, were Vm = -46 mV, Rin = 27 M omega, Rm = 1665 omega cm2, tau m = 5 msec, and Cm = 3.2 microF/cm2. Bath perfusion of the cholinergic agonist muscarine depolarized most neurons with an increase in input resistance, while carbachol depolarized neurons with both increases and decreases in input resistance. GABA depolarized all neurons tested with a decreased resistance. High concentrations of catecholamines (2-5 mM) generally hyperpolarized explanted neurons, usually in association with an increased resistance. Extracellularly or intracellularly applied cyclic AMP and two other analogues produced weak and inconsistent hyperpolarizations. In contrast, perfusion or iontophoresis of most non-cyclic purine and pyrimidine nucleotides markedly depolarized most neurons in association with an increased input resistance. UTP and UDP were most potent, revealing threshold concentrations of about 10(-8) to 5 x 10(-8) M. The related nucleosides were largely ineffective. The nucleotide-evoked depolarizations were similar to the muscarine responses but were not blocked by atropine. These results suggest that the purine or pyrimidine nucleotides should be considered for a possible involvement in neurotransmission in sympathetic ganglia.

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1 Dendrotoxin (DTOX)6, 6a and 5,6-1, fractions of the venom isolated from the green mamba (Dendroaspis viridis) promoted both spontaneous and stimulus-coupled rhythmic activity and antagonized the cholinergically mediated ventral root-dorsal root potential (VR-DRP) of frog spinal cord. The different time course and reversibility of these two effects indicates that the toxin has two entirely separate sites of action on the frog spinal cord. 2 Since DTOX 6 neither blocked nor enhanced responses of ventral and dorsal roots to glutamate, gamma-aminobutyric acid (GABA), beta-alanine, glycine or aspartate, it is unlikely that its convulsant action resulted from an alteration of the postsynaptic actions of inhibitory or excitatory amino acids. 3 An alteration in the threshold for action potential generation could perhaps contribute to the convulsant action of DTOX 6, although other mechanisms such as blockade of the release of unspecified inhibitory substances cannot be excluded. 4 In addition to the lack of effect on amino acid responses, DTOX failed to block the polysynaptic DR-VRP or DR-DRP pathways, which are mediated at least in part by amino acid neurotransmitters. Although this would be consistent with a specific action of DTOX at the cholinergic synapse of the VR-DRP pathway, this site of action has not yet been demonstrated unequivocally. Other possible mechanisms whereby DTOX could block VR-DRP are discussed.

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