Paradoxical role of GABA in a chronic model of petit mal (absence)-like epilepsy in the rat.

Neonatal Long-Evans hooded rats were treated with AY-9944, a cholesterol biosynthesis inhibitor, every 6 days for 7 weeks to induce a permanent absence-like epileptic condition. AY-9944-treated rats averaged 50 +/- 15 generalized non-motor seizures per hour of 2-15 s duration as monitored by electrocorticography. Clinically effective anti-absence drugs were observed to reduce seizure occurrence in a dose-dependent manner. Paradoxically, GABA agonists increased seizure occurrence while GABA antagonists decreased seizure occurrence. Evaluation of the benzodiazepines, diazepam and clonazepam, in this model revealed inhibition of seizure activity by GABA-independent mechanisms. Valproic acid produced a biphasic effect suggesting a GABA-independent, antiabsence action at low doses and GABAergic augmentation of seizure occurrence at higher doses. The results of this study support the hypothesis that increased GABAergic stimulation may induce inhibitory seizures in absence epilepsy.

Are postsynaptic nicotinic end-plate receptors involved in lead toxicity?

The effects of Pb2+ on the postsynaptic nicotinic end-plate receptor were examined in the perfused mouse hemidiaphragm preparation. Postsynaptic nicotinic responses, evoked by pressure ejection of ACh, were blocked by Pb2+ in a transient way. After 9-12 min of exposure to 1 microM Pb2+ the amplitude of the depolarization induced by 1 mM ACh was reduced to 39.5 +/- 11% of the control value. During continued exposure to Pb2+ this blocking effect was reversed and after 30 min of exposure to 1 microM Pb2+ the amplitude of the ACh-induced depolarization had returned to the control value. The amplitude and the frequency of miniature end-plate potentials were not altered in the presence of 1 microM Pb2+. Under voltage clamp conditions the effects of Pb2+ on the ACh-induced inward current were similar to those of Pb2+ on the ACh-induced depolarization. After 12 min of exposure to 1 microM Pb2+ the inward current induced by 1 mM ACh was reduced to 44% of the control value and after 30 min the ACh-induced inward current had recovered to 94% of the control value. It is concluded that, in addition to the generally established mechanism of action of Pb2+ at the muscle end-plate, Pb2+ blocks the postsynaptic nicotinic receptor-mediated response at a relative low concentration. The contribution of these postsynaptic effects to the neurotoxic symptoms of Pb2+ remains to be established.

Adaptation of a perfused rat hemidiaphragm preparation to the study of intermediary metabolism.

Glucose catabolism of a vascular perfused rat hemidiaphragm was determined at rest and during stimulation of the phrenic nerve with trains of either 5 (T5) or 15 (T15) pulses (20 msec intervals) per second. Tissues were perfused and bathed in HEPES-buffered medium containing 11 mM D-[U-14C, 5-3H]glucose, equilibrated with 100% O2. Resting glucose catabolism via the Emden-Meyerhof pathway was indicated by a 3H2O production rate per hemidiaphragm of 1.45 +/- 0.07 mumol/h, of which 47% was recovered as [14C]lactate with the remainder assumed to be metabolised by mitochondria. During the first 30 min of T5 and T15 stimulation, peak isometric tension declined from an initial value of 105 +/- 8 g by 54% and 79%, respectively. The resulting peak tensions of 48 and 22 g remained constant for the next 60 min. These tensions were associated with linear rates of 3H2O production of 2.93 +/- 0.41 and 2.84 +/- 0.25 mumol/h. Stimulation by T5 and T15 increased mitochondrial metabolism of glucose by 64% and 95%, respectively, with no significant alterations in lactate formation from either exogenous or endogenous sources. The results suggest that the initial decline in tension is due to fatigue of the fast anaerobic myofibers; whereas, the sustained force beyond 30 min is attributable to the mitochondrial-rich slow myofibers.

Anti-curare effect of plasma from patients with thermal injury.

Following severe thermal injury, patients are resistant to non-depolarizing muscle relaxants. Although this resistance has been well documented clinically, little is known about its etiology. We have tested the hypothesis that circulating factors contribute to the decreased potency of neuromuscular blockers following burns. The potencies of d-tubocurarine (2 microM) or pancuronium (2 microM) dissolved in plasma from either burned or control human subjects were tested on the indirectly stimulated (0.2 Hz) rat phrenic nerve-hemidiaphragm preparation. The muscle relaxants produced less neuromuscular blockade when dissolved in plasma from burned patients than when they were dissolved in plasma from controls. Thus, circulating factors are involved in the decreased potency of non-depolarizing neuromuscular blocking drugs.

A comparison of chemiluminescent and radioenzymatic methods for the measurement of acetylcholine released from a rat phrenic nerve-hemidiaphragm preparation.

A chemiluminescent assay coupled to a periodide extraction method is described for the measurement of acetylcholine release from the vascular perfused rat phrenic nerve-hemidiaphragm preparation. A direct comparison of the chemiluminescent assay with an established radioenzymatic assay for acetylcholine demonstrates that the two assays are quantitatively equivalent and yield similar limits of sensitivity of approximately 2 pmol, and that the periodide extraction/chemiluminescent assay method is more consistent than the tetraphenylboron extraction/radioenzymatic assay method. Additionally, cholinergic drug interference with the chemiluminescent assay is minimal. The absence of radioactivity and the reduced cost of the chemiluminescent assay make it an attractive alternative to the radioenzymatic assay.

Frequency-dependence of neuromuscular blockade in the presence of d-tubocurarine.

The frequency-dependence of neuromuscular blockade in the presence of d-tubocurarine (dTC) was studied in the vascular perfused rat phrenic nerve-hemidiaphragm preparation by intracellular recording. Endplate potential (EPP) amplitude decreased as the frequency of stimulation increased (2.5-40 Hz). Additionally, the slopes of the regression lines of EPP amplitude versus the log of stimulation frequency decreased when the dTC concentration was increased. Since the dependence of neuromuscular blockade on frequency was not enhanced by higher concentrations of dTC, the postjunctional channel-blocking hypothesis does not sufficiently explain frequency-dependent neuromuscular blockade in the presence of dTC. However, the present results are compatible with the hypothesis that dTC influences neuromuscular blockade via a prejunctional action, affecting acetylcholine release.

Botulinum toxin prevents stimulus-induced backfiring produced by neostigmine in the mouse phrenic nerve-diaphragm.

The origin of motor nerve antidromic activity (backfiring) induced by anticholinesterase treatment was examined in the mouse phrenic nerve-hemidiaphragm preparation. Botulinum toxin was used to determine whether backfiring is due to (a) a direct effect of the cholinesterase inhibitor on the nerve terminal, or (b) an indirect effect via the prolongation of the action of acetylcholine. In previously untreated control preparations, neostigmine produced spontaneous and stimulus-induced antidromic activity in the phrenic nerve when rapidly introduced into the diaphragm via its vasculature. This activity could be reversibly blocked by d-tubocurarine and decamethonium, but not by atropine. Neostigmine-induced backfiring did not occur in preparations in which transmitter release was blocked with botulinum toxin. Infusion of a small bolus of a high concentration of acetylcholine following neostigmine treatment resulted in a short-term increase in the incidence of antidromic activity, followed by block, in both controls and botulinum toxin-treated preparations. It is concluded that transmitter release is necessary for the production of backfiring following cholinesterase inhibition since neostigmine alone does not elicit antidromic activity in botulinum toxin-treated preparations at concentrations which are effective in controls. Our results support the hypothesis that the effects of neostigmine on the motoneurone terminal are mediated by the prolonged action of acetylcholine that occurs with inhibition of acetylcholinesterase.

Diisopropylfluorophosphate inhibits choline efflux from the perfused rat hemidiaphragm.

Acetylcholine (ACh) synthesis by motor nerve terminals requires an adequate supply of its precursor, choline. The results reported here show that diisopropylfluorophosphate (DFP), an acetylcholinesterase inhibitor commonly used in ACh release studies, reduces the rate of endogenous choline efflux from the perfused rat hemidiaphragm. Perfusion of the isolated hemidiaphragm with 10 microM or 100 microM DFP reduced choline efflux by 39% and 69% respectively. DFP administration to rats (6 mg/kg) also lowered the in vitro release of choline by 33%. The rate of ACh release from hemidiaphragm preparations perfused with DFP was significantly lower than the rate of release from preparations perfused with physostigmine, an acetylcholinesterase inhibitor which had no effect on choline efflux. The addition of choline (10-30 micron) to the perfusion medium restored the rate of ACh release from DFP-treated hemidiaphragms but did not further elevated ACh release from physostigmine-treated preparations. These results demonstrate that DFP inhibits choline efflux from the isolated hemidiaphragm and further suggest that, by limiting the availability of choline for ACh synthesis, DFP reduces the rate of ACh release.

Axonal transport of alpha-bungarotoxin binding sites in rat sciatic nerve.

[125I]alpha-Bungarotoxin (alpha-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [125I]alpha-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [125I]alpha-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature. D-Tubocurarine, nicotine, decamethonium and atropine displaced [125]alpha-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [125I]alpha-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.

Histopathology of spinal cord, peripheral nerve, and soleus muscle of rats treated with triethyltin bromide.

Mammalian exposure to triethyltin (TET) leads to severe impairment of motor function. Spinal cord, rootlets of spinal nerve, sciatic nerve, and soleus muscle of TET-treated rats were examined to assess their involvement. Adult rats were exposed to TET bromide in drinking water (30 mg/liter) for 3 weeks. The spinal cord became edematous with vacuolar formation in the myelin of white and gray matter. Chromatolysis occurred in motor neurons of the ventral root. Myelin of ventral and dorsal rootlets of spinal nerve was also affected with ventral rootlets showing greater involvement. Minimal effect was seen in myelin of sciatic nerve. Soleus muscle contained atrophic myofibers and, after 3 weeks recovery, type-grouping of fast-twitch myofibers. Chromatolysis and type-grouping were regarded as indications of a degenerative effect of TET-treatment on peripheral axons. We conclude that the neuromuscular toxicity of TET has a myopathic and neuropathic component and that the involvement of the neuronal cell body and the denervation of myofibers may contribute to the peripheral motor dysfunction.

Responses of in vitro rat diaphragm to changes in acid-base environment.

In vitro rat diaphragms initially demonstrated a decrease in the force of the twitch contraction (FC) in response to field electrode stimulation when exposed to an unbuffered increase in PCO2 (UIPCO2). These diaphragms tended to regain their initial FC upon addition of a beta-agonist even while the increased PCO2 perdured. The effect of the agonist could be reversed by propranolol. Four hemidiaphragms were bathed in a medium containing curare and exposed to UIPCO2. Their tension values were compared to the opposite sides bathed without curare and exposed to UIPCO2 of the same intensity and duration. There was no statistically significant difference in the response. Subsequently 10 rat diaphragms were each systematically challenged by UIPCO2, buffered increases in PCO2 (BIPCO2), unbuffered decreases in bicarbonate (UDHCO3), and buffered decreases in bicarbonate (BDHCO3) first without and then with isoproterenol (10(-6) M). Without isoproterenol all four challenges after 15-min exposure produced a decrease in FC, the least by BIPCO2; the largest, by UDHCO3. Upon addition of isoproterenol, FC actually increased during BIPCO2; the decreases in FC in response to UIPCO2 and UDHCO3 were abolished; the FC in response to BDHCO3 was still decreased, but less severely. The effect of the isoproterenol was not due to its following the four challenges without isoproterenol. The different magnitudes in the FC response and the presumed lack of uniform change in intracellular pH during the four challenges suggest the possibility that different components in the sarcolemma, or in the excitation-contraction coupling mechanisms responsible for the genesis of the FC are affected by the four challenges, but the nerve or neuromuscular junction may also be affected.

Neuromuscular function and organotin compounds.

Mammalian exposure to toxic levels of the trialkyltin compounds, triethyltin (TET) and trimethyltin, results in pathological manifestations largely restricted to the nervous system. The remarkable features of TET toxicity are cerebral edema and muscular weakness. Rats exposed orally to TET (10-30mg TET Br/l of drinking water) progress through an increasingly compromised state beginning with mild ataxia and hindlimb weakness after one week, spastic paresis of the hindlimbs by two weeks; and hindlimb paraplegia and sensory changes by 3 weeks. Histopathological studies of chronic TET-exposed rats report minimal ultrastructural damage to distal peripheral nerves, myelin, and muscle. Chromatolytic reactions are observed in some alpha motor neurons; intramyelinic vacuolization in the ventral roots and horn is substantial by 3 weeks. Myelin vacuolization and degeneration are observed to a lesser extent in the dorsal roots of the spinal cord. Wet weights and myofiber diameters of EDL and soleus muscles are reduced during chronic TET intoxication, but no histopathology is evident using light microscopy. Conduction of compound action potentials in vivo along distal sensory fibers, ventral roots and distal motor fibers (in sciatic n.) is normal in 3 week TET rats as compared to control; however, nerve conduction velocity is decreased in the segment of the H-reflex arc involving the dorsal roots. Earlier studies by Stoner and coworkers led to suggestions that the neuromuscular junction may be preferentially affected by TET and could contribute, in part, to the symptoms of muscular weakness. In support of this hypothesis preliminary studies from our laboratory and others indicate that neurotransmission is functionally depressed at the myoneural junction following chronic TET treatment in vivo or when applied in vitro to isolated muscle preparations. Stimulated, but not unstimulated, release of acetylcholine from the vascular perfused rat phrenic nerve-hemidiaphragm preparation is decreased by TET especially at higher stimulation rates (20 Hz). In vitro administration of TET Br (10(-6)M) results in an irreversible decrease in the amplitude of evoked endplate potentials; chronic in vivo exposure to TET causes a decrease in the resting membrane potential of soleus muscle (in situ recordings) and provokes a peculiar post-stimulus (200Hz bursts) elevation of spontaneous miniature endplate potentials in isolated cut diaphragm preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro assessment of neuromuscular toxicity.

This report has presented an overview of my approach to assessing neuromuscular toxicity using primarily in vitro techniques. Our current work on triethyltin toxicity exemplifies the use of these in vitro methodologies and the data they yield. Our neurophysiological and neurochemical experiments along with reports in the literature have directed our attention to several possible mechanisms for explaining the myopathic and neuropathic manifestation of TET intoxication. These mechanisms include (a) a compromise in the bioenergetic capacity of neurons and/or myofibers via disruption in mitochondrial function and (b) the direct effect of TET on the cell membrane and/or Na+-K+-ATPase. These hypotheses are currently being explored using additional biochemical and electrophysiological techniques in the isolated vascular perfused phrenic nerve-hemidiaphragm.