Search

To reduce the recurrence of keratocysts, tanning of the epithelial lining with modified Carnoy's solution has been advocated as an ancillary procedure. This agent has occasionally been reported to induce long-lasting local neurotoxicity, when the inferior alveolar nerve (IAN) was located within the bony cavity of larger cysts. As the severity of the neurologic damage depends on the tissue penetration of the solution, a critical exposure time must be assumed. To substantiate this hypothesis, rabbit IANs were decorticated over an approximate length of 1 cm and soaked with modified Carnoy's solution for periods from 30 seconds to 10 minutes. Sensory nerve function was monitored using somatosensory evoked potentials. Exposures up to 2 minutes did not result in any electrophysiologic abnormality. Exposure for 3 minutes led to either normal or rudimentary evoked potentials. After exposure of 5 minutes, and invariably after 10 minutes, the evoked potentials from the IAN were absent. Nerve segments were removed for histologic examination and the penetration depth of the Carnoy's solution was identified by staining with the Berlin-blue reaction. The involved areas were morphometrically evaluated and they reflected the electrophysiological findings. Transmission electron microscopy showed morphologic changes confined to the outer nerve sheaths (epineurium and perineurium) after exposure of 3 minutes. Exposure of 5 minutes and longer resulted in involvement of both the nerve sheaths and their axonal contents, with disruption and disintegration of the neural tissue. This study clearly supports the hypothesis that contact of a peripheral nerve (ie, IAN) with Carnoy's solution carries a time-related risk to produce acute sensory impairment.(ABSTRACT TRUNCATED AT 250 WORDS)

The beta-2 agonist clenbuterol was tested for its effect on the proliferation of satellite cells in transplanted skeletal muscles. Using autoradiographic techniques it was found that satellite cells in clenbuterol treated transplants began proliferating earlier than in control animals. The effect of clenbuterol on the growth of regenerating muscle fibres was also examined using morphometric techniques, which manifested itself as hypertrophy of the fibres when compared with the controls.

In voltage clamp experiments on the frog Ranvier node, the specific protein reagent, N-bromacetamide, significantly decelerates the sodium inactivation kinetics and makes it incomplete. After treatment with N-bromacetamide, both fast and slow inactivation time constants are increased and the proportion of inactivation components is changed favouring the slowly inactivating one in the wide range of membrane potentials. The results are consistent with a single channel population following the 3-state model of inactivation.

Ionic currents of normal and aconitine modified sodium channels of the Ranvier node membrane were measured under voltage clamp conditions. The experiments with local anesthetics in the external Ringer solution have showed that dissociation constant (Kdis) of normal channel-anesthetic complex for procaine is 0.27 + 0.03 mM, and for benzocaine is 0.68 +/- 0.04 mM. With aconitine modified channels, Kdis increases and becomes 1.32 +/- 0.5 mM and 1.52 +/- 0.3 mM for procaine and benzocaine, respectively. It is ascertained that the development of aconitine effect is inhibited by neutral benzocaine to a lesser extent than by procaine. It is shown that the aconitine effect cannot be reversed by a high concentration of anesthetic. Hence, it appears that aconitine and anesthetic receptors do not coincide.

The general part consists of a review on nerve stimulus mechanism as well as the nerve structure and the function of nerve membrane as the site of action of local anesthetics. Furthermore a possibility of interaction between local anesthetics and the membrane components especially phospholipids is discussed. These phospholipids show interesting properties with respect to ions as well as local-anesthetics, which allow us to suppose that they are important participants in nervous stimulus transmission. In the experimental part two methods are described. The first deals with the measurement of electrical resistance as function of time at cephalin and cholesterin impregnated filter membrane in solutions of cinchocain homologues. An increase in the resistance with the increase in concentration of test substances was observed. The same was the effect of increasing chain length in alkoxy group where after butoxy derivative, a deformation of membrane was observed. In the second method the drug binding capacity of cephalin dispersed in aqueous medium was measured. Here, too, the increase in the binding capacity with the increase in alkoxy chain was observed. The large difference in free binding energy between two subsequent homologues is explained as the effect of increase in van der Waals' forces and hydrophobic interactions on one hand, and a change in colloidal form of cephalin dispersion on the other hand.

Influence of different metabolic poisons on cAMP-evoked neuron membrane permeability is investigated. Drugs preventing cAMP binding with R subunits of protein kinase decrease the cAMP-evoked current, but the inhibitor of the C subunit. H8, has no effect. The cAMP-dependent current is increased by uncouplers and decreased by inhibitors of glycolysis and oxidative phosphorylation. The mechanism of cAMP action on neuron permeability is discussed.

During incubation in vivo, exogenously applied ionic lanthanum comes to surround the numerous neurosecretory terminals which are found lying within or immediately beneath the acellular neural lamella ensheathing the nerves from fifth instar and adult specimens of Rhodnius prolixus. The lanthanum does not penetrate beyond the cellular perineurium, which completely surrounds the non-neurosecretory axons in these nerves and constitutes a form of 'blood-brain barrier'. In some cases, however, lanthanum is found in the vicinity of a neurosecretory axon lying beneath the perineurium, where it can be assumed to have leaked in from the neurosecretory terminal lying free in the neural lamella. When nerves are incubated in calcium-free media, regions with an attenuated perineurium become 'leaky', in that lanthanum is found lying in those extracellular spaces between axons and glia which lie immediately below the thin part of the perineurial layer. Bathing solutions made slightly hyperosmotic to the haemolymph with sucrose have no apparent disruptive effects on the barrier. When the tissues are incubated in more hypertonic solutions, the perineurial barrier becomes 'leaky' throughout, and tracer pervades beyond its cells into all the intercellular spaced between glia and axons. The possible role of the zonulae occludentes in both the maintenance of the perineurial barrier and in the formation of interglial occlusions to local penetration of exogenous substances is considered.

Previous work from this laboratory has shown that in utero exposure to ethanol significantly alters the synthesis of glycoproteins in synaptic, axolemmal, and myelin membranes from developing rats. In an attempt to determine whether in utero exposure to ethanol similarly alters the synthesis of other glycoconjugates involved with cell-cell interactions, the present study examined the influence of chronic maternal ethanol consumption prior to parturition on the content and synthesis of gangliosides in axolemmal and synaptic plasma membranes from developing rats. The results demonstrate that, in contrast to central nervous system glycoproteins, synaptic and axolemmal glycolipids are minimally affected by in utero exposure to ethanol. At all ages examined (17 to 34 days of age), the offspring of control and ethanol-treated rats had a comparable distribution of radiolabel among synaptic and axolemmal gangliosides, a normal concentration of ganglioside sialic acid in synaptic plasma membranes, and a near-normal distribution of sialic acid among synaptic gangliosides. The present study provides evidence which indicates that the radiolabeling patterns of axolemmal and synaptic membrane gangliosides are similar. Specifically, the most heavily labeled synaptic and axolemmal gangliosides were GT1b (20-37% of the total radioactivity) and GD1a (20-32%). A smaller proportion of radioactivity was associated with GD1b (approximately 11-16%), GM1 (5-10%), and GQ1b (4-11%), as well as with GD3 and the other monosialogangliosides (less than 5%). During the age period examined the proportion of radioactive GT1b increased in both membrane fractions.

Compounds with local anaesthetic properties may be classified according to their action upon the nerve membrane. Four classes of agent are recognized: A. Compounds acting at the exterior of the sodium channels; B. Compounds acting at the axoplasmic part of the sodium channels; C. Compounds acting through a physicochemical mechanism; BC. Compounds acting by a combination of B and C mechanisms. The chemical basis for these different mechanisms is discussed.

Ultrastructural changes of the terminal plates of the bushy receptors in the frog urinary bladder have been studied after two hours' exposition in 0.05% novocaine solution and one hour's exposition in 0.05% dicaine and trimecaine solution. During these periods a steady block of the receptor impulse activity develops. The local anesthetics essentially change ultramicroscopic structure of the terminals. The reaction to the anesthetics investigated has both some features in common and certain peculiarities. At each effect three types of changes can be determined, characterized with various degree of rearrangement in neurilemma, neuroplasm and organelles. Each type of the changes is supposed to reflect a certain phase of the plate reactive response. Specificities of the reaction to novocaine are minimal changes of mitochondria, accumulation of glycogen granules, deformity and decreasing amount of vesicles. Under dicaine effect mitochondria do not change, amount of vesicles increases, their form does not change; under trimecaine effect mitochondria undergo most noticeable alterations. The changes of the terminal plates observed are interpreted as adaptive. The effect of the local anesthetics on the receptors is not limited with the blockade of the sodium canals of the afferent fibers, in parallel, biochemical processes, occurring in cytosol of the terminals also change; their morphological manifestations are the ultrastructural changes observed.

Studies have been made on the effect of trypsin, chymotrypsin, pronase, lipases, hyaluronidase and digitonin on electrophysiological properties of the neurons of the snail H. pomatia under external application. Proteases and lipases gradually depolarize the neuronal membrane, decrease the amplitude and prevent the onset of action potentials, initially increase and then decrease the membrane resistance. The decrease in the membrane resistance coincides with the period of maximum inhibition of resting and action potentials in the neurons. The enzymes studied do not affect the membrane capacitance. Changes in electrophysiological characteristics induced by the enzymes are partially reversible provided the preparation is soaked in Ringer's solution for a sufficient time. Digitonin rapidly and irreversibly depolarizes the membrane, decreases its resistance and blocks action potentials. Hyaluronidase does not significantly affect neuronal electrophysiological properties when applied solely, but facilitates the development of changes during subsequent effect of proteases.

The depressing action of epinephrine on the giant neurons is due to a progressively increasing passive potassium conductivity of the membrane, hyperpolarization and derangement of its generating properties. The triggering factor in the mechanism of the developing epinephrine hyperpolarization is an epinephrine induced increment of passive potassium conduction of the neuronal membrane.

The early time course of the current passing across the membrane in squid giant axons in which the ionic currents have been blocked reveals substantial asymmetries during and after the application of hyperpolarizing and depolarizing voltage-clamp pulses of identical size. Since the integral of the 'on' and 'off' current transients is zero, these currents must result from charge movements confined to the membrane and, therefore, they are nonlinear displacement currents. The steady state rearrangement of the charges as a consequence of sudden displacements of the membrane potential is consistent with a Boltzmann distribution of charges between two states characterized by different energy levels. Following changes in membrane potential the charges undergo a first order transition between these states. The relaxation time constant for the transition at a given temperature is a function of membrane potential. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.

The effects of the epileptogenic agent, picrotoxin, on inhibitory postsynaptic potentials (IPSPs) were studied in the amygdala in vitro slice preparation. Picrotoxin was superfused onto the tissue and intracellular recordings were obtained from basolateral amygdaloid neurons (BLANs). Stimulation of the stria terminalis pathway evoked an excitatory postsynaptic potential (EPSP)--IPSP sequence. Depolarization of the membrane potential increased the amplitude of the IPSP, whereas hyperpolarization of the membrane potential decreased the amplitude of the IPSP. The IPSP reversed polarity at -74 mV. Application of picrotoxin reduced the IPSP and resulted in the development of paroxysmal depolarizing shifts. Picrotoxin itself had no apparent effect on the resting membrane potential or input resistance. These results indicate that epileptiform activity induced by picrotoxin in the basolateral amygdaloid neuron is of synaptic origin.

The effect of external and internal K+ on Na+o-dependent Ca2+ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K+ (up to 70 mM) has no effect on Na+-Ca2+ exchange. Removal of Ki+ causes a marked inhibition in the Na+o-dependent Ca2+ efflux component. Internal K+ activates the Na+-Ca2+ exchange with low affinity (K 1/2 = 90 mM). Activation by Ki+ is similar in the presence or in the absence of Na+i, thus ruling out a displacement of Na+i from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca2+ efflux on Ki+. The present results demonstrate that Ki+ is an important cofactor (partially required) for the proper functioning of the forward Na+-Ca2+ exchange.

Voltage-clamped single nerve fibers of the frog Rana esculenta were treated with the carboxyl group activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of different primary amines and without added amine. Carboxyl groups form stable amide bonds with primary amines in the presence of EEDQ. EEDQ treatment reduced the sodium current considerably and irreversibly, regardless of the presence of a primary amine in the Ringer's solution. The potassium current was also reduced. After modification the reduced sodium currents inactivated slowly and incompletely. The descending branch of the sodium current-voltage relation, INa(E), was shifted along the voltage axis in the depolarizing direction. The size of the shift was strongly dependent on the amine present during modification with EEDQ. The voltage-dependence of sodium inactivation, h infinity (E), was shifted to more positive values of membrane potential by EEDQ in the presence of ethylenediamine (11 mV) and glucosamine (3 mV). In contrast, a small shift to more negative potentials occurred in the presence of taurine (-3 mV) or without the addition of an amine (-2 mV). A tenfold increase of the calcium concentration still shifted the INa(E) and h infinity (E) curves of the chemically modified fibers. However, these shifts were smaller than those observed on untreated fibers. The currents remaining after the modification were completely blocked by tetrodotoxin; no change of the reversal potential occurred.

Asymmetrical displacement currents in the Ranvier node membrane were recorded in the normal Ringer and under the action of a neurotoxin from the scorpion Buthus eupeus venom and of the alcaloid aconitine. In the total displacement current (DC) at the beginning of the pulse (Ion), two-exponentially decaying components were extracted: a rapid component with the maximum time constant of decay about 70 microns, and a slower one with the time constant of decay--300 microns. At high negative levels of conditioning prepulse (Vc) (-130 divided by -145 mV), the maximum value of the charge transferred by the fast and slow components was estimated to be 2.6 x 10(-14) and 4.8 x 10(-14) C, respectively. The slow component of Ion decreased with lowering Vc and was inhibited by the scorpion neurotoxin and aconitine. The fast phase of Ion remained unchanged with decreasing Vc from -145 to -100 mV and toxin-treated axons. Aconitine evoked a shift in the curve relating the change displaced by the fast component to the membrane potential in the direction of hyperpolarization by 40--50 mV. The results obtained suggest that the fast and the slow components of DC are associated with the initial potential-dependent stages of the processes of channel activation and inactivation, respectively.

The ionic currents of the nodal membrane were measured under voltage clamp conditions. The membrane being +40 mv. The replacing of the external Na+-ions to K+- and NH4+-ions have showed that the relative pearmeabilities of the veratrine-modified channels calculated from the constant field theory are arranged in the following row: PNa:PK:PNH4 = 1:0.29:0.61, which differs from the same row for the normal channels. The decreasing of the slope of current-voltage relations of the modified channels with the replacing of Na+-ions to K+- and NH4+-ions is the evidence of a more strong binding of these ions to external mouth of the modified channel compared to the binding of Na+-ions.

The time course of recovery from use-dependent block of sodium channels caused by local anesthetics was studied in squid axons. In the presence of lidocaine or its quaternary derivatives, QX-222 and QX-314, or 9-aminoacridine (9-AA), recovery from use-dependent block occurred in two phases: a fast phase and a slow phase. Only the fast phase was observed in the presence of benzocaine. The fast phase had a time constant of several milliseconds and resembled recovery from the fast Na inactivation in the absence of drug. Depending on the drug present, the magnitude of the time constant of the slow phase varied (for example at -80 mV): lidocaine, 270 ms; QX-222, 4.4 s; QX-314, 17 s; and 9-AA, 14 s. The two phases differed in the voltage dependence of recovery time constants. When the membrane was hyperpolarized, the recovery time constant for the fast phase was decreased, whereas that for the slow phase was increased for QX-compounds and 9-AA or unchanged for lidocaine. The fast phase is interpreted as representing the unblocked channels recovering from the fast Na inactivation, and the slow phase as representing the bound and blocked channels recovering from the use-dependent block accumulated by repetitive depolarizing pulse. The voltage dependence of time constants for the slow recovery is consistent with the m-gate trapping hypothesis. According to this hypothesis, the drug molecule is trapped by the activation gate (the m-gate) of the channel. The cationic form of drug molecule leaves the channel through the hydrophilic pathway, when the channel is open. However, lidocaine, after losing its proton, may leave the closed channel rapidly through the hydrophobic pathway.

Acetylcholine (10(-6)--10(-3) M) added to the rat brain homogenate increased that activity of microsomal Na, K-ATPase and (14C)-amino acid incorporation in microsomal proteins. Actinomicin D (5.10(-5) M) eliminated the effect of acetylcholine. It is concluded that acetylcholine induced the synthesis of either Na, K-ATPase itself or some other proteins involved in the enzyme activity regulation.

Completely isolated identified neurones (LPed-2 and LPed-3) of the left pedal ganglion of Planorbarius corneus were shown to have two kinds of cholinoreceptors (ChR) on their membrane. One kind of ChR is a classical nicotinic receptor which is sensitive to nicotinomimetics and can be blocked by tubocurarine; the depolarization caused by activation of this ChR type is chloride-dependent. The other kind of ChR, which mediates a potassium-dependent hyperpolarization, has some common features with muscarinic (M) ChR of vertebrates, being sensitive to the muscarinomimetics, dioxolane F-2268, methylfurmethide, mecholyl, and arecoline, although insensitive to oxotremorine. The sensitivity of this receptor to muscarine, itself, was not tested. Like the muscarinic ChR of vertebrates, it can be blocked by benzilylcholine mustard, but, in contrast to vertebrate muscarinic receptors, it cannot be blocked by atropine and cannot distinguish between the optical isomers of F-2268. TEA, mytolon, and cooling prevent the hyperpolarization caused by ACh and muscarinomimetics. The two kinds of ChR in P. corneus neurones seem to be similar to those found by Kehoe24-26 on the medial cells of Aplysia pleural ganglia, both in their pharmacological characteristics and in the ionic permeability changes which they control.

Squid giant axons were treated with tetrodotoxin (TTX) in concentrations ranging from 1 nM to 25 nM and the resulting decrease in sodium current was followed in time using the voltage clamp technique. The removal of TTX from the bathing solution produced only partial recovery of the sodium current. This suggests that the over-all interaction is more complex than just a reversible reaction. By correcting for the partial irreversibility of the decrease in sodium current, a dissociation constant of 3.31 x 10(-9)M was calculated for the reaction between TTX and the reactive site of the membrane. The data obtained fit a dose-response curve modified to incorporate the correction for partial irreversibility when calculated for a one-to-one stoichiometry. The fit disagreed with that calculated for a reaction between two molecules of TTX with a single membrane-reactive site, but neither supported nor disproved the possibility of a complex formed by two reactive sites with one molecule of TTX. Values of the rate constants for the formation and dissociation of the TTX-membrane complex, k(1) and k(2), respectively, were obtained from the kinetic data. The values are: k(1) = 0.202 x 10(8)M(-1), and k(2) = 0.116 min(-1). The magnitude of the dissociation constant derived from these values is 5.74 x 10(-9)M, which has the same order of magnitude as that obtained from equilibrium measurements. Arrhenius plots of the rate constants gave values for the thermodynamic quantities of activation.

Saxitoxin (STX) and tetrodotoxin (TTX) have the same striking property of blocking the Na(+) channels in the axolemma. Experiments with nerve plasma membrane components of the squid Dosidicus gigas have shown that TTX interacts with cholesterol monolayers. Similar experiments were carried out with STX. The effect of STX on the surface pressure-area diagrams of lipid monolayers and on the fluorescence emission spectra of sonicated nerve membranes was studied. The results indicate a TTX-like interaction of STX with cholesterol monolayers. The expansion of the monolayers caused by 10(-6)M STX was 2.2 A(2)/cholesterol molecule at 25 degrees C. From surface pressure measurements at constant cholesterol area (39 A(2)/molecule) in media with various STX concentrations, it was calculated that the STX/cholesterol surface concentration ratio is 0.54. The apparent dissociation constant of the STX-cholesterol monolayer complex is 4.0 x 10(-7)M. The STX/cholesterol ratio and the apparent dissociation constant are similar to those determined for TTX. The presence of other lipids in the monolayers affects the STX-cholesterol association. The interactions of STX and TTX with cholesterol monolayers suggest (a) that cholesterol molecules may be part of the nerve membrane Na(+) channels, or (b) that the toxin receptor at the nerve membrane shares similar chemical features with the cholesterol monolayers.