Differential effects of delta-9-tetrahydrocannabinol and its 11-hydroxy metabolite on sodium current in neuroblastoma cells.

Whole-cell voltage-clamp techniques were used to study the comparative effects of delta-9-tetrahydrocannabinol (THC) and its principal metabolite, 11-hydroxy-delta-9-tetrahydrocannabinol (11-OH-THC), on the voltage-gated sodium current in neuroblastoma cells. The parent compound markedly depressed the inward sodium current with minimal reduction of the outward current, demonstrating that the effects of the drug were related to the membrane potential. In addition, THC reduced the reversal potential, indicating that the drug modified the ion selectivity of the channel. 11-OH-THC similarly depressed inward sodium current; however, in marked contrast to the effects of the parent compound, the drug equally depressed the outward voltage-gated sodium current, indicating that its effects were not related to the membrane potential. Furthermore, 11-OH-THC differed from THC in that it did not alter the reversal potential. The results demonstrate that THC and its 11-OH metabolite both reduce inward sodium current, but their effects on the outward current and ion selectivity are distinctly different. The sum of the actions of these two cannabinoids on the voltage-gated sodium channel provides a plausible cellular basis for THC's depression of action potentials in vivo and for some of its central depressant effects.

Calcium channel blockers and excitatory amino acids.

The calcium channel blockers (CCB), diltiazem, verapamil and nifedipine, antagonize in mice both N-methyl-DL-aspartate- (NMDLA) and kainate-induced convulsions, which were not affected by carbamazepine and ethosuximide. The CCB, on the other hand, were ineffective against convulsions induced by bicuculline, pentylenetetrazol and electroshock. The results suggest that the CCB may be efficacious in the treatment of those neurodegenerative diseases putatively caused by the excitatory amino acids.

DNQX blockade of amphetamine behavioral sensitization.

The role of the N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid (EAA) receptors in the mechanism of behavioral sensitization to amphetamine-induced sterotypy was investigated in mice. The results confirm previous observations that NMDA antagonists can block the induction of the phenomenon but not the expression; in contrast, DNQX, a non-NMDA receptor antagonist, can block both the induction and the expression of the sensitization. The differential effects of the two classes of antagonists suggest that the induction and the expression are the result of different mechanisms, both of which involve the EAA system. The DNQX results differ from those of haloperidol, which can also block both the induction and expression, because haloperidol can completely block the amphetamine-induced responses in naive and in sensitized animals; whereas DNQX is without effect on the amphetamine activity in naive animals and, in the sensitized animal, can block only that portion of the response that is derived from the sensitization phenomenon. The effects of the EAA antagonists support the hypothesis that the enhanced responsiveness in the sensitized animals is derived from the activation of EAA receptors, which, in turn, increases the release of dopamine in the striatum. Finally, the involvement of the non-NMDA receptors in the expression of the behavioral sensitization further substantiates the postulate that the amphetamine-induced sensitization is a behavioral manifestation of long-term potentiation (LTP).

Calcium channel blockers and behavioral sensitization.

Behavioral sensitization to amphetamine-induced stereotypy was previously shown to consist of two separable phenomena, induction and expression, both of which involve the excitatory amino acids (EAA). In the present experiments, the calcium channel blockers (CCB), nifedipine, diltiazem and verapamil, were shown to block both phenomena; these results are similar to those reported earlier for DNQX, an antagonist of the non-N-methyl-D-aspartate receptors for the EAA. The CCB, like DNQX, affect only that percentage of the stereotypic response which results from the sensitization reaction, without affecting the quantitative portion of the response attributable to the acute effect of amphetamine. The results support previous conclusions that the sensitization response consists of two quantitative components, only one of which involves the EAA. The antagonism exhibited by the CCB suggests that behavioral sensitization involves Ca++ and L-type calcium channels.

Delta-9-tetrahydrocannabinol depresses inward sodium current in mouse neuroblastoma cells.

Whole-cell voltage-clamp techniques were used in order to define the effects of delta-9-tetrahydrocannabinol (THC) on the voltage-gated sodium current in neuroblastoma cells. With regard to the inward sodium current, THC decreased the peak amplitude and increased both the time to peak and tau for recovery. The reversal potential was unchanged, suggesting that channel selectivity for sodium was not altered by the drug. With regard to the outward sodium current, THC had no effect on the peak amplitude, time to peak or tau for recovery. This functional alteration of the voltage-gated sodium channel may contribute to the depressant effects of the cannabinoid.

Amphetamine behavioral sensitization and the excitatory amino acids.

The effects of 8 selective neuroeffector agonists and antagonists were measured in mice in order to identify specific functional changes associated with behavioral sensitization to amphetamine-induced stereotypy. The changes observed included a decreased convulsive threshold to N-methyl-DL-aspartic acid, an increased convulsive threshold to bicuculline, and an increased head-twitch response to 5-hydroxytryptophan. Of these effects, only the persistence of the two convulsive threshold changes correlated with the persistence of the behavioral sensitization. The induction of behavioral sensitization was blocked by haloperidol, dizocilpine or ketamine, but not affected by cyproheptadine or diazepam: therefore, the mechanism of the enhanced responsiveness involves not only a functional dopamine system, but also a functional N-methyl-D-aspartic acid component of the excitatory amino acid system. The block of behavioral sensitization also prevented the amphetamine-induced changes in the convulsive thresholds. Finally, neither ketamine nor dizocilpine affected the expression of the enhanced response in the behaviorally sensitized animals. The data indicate that the characteristics of amphetamine behavioral sensitization to stereotypy are similar to those of long-term potentiation.

Whole-cell voltage-clamp study of sodium current in neuroblastoma cells: effects of inhibition of neurite outgrowth by colchicine.

A method is described for the production of large numbers of neurite-free neuroblastoma cells that are especially suitable for studies involving whole-cell voltage clamp. Differentiation in the presence of colchicine yielded cells having abundant sodium channels, highly reproducible peak currents and no space-clamp problems. Treatment with this drug did not alter the electrophysiological properties of the cells. Colchicine might be similarly advantageous in voltage-clamp studies of different ion channels and other types of cultured neurons.

Reverse tolerance to amphetamine evokes reverse tolerance to 5-hydroxytryptophan.

Repeated intermittent administration of amphetamine in mice caused reverse tolerance to 5-hydroxy-L-tryptophan (5-HTP)-induced head twitch, as well as to amphetamine-induced stereotypy. The repeated administration of 5-HTP alone also resulted in reverse tolerance in the head-twitch test. Daily pretreatment with haloperidol prior to amphetamine administration blocked the development of both reverse tolerance to amphetamine and to 5-HTP, whereas daily pretreatment with cyproheptadine prior to amphetamine blocked only the reverse tolerance to 5-HTP. On the other hand, 5-HTP-induced reverse tolerance was blocked by daily pretreatment with cyproheptadine, but not with haloperidol. There appears to be no difference in the persistence of the reverse tolerance to 5-HTP, whether induced by amphetamine or by 5-HTP; in both instances, the persistence does not correlate with the persistence of reverse tolerance to amphetamine. The data suggest that the reverse tolerance to amphetamine and the associated reverse tolerance to 5-HTP are independent events, both of which are mediated by dopaminergic mechanisms.

Effects of cocaine on neuromuscular transmission in the lobster.

In vitro electrophysiological techniques were used on an excitatory neuromuscular junction of a walking-limb stretcher muscle of the lobster in order to define the pharmacology of cocaine on a glutamatergic synapse. Cocaine reduced the amplitudes of the excitatory and the miniature junction potentials, as well as the mean quantum content of the muscle fiber. The effect on mean quantum content points to a presynaptic site of action of the drug, and the effect on miniature junctional potential suggests a postsynaptic site of action of the drug. In addition, cocaine shifted the concentration-depolarization curve for glutamate to the right, demonstrating a postsynaptic depression of the response to glutamate. Cocaine exerted no effect on the resting membrane potential or the effective membrane resistance of the muscle fibers. The data indicate that the drug concomitantly exerted pre- and postsynaptic depression of a glutamatergic junction.

Pentylenetetrazol kindling in mice.

Kindling with pentylenetetrazol to produce minimal and maximal convulsions was investigated in CF-1 mice. Like electrical kindling, the kindling effect was directly proportional to the dose or the intensity of the kindling stimulus. Similarly, the kindling effect was persistent, as was emphasized by the ability to kindle with an interdose interval of 3 days and by the convulsions produced by a challenge with pentylenetetrazol 30 days after withdrawal from the kindling treatment. The changes in excitability, associated with the kindling state, appeared to be relatively selective for pentylenetetrazol, because no changes in thresholds to either electroshock or administration of picrotoxin or N-methyl-DL-aspartate correlated temporally with the persistence of kindling. The influence of two anticonvulsant drugs, ethosuximide and cannabidiol, on kindling was also investigated. Both drugs blocked the development of kindling to pentylenetetrazol-induced minimal convulsions. Of these drugs, only ethosuximide raised the minimal convulsive threshold to pentylenetetrazol. Against pentylenetetrazol-induced kindling to maximal convulsions, only cannabidiol blocked kindling and only cannabidiol raised the maximal seizure threshold for pentylenetetrazol. Although the drugs modified the kindling effect, the mechanism of the interaction is not clear.

Proconvulsant and anticonvulsant effects in mice of acute and chronic treatment with cocaine.

The proconvulsant and anticonvulsant effects of acute and chronic exposure to cocaine were investigated in adult, male, CF-1 mice. The proconvulsant effects of cocaine in mice only manifested themselves after daily exposure to motor-stimulant doses. Although daily treatment decreased electroshock convulsion threshold, no motor convulsions were observed. Animals in the proconvulsant state, however, kindled to electrically-induced convulsions more rapidly than did controls. Furthermore, daily treatment with cocaine and electroshock also enhanced the development of electrical kindling. These results illustrate that the excitatory properties of cocaine in the CNS can enhance phenomena which cause a persistent increase in excitability of the CNS. In contrast to the proconvulsant activity after chronic exposure, cocaine, administered acutely, in motor-stimulant doses, was anticonvulsant in a variety of tests using electroshock and chemically-induced convulsions. The drug elevated electroshock thresholds for both minimal and maximal convulsions and these responses were not blocked by haloperidol. In tests for minimal chemically-induced convulsions, cocaine elevated the threshold to N-methyl-DL-aspartate, but not to bicuculline; against maximal convulsions, the drug was anticonvulsant against both N-methyl-DL-aspartate and bicuculline. Cocaine did not affect convulsion thresholds for strychnine, arecoline or aminophylline; these data suggest that the anticonvulsant action of cocaine is relatively selective for the gamma-aminobutyric acid (GABA) and glutamate systems.

Blockade of "reverse tolerance" to cocaine and amphetamine by MK-801.

"Reverse tolerance" was produced in rats and mice by repeated exposure to either cocaine or amphetamine. The locomotorstimulant effect was studied in mice; stereotypy and convulsions in rats. MK-801, the NMDA antagonist, blocked the development of "reverse tolerance" to all three effects. In contrast, haloperidol selectively blocked "reverse tolerance" to cocaine-induced stereotypy but not to convulsions. The data suggest that the glutamate system participates in the mechanism of "reverse tolerance" to the dopaminergic effects of cocaine and amphetamine, as well as to the convulsant effect of cocaine.

The effects of amphetamine on neuromuscular transmission in the lobster.

In vitro electrophysiological techniques were used on an excitatory neuromuscular junction of a walking-limb stretcher muscle of the lobster in order to define the pharmacology of amphetamine on a glutamatergic synapse. A single electrical stimulus in the presence of the drug produced a train of action potentials in the axon innervating the neuromuscular junction, resulting in a staircase-like enhancement of the evoked excitatory junction potential; both effects were antagonized by relatively small doses of haloperidol. In contrast, amphetamine reduced the amplitude of the spontaneous miniature junctional potential, suggesting a postsynaptic site of action; this depressant response to the drug was not antagonized by haloperidol. The data demonstrate that amphetamine concomitantly produces both excitation and depression at a single synapse.

Characteristics of "reverse tolerance" to amphetamine-induced locomotor stimulation in mice.

The characteristics of chronically administered amphetamine on the locomotor and anticonvulsant effects were studied in adult CF-1 mice. The influence of dose of the drug and interdose interval on the development of "reverse tolerance" to the locomotor stimulation was investigated, in addition to the selectivity of the response and the persistence of the change in pharmacodynamics. Once-daily treatment with 6 mg/kg amphetamine for 4 weeks resulted in a 2-3 fold increase in locomotor activity. The increase in responsiveness, however, was limited to the first period of 2 weeks and there was no subsequent change in pharmacodynamics during the last 2 weeks of treatment. After 36 days of withdrawal, the response had not returned to that of control, illustrating the persistence of the effect. The results of varying the interdose interval indicated that "reverse tolerance" occurred even when the interval was as long as 14 days. These results represent additional evidence of the persistence of the phenomenon. Selectivity of the changes in the CNS was illustrated by the cross-reactivity with a motor-stimulant dose of cocaine but not with that of morphine. Selectivity was also demonstrated by the failure of "reverse tolerance" to develop to the anticonvulsant effects of amphetamine, which also appear to be mediated dopaminergically.

Changes in neurotransmitter release at a neuromuscular junction of the lobster caused by cannabinoids.

In vitro intracellular recording techniques were used on an excitatory neuromuscular junction of a walking-limb stretcher muscle of the lobster in order to define the synaptic pharmacology of delta-9-tetrahydrocannabinol (THC), 11-hydroxy-THC and cannabidiol. Delta-9-tetrahydrocannabinol and 11-hydroxy-THC, in relatively small concentrations, increased the amplitude of the excitatory junctional potential and the mean quantum content of a muscle fiber, whereas larger concentrations produced depression. In contrast, cannabidiol reduced the excitatory junctional potential and the mean quantum content. All three cannabinoids, however, depressed the amplitude of the spontaneous miniature junctional potential. The changes in mean quantum content point to a presynaptic site of action for the drug, while the reduction of the amplitude of the miniature junctional potential presumes a postsynaptic site. Such findings suggest synaptic mechanisms and sites of action for the central excitatory and depressant properties of the cannabinoids.

Effects of delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol and cannabidiol on neuromuscular transmission in the frog.

Intracellular recording techniques were used on neuromuscular junctions of the sartorius muscle of the frog, in vitro, to define the synaptic pharmacology of delta-9-tetrahydrocannabinol (THC), 11-hydroxy-THC and cannabidiol (CBD). The frequency of miniature endplate potentials was increased by THC, decreased by CBD and was unaffected by 11-hydroxy-THC, whereas the amplitude of the miniature endplate potentials was depressed by all three cannabinoids. In addition, the mean quantum content of the endplate potential (m) was first increased and then decreased by THC and 11-hydroxy-THC, but CBD produced only depression. Changes in m and the frequency of the miniature endplate potential indicated presynaptic sites of drug action and reduction of the amplitude of the miniature endplate potential suggested a postsynaptic site. The findings suggest possible mechanisms of action for the central excitatory and depressant properties of the cannabinoids.

Cannabidiol-caused depression of spinal motoneuron responses in cats.

Intracellular recording techniques were used on spinal motoneurons in the cat in order to define the synaptic pharmacology of cannabidiol (CBD). The cannabinoid produces only depression of electrophysiological responses of the motoneurons: For instance, the drug decreases the amplitude of excitatory postsynaptic potentials (EPSPs); this reduction does not appear to be the result of a change in the afferent input. In addition, CBD raises the firing threshold and decreases the amplitude of motoneuron action potentials; the effects on action potentials are related to changes in postsynaptic membrane conductances, probably involving at least sodium conductance. The spinal motoneuron effects provide potential electrophysiological mechanisms for CBD's central depressant actions.

Prolonged CNS hyperexcitability in mice after a single exposure to delta-9-tetrahydrocannabinol.

A single exposure to delta-9-tetrahydrocannabinol (THC) resulted in a "rebound" hyperexcitability in the CNS in mice, which was assessed in terms of the susceptibility of the CNS to electrically-induced convulsions. The magnitude of the hyperexcitability was dose-related (25-150 mg/kg, i.p.), as measured 24 hr after treatment. The time-course study of the effect indicated a peak-effect at 24 hr after administration of the drug, with a duration of the effect for as long as 196 hr. The time course of the rebound hyperexcitability to THC was compared to that for phenobarbital, which peaked at 48 hr after administration of the drug and returned to the control value by 96 hr. Tolerance developed rapidly to the motor-toxic effect of THC, but after 23 days of daily treatment there was no evidence of tolerance to the rebound hyperexcitability. The functional significance of the hyperexcitable state was assessed in two tests; electrical kindling to minimal convulsions was enhanced, even when the kindling procedure was initiated 120 hr after exposure to the drug; and the anticonvulsant activity of phenytoin was blocked when mice were treated with the anticonvulsant 96 hr after a single exposure to THC. The results suggest that the rebound response from a single exposure to THC represents a functionally significant prolonged increase in excitability of the CNS.