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

Neuropharmacolagy Vol. 27, No. 7, pp. 737-742, 1988 Printed in Great Britain. All rights reserved

Copyright 6

OOZE-3908/88 53.00 + 0.00 1988 Rrgamoa Press pk

CHANGES IN NEUROTRANSMITTER RELEASE AT A NEUROMUSCULAR JUNCTION OF THE LOBSTER CAUSED BY CA~ABINOIDS Department

of Pharmacology,

S. A. TURKANIS* and R. KARLER University of Utah School of Medicine, Salt Lake City, Utah 84132, U.S.A. (Accepted

7 December

1987)

Summar--la 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 (‘T’HC), 1l-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 ~nnabinoids. Key words: cannabinoids, neuromuscular junction, lobster, mechanism of action, site of action.

Various el~~ophysi~logical investigations have demonstrated that delta-9-tetrahydrocannabinol (THC) produces excitation and depression in many areas of the central nervous system, such as the cerebral cortex, the hmbic system and the spinal cord; these effects are manifested as modifications in the amplitudes of various synaptic or evoked potentials (Boyd, Boyd, Muchmore and Brown, 1971; Boyd,

Boyd and Brown, 1974; Turkanis and Karler, 1981, 1983; Deadwyler, Hampson and Marlow, 1986). S~ifi~lly, THC increases the ~plitude of excitatory postsynaptic potentials of spinal motoneurons in the cat and hippocampal neurons in the rat (Turkanis and Karler, 1983; Deadwyler et al., 1986). The increases in excitatory postsynaptic potentials are at least partially att~bu~ble to a rise in the resistance of the postsynaptic membrane, but increases in release of neurotransmitter may also contribute to changes in potential amplitude. Because the excitatory postsynaptic potential of the spinal motoneuron appears to be mediated by glutamate (McGeer, Eccles and McGeer, 1978), in the present investigation the effects of three cannabinoids, THC, 1l-hydroxy-THC [the principal pharmacologically active metabolite of THC (Burstein, 197311and cannabidiol [a major but nonpsychoactive constituent af marijuana (Perez-Reyes, Timmons, Davis and Wall, 197311, were assessed electrophysiologically on a glutamate-mediated neuromuscular junction of the lobster. A neuromuscular test system was selected, for it is one of the limited *To whom correspondence should be addressed.

number of preparations in which the mean quantum content, an electrophysiological measure of release of neurotransmitter (Katz, 1969), can be determined. METHODS Preparations and bathing solution

The present investigation was carried out with 425-500 g lobsters, Homarus americanus. The lobsters were maintained in artificial sea water (Instant Oceans, Aquarium Systems) at about 14°C on a diet of shrimp. The animals were kept for at least 3 weeks prior to their use in the neuromuscular studies. The walking-limb stretcher muscle, with its excitor axon, was dissected as described in detail previously (Grundfest, Reuben and Rickles, 1959; Colton and Freeman, 1975; Colton and Colton, 1982). Preparations were perfused continuously with artificial sea water containing (mM): NaCl, 455; KCl, 5; CaCl, 20; MgCI, 8; Hepes buffer, 15. The perfusion medium was maintained at a temperature of approximately 18°C and a pH of about 7.4. Equipment and experimental design

Evoked excitatory junctional potentials and spontaneous miniature junctional potentials were recorded intracellularly with l-5 MR glass microelectrodes, filled with 3 M potassium-acetate solution. Excitatory junctional potentials were evoked by stimulating the motor nerve supram~mally at intervals of 5 see for 16 or 32 stimuli per test. In addition, a series of 64 miniature junctional potentials were recorded for determination of their mean amplitude. 737

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A. TURKANISand R. KARLER

The evoked and spontaneous potentials were summed by a Nicolet 1174 signal processor and the summed potentials were drawn by an X-Y plotter (Honeywell 550). The spontaneous potentials were initially stored in a delay circuit. The mean quantum content of a muscle fiber was calculated from the ratio of the mean amplitude of the excitatory junctional potential to that of the miniature junctional potential (Atwood and Bittner, 1971). As described by Atwood and Bittner (1971), two classes of excitatory junctional potentials were observed, that is small amplitude (< 5 mV), exhibiting a high level of synaptic facilitation and large amplitude (> 6 mV), exhibiting a low level of synaptic facilitation. An initial study demonstrated that junctions exhibiting excitatory junctional potentials of 2-3 mV, were more sensitive to THC than those with 8-10 mV potentials (see Results, below); therefore, the data in Figures l-6 and Table 1 were obtained only from junctions with small amplitude excitatory junctional potentials. Effective membrane resistance was determined by two intracellular microelectrodes inserted into a muscle fiber, less than 100pm apart; one electrode was used for passing current, the second for recording the resulting change in potential. Effects of the cannabinoids on evoked and spontaneous synaptic potentials and effective membrane resistance were determined in the following manner: initially, the preparation was perfused with control

.A

E’

I

I

0

I

10 20

I3

0 CONTROL

I

I

I

I

2.5 5

I

I

IO

I5

PM THC

Fig. 1. Dose-effect relationships for THC on an excitatory neuromuscular junction. All three dose-effect relationships were obtained from the same junction. 0 = Control values; l = values 20 min after drug. Bach mean amplitude value of the excitatory junctional potentials (e.j.p.s) and miniature junctional potentials (m.j.p.s) was determined from 32 and 64 responses, respectively. Control resting membrane potential, 86 mV; resting membrane potential after drug, 87 mV. Control effective membrane resistance, 0.9 Ma; effective membrane resistance after drug, 0.9 MR.

I

I

30

0

I

I

I

I

I

I

I

IO 20 30 40 50 60 70 MINUTES

Fig. 2. Time-effect relationships for THC on an excitatory neuromuscular junction. All three time courses were obtained from the same junction. 0 = Control values; l = values after drug; 7 indicates the beginning of treatment with vehicle or drug. Bach mean-amplitude value of the excitatory junctional potentials and miniature junctional potentials was determined from 32 and 64 responses, respectively. Control resting membrane potential, 88 mV, resting membrane potential after drug, 91 mV. Control effective membrane resistance, 1 .O MR, effective membrane resistance after drug, 1.0 MR.

A

Y-1

I

CA

n n

CONTROL

IJJM THC

IOyM

THC

Fig. 3. Increase and decrease of amplitude of excitatory junctional potential caused by THC. Each response is the electronically-obtained average of 32 excitatory junctional potentials. (A) Control response; (B) response 30 min after 1 PM drug; (C) response 30 min after 10 p M drug. Control resting membrane potential, 93 mV; resting membrane potential after drug, 94mV. Control effective membrane resistance, 0.92 Ma; effective membrane resistance after drug, 0.92 MR.

Cannabinoid junctional effects

3 0’ 0

IO

I 20

I,

30

0

I

I

I

I,

IO

20

30

40

50

I

I

60

70

MINUTES

:

Fig. 4. Time-effect relationships for 1I-hydroxy-THC on an excitatory neuromuscular junction. All three time-courses were obtained from the same junction. 0 = Control values; l = values after drug; t indicates the beginning of treatment with vehicle or drug. Each mean-amplitude value of the excitatory junctional potentials and miniature junctional potentials was determined from 32 and 64 responses, respectively. Control resting membrane potential, 86 mV, resting membrane potential after drug, 85mV. Control effective membrane resistance, 0.83 MQ effective membrane resistance after drug, 0.84 MfL artificial sea water and, as illustrated in Figures 2, 4 and 6, at least 30min of relatively stable control electrophysiological values were obtained at about IO-min intervals. The preparation was then perfused with a cannabinoid-containing medium for 1Omin and a control medium for the remainder of the

CONTROL

IOyM

THC

A

Fig. 5. Depression of the amplitude of miniature junctional potential by THC. Each response is the electronically-obtained average of 64 spontaneous potentials. (A) Miniature junctional potential 20min after vehicle; (B) miniature junctional potential 20 min after 8 PM THC. Control resting membrane potential, 87 mV; resting membrane potential after drug, 86mV. Control effective membrane resistance, 0.92 MQ; effective membrane resistance after drug, 0.93 MD.

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51, 0

IO 20

30

0

IO

20

30

40

50

MINUTES

Fig. 6. Time-effect relationships of an excitatory neuromuscular junction for cannabidiol. All three time-courses were obtained from the same junction. 0 = Control values; l = values after drug; 1 indicates the beginning of treatment with vehicle or drug. Each mean-amplitude value of the excitatory junctional potentials and miniature junctional potentials was determined from 32 and 64 responses, respectively. Control resting membrane potential, 9OmV, resting membrane potential after drug, 89mV. Control effective membrane resistance, 0.92 MR, effective membrane resistance after drug, 0.91 MR.

test period. At the start of administration of drug, electrophysiological measurements were made at about IO-min intervals for 20-30min for the concentration-effect studies and until maximum responses were attained in the time-effect studies. The perfusion time for the cannabinoids were limited to 10min because perfusion times of 30-60 min can produce concentrations of drugs in the tissue that are greater than that of the perfusion medium (Smiley, Karler and Turkanis, 1976), probably as the result of the high lipid-solubility of the drugs. Perfusion with control medium did not reverse the effects of the drugs. The maximum change in electrophysiological responses after the drugs was expressed as a percentage of the mean control values obtained in each experiment. Such an experimental design permitted each junction to be. employed as its own control; for this reason, only one junction was studied in each muscle preparation. The resting membrane potential and the effective membrane resistance were used as indicators of the physiological status of the muscle fibers because these parameters are not affected by the cannabinoids (Table 1). To be used in the present study, a muscle fiber had to exhibit a resting membrane potential of at least 80 mV and an effective membrane resistance

S.

740 Table 1. Summary

A. TIJRKANISand

R. KARLER

of data of the effects of cannabinoids on parameters the lobster Means

Treatment THC l-5 PM 7.5-12pM I I-hydroxy-THC I-5pM 7.5-12pM Cannabidiolt 1-12pM

junction

of

and standard deviations (% of control)

Amplitude excitatory junctional potentials

Amplitude of miniature junctional potentials

Mean quantum content of a muscle fiber

142 f 5* 38 + a*

75 + 4* 64 * 21

165 i_ 3* 32 + 6’ 41 * 13f

of the neuromuscular

Effective membrane resistance

Resting membrane potential

180 f 9* 57* II*

101 * 2 98 i 2

101 t2 99 * 3

a2 k 4* 71 i4’

197 *a* 45 + IO’

98 k 3 10252

lOOi I 99 * 3

65 f 3*

59 + 151

99 * 3

102+2

Data are means and their standard deviations of the maximum responses to drug obtained from time-effect experiments and are expressed as a percentage of initial control values obtained in each experiment. Each mean value was calculated from the results from 5 different muscle fibers in 5 different preparations. Mean control values and their standard deviations (n = 15): excitatory junctional potential amplitude, 2.5 If- 0.4 mV; miniature junctional potential amplitude, 0.12 f 0.08 mV; mean quantum content of a muscle fiber, 20 f 3; effective membrane resistance, 0.94 f 0.14 MQ resting membrane potential, 86 * 3 mv. Concentrations of cannabinoids less than I M usually produced minimal responses. *Significantly different from initial mean vehicle control values obtained in each experiment, as determined by a r-test for the data for cannabidiol and by analysis of variance and the Newman-Kuels multiple means test for the THC and 1 I-hydroxy THC data (P < 0.05; Snedecor and Cochran, 1967). tcannabidiol (0.1-12 PM) did not increase mean quantum content.

of 0.2-1.0 MR, and the results were discarded if the resting membrane potential decreased by 5 mV or the effective membrane resistance changed by 10%. Preparation of drugs

The cannabinoids were dispersed in artificial sea water by the use of the nonionic surfactant Pluronic F68 (BASF Wyandote Corporation) (Turkanis and Karler, 1975). The molar ratio of cannabinoid to Pluronic F68 in the perfusion medium was 1.O: 0.075; as in previous investigations (Turkanis and Karler, 1986b), initial experiments demonstrated that the control perfusion solution containing surfactant had no measurable effect on the electrophysiological parameters studied. RESULTS

Concentration-response relationships for the effects of THC on an excitatory junction are shown in Figure 1, Delta-9-tetrahydrocannabinol produced dual effects on the mean quantum content of the muscle fiber and the amplitude of the excitatory junctional potential; that is, l-5 PM THC increased these parameters in a concentration-related manner, whereas larger concentrations caused only a decrease. In contrast, 1-15 PM only depressed the amplitudes of the miniature junctional potentials. In a series of three comparable concentration-effect experiments, concentrations of drug less than 1 PM elicited minimal responses. In addition, more than 15 PM of the cannabinoid usually abolished the synaptic potentials, precluding the electrophysiological measure of synaptic function. Over the same concentration yielded THC-like 11-hydroxy-THC range, concentration-effect relationships, but cannabidiol elicited concentration-related depression.

From the concentration-effect relationships for the cannabinoids, the concentrations for the time-effect studies described below were chosen. Examples of individual time-effect curves are illustrated in Figures 2, 4 and 6, and all of the data are summarized in Table 1. The responses to drugs, obtained with the timeeffect studies, confirmed those in the concentration-effect experiments; that is, in relatively small concentrations (l-5 PM), both THC and its 1I-hydroxy metabolite increased the amplitude of the excitatory junctional potentials, whereas larger concentrations (7.5-12 p M) produced only a decrease (Figs 2-4 and Table 1). The enhancement caused by THC appeared to be related to the amplitude of the control excitatory junctional potential, for the means and standard deviations of the maximum amplitudes, l&30 min after l-5 PM THC (expressed as a percentage of control) were 142 f 5 (n = 5) for 2-3 mV potentials and 109 f 2 (n = 5) for 8-10 mV potentials. The two mean values were significantly different, as determined by a t-test (P < 0.05; Snedecor and Cochran, 1967). Because junctions with excitatory junctional potentials of small amplitude (i.e. l-5 mV) exhibit a greater degree of synaptic facilitation than do those with large amplitude (>6mV) (Atwood and Bittner, 1971) the increase caused by THC may depend upon the ability of a junction to produce synaptic facilitation. The data reported here support similar findings with a watersoluble derivative of THC, SP 1llA (THC ester of 4-N-morpholinol butanoic acid hydrochloride), at neuromuscular junctions of the crayfish (Aldridge and Pomeranz, 1977). In addition, THC and its 11-hydroxy metabolite reduced the amplitude of the miniature junctional potentials (Figs 2, 4, 5 and Table 1); again, the

Cannabinoid junctional effects reduction appeared to be dose-related. This effect suggests that the cannabinoids produce a postsynaptic depression. In support of a postsynaptic site of action, the reduction occurred within a few minutes at junctions at which the motor axon was not electrically stimulated. Under these conditions, the turnover of neurotransmitter is minimal; thus, a presynaptic effect of the drug on synthesis of neurotransmitter is highly unlikely (Hubbard, Llinas and Quastel, 1969). In addition, it is clear that the changes in the amplitudes of excitatory junctional potentials and miniature junctional potentials were not due to modifications in either the resting membrane potential or the effective membrane resistance of the muscle fibers (Figs l-6 and Table 1). As for the discharge frequency of the spontaneous miniature junctional potentials, THC had little or no effect: in five experiments the cannabinoid caused a slight increase twice, a slight decrease twice and no change once; the 1I-hydroxy metabolite and cannabidiol yielded similar results. Both THC and its metabolite also affected the mean quantum content of a muscle fiber: concentrations of l-5 FM caused an increase, whereas larger concentrations (7.5-12 PM) produced a decrease (Figs 2 and 4 and Table 1). These effects reflect presynaptic actions; that is, increases and decreases in release of glutamate. The cannabinoid-induced reduction of the amplitude of the miniature junctional potential, described above, is important in assessing changes in mean quantum content, because miniature junctional potentials of small amplitude after treatment with drug may be missed, leading to an underestimation of the increase and an overestimation of the decrease in mean quantum content. To minimize this problem, only junctions displaying relatively large control values for the amplitude of the miniature junctional potential were selected: a mean and standard deviation of 0.12 f 0.14mV (n = 15) (Table 1). In contrast to THC and its metabolite, cannabidiol elicited only depression: a reduction of the amplitudes of excitatory and miniature junctional potentials and the mean quantum content (Fig. 6 and Table 1). The possibility existed that the concentrations of cannabidiol were too large to elicit to excitation, but reducing the concentrations 0.1-0.9 p M did not cause excitation, which confirmed earlier reports that cannabidiol does not share excitatory properties with THC (Karler and Turkanis, 1981; Turkanis and Karler, 1981, 1986a, b). Like THC and its 1 1-hydroxy metabolite, cannabidiol had no effect on the frequency of the spontaneous miniature junctional potential.

DISCUSSION

The above findings with cannabinoids with the lobster neuromuscular junction are consistent with N.P.27,7--F

741

previous electrophysiological studies: at every level of organization studied, from pools of cortical neurons to individual spinal motoneurons and neuromuscular junctions, THC produced excitation and/or depression (Turkanis and Karler, 1981, 1983, 1986a, b), as did 1 1-hydroxy-THC (Turkanis and Karler, 1984); in contrast, cannabidiol was only depressant. These effects at individual synapses reflect the responses of the cannabinoids in conscious animals; therefore, electrophysiological studies with single junctions may point to synaptic sites and mechanisms of action for the central excitatory and depressant properties of the cannabinoids. The effects on the lobster preparation partly support those reported earlier for an excitatory neuromuscular junction in the crayfish, in vitro (Aldridge and Pomeranz, 1977). Like THC at junctions in the lobster, SP 111A, a water-soluble derivative of THC, increased the amplitude of excitatory junctional potentials and the increase was greater with small amplitude potentials than with large amplitude potentials. In addition, the conclusion was drawn that the enhancement of the excitatory junctional potential was a presynaptic response to the drug; that is, an increase in release of neurotransmitter. In contrast to the present investigation, no depression caused by the cannabinoid was seen. There are two possible reasons for this discrepancy: first, the crayfish synapse may not be depressed by the cannabinoids; secondly, SP 111A,unlike THC, may lack depressant properties. The results of experiments in the lobster are also consistent with studies of cannabinoids on neurotransmission in sartorius muscle of the frog in vitro (Turkanis and Karler, 1986b), which showed that THC and its 11-hydroxy metabolite first increased and then decreased mean quantum content of the endplate potential; in addition, both drugs reduced the amplitude of spontaneous miniature endplate potentials. Again, cannabidiol elicited only depression. The results, therefore, with three different synaptic preparations, frog, lobster and crayfish neuromuscular junctions, demonstrate that cannabinoids can modify release of neurotransmitter, and, indeed, produce similar responses with both acetylcholine and glutamate release. The consistency of the effects suggests that the drugs affect a basic mechanism of release of neurotransmitter; for instance, it is possible that they alter a voltage-gated calcium conductance in the nerve terminal (Katz, 1969). Acetylcholine and glutamate are among the many neurotransmitters that have been implicated in the central effects of THC (Paton, 1975; Turkanis and Karler, 1983; Martin, 1986). Delta-9-tetrahydrocannabinol for example, has been reported to cause an increase in the release of acetylcholine, a decrease in the turnover of acetylcholine and an anticholinergic response (Drew and Miller, 1974; Paton, 1975; Revuelta, Moroni, Cheney and Costa, 1978). Delta-9-tetrahydrocannabinol also appears to alter

S.

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A. TURKANISand R. KARLER

glutamate-median ne~otr~s~ssion in u&o; in this case, the drug enhances the amplitudes of excitatory postsynaptic potentials at spinal motoneurons of the cat (T’urkanis and Karler, 1983). The results of studies with THC on various ne~omu~ular junctions, therefore, may be relevant to the central excitatory and depressant properties of the drug. Another finding of relevance to the central nervous system is that the effective con~ntrations of cannabinoid at the isolated junctions of the lobster are similar to those that are active centrally in vivo in several different species. Specifically, the concentrations of drug in brain in viva in rats, mice and frogs, after the administration of excitatory and depressant doses of cannabinoid, ranged 0.3-30 p M (Karler, Cely and Turkanis, 1974; Karler and Turkanis, 1981, unpublished). These data indicate that the con~ntrations used in vitro are also attainable in vivo. In summary, at excitatory (glutamate) neuromuscular junctions in the lobster, the cannabinoids exerted excitation by increasing the mean quantum content and the excitatory junctional potentials, and depression by decreasing the mean quantum content, the excitatory and the miniature junctional potentiats. In addition, the effects on the mean quantum content indicate a presynaptic site of action of cannabinoids, possibly involving a voltage-gated calcium conductance, while the reduction in the miniature junctional potential suggests a postsynaptic response to the drug, which may be due to depression of a neurotransmitter-gated conductance. These findings suggest synaptic sites and mechanisms of action that may contribute to the central excitatory and depressant effects of the cannabinoids; that is, postsynaptic depression and alterations in release of neurotransmitter caused by cannabinoids may contribute to changes in amplitude of central evoked and synaptic potentials. Acknowledgements-This work was supported by NIDA research grant DA-00346 and a University of Utah Research Committee grant. REFERENCES

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