Since cannabinoid receptors inhibit excitatory synaptic transmission by reducing glutamate release, we have examined whether this might occur through the direct inhibition of presynaptic Ca2+ channels. In cerebrocortical nerve terminals, activation of cannabinoid receptors with WIN55,212‐2 reduces the KCl‐evoked release of glutamate. However, this inhibition is attenuated when N‐ and P/Q‐type Ca2+ channels are blocked. Through Ca2+ imaging in single nerve terminals, we found that WIN55,212‐2 reduced the influx of Ca2+ both in nerve terminals that contain N‐type Ca2+ channels and those that contain P/Q‐type Ca2+ channels. Thus, cannabinoid receptors modulate the two major Ca2+ channels coupled to glutamate release in the cerebral cortex.
Endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors in many synapses, including those in the cerebral cortex 1-6. It has been suggested that inhibition of presynaptic Ca2+ channels is responsible for the reduction of glutamate release by cannabinoid receptors. However, it has not been clearly established which types of Ca2+ channel can be modulated by this presynaptic mechanism. In cultured rat hippocampal neurons, cannabinoids inhibit somatic N‐ and P/Q‐type calcium channels 7. However, studies of synaptic transmission have indicated a selective inhibition of N‐type Ca2+ channels, since blockage of these Ca2+ channels subtype with toxins suppresses the response to cannabinoids 1, 8, 9.
Direct measurement of the effects of cannabinoid on presynaptic Ca2+ channels has been limited to synapses with a large presynaptic bouton like those at the Calyx of Held 10. However, monitoring the presynaptic Ca2+ influx and measuring the postsynaptic current amplitude in brain slices reflect the activation of many synaptic terminals that may or may not express the presynaptic receptor under study 11. Here, we have used a preparation of cerebrocortical nerve terminals to determine the effect that cannabinoids have on glutamate release. In addition, by imaging Ca2+ in individual nerve terminals we determined the impact that the activation of cannabinoid receptors has on the Ca2+ response evoked by depolarization, identifying the type of Ca2+ channel involved. The results show that cannabinoid receptors reduce the depolarization‐induced influx of Ca2+ in nerve terminals that contain N‐ or P/Q‐type of Ca2+ channels. As a consequence, they reduce the glutamate release component associated with the activation of these two Ca2+ channels.
2 Materials and methods
2.1 Synaptosomal preparation
The cerebral cortex was isolated from adult male Wistar rats (2–3 months) and the synaptosomes were purified as described previously 12, 13 on discontinuous Percoll gradients (Amersham–Pharmacia Biotech, Uppsala, Sweden). Following the final centrifugation at 22 000 × g for 10 min, the synaptosomes were resuspended in 8 ml of HEPES buffer medium (HBM): 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM NaH2PO4, 1 mM MgCl2, 10 mM glucose and 10 mM HEPES, pH 7.4. The protein content was determined by the Biuret method, and 1 mg of the synaptosomal suspension was diluted in 8 ml of HBM and spun at 3000 × g for 10 min. The supernatant was discarded and the pellet containing the synaptosomes was stored on ice. Under these conditions, the synaptosomes remain fully viable for at least 4–6 h, as judged by the extent of KCl evoked glutamate release.
2.2 Glutamate release
Glutamate release was assayed by on‐line fluorimetry. Synaptosomal pellets were resuspended in HBM (0.67 mg/ml) and preincubated at 37 °C for 1 h in the presence of 16 μM bovine serum albumin (BSA). The BSA served to bind any free fatty acids released from synaptosomes during the preincubation. A 1 ml aliquot was transferred to a stirred cuvette and the release of glutamate was measured as previously described 14. The Ca2+‐dependent release was calculated by subtracting the release obtained during a 5 min period of depolarization in the presence of 200 nM free [Ca2+] from the release at 1.33 mM CaCl2.
2.3 Plasma membrane potential
The plasma membrane potential was determined with 3,3′‐dipropylthiadicarbocyanine iodide, DiSC3(5). Synaptosomes were resuspended (0.67 mg/ml) in HBM without BSA, and 1 ml aliquots were transferred to a stirred cuvette containing 5 μM DiSC3(5) and 1.33 mM CaCl2. After allowing the mixture to equilibrate for 1 min, the fluorescence was determined at 651 and 675 nm. Data were collected at 1 s intervals.
2.4 Ca2+ imaging of the responses in single synaptosomes
Synaptosomes in HBM (2 mg/ml) with 16 μM BSA were preincubated with 5 μM fura‐2 AM and 1.33 mM CaCl2 for 40 min and the synaptosomal suspension was then attached to a polylysine‐coated coverslip for another hour. Synaptosomes were illuminated alternately at 340 and 380 nm for 0.8 s through a 100× objective with the aid of a monochromator (Kinetic Imaging, Ltd., UK). The fluorescence emitted from the nerve terminals was collected through a band‐pass filter centered at 510 nm and images were obtained using a slow‐scan CCD camera (Hamamatsu C4880) operating at 12‐bit digitalization (4096 levels). The output from the camera was stored by the computerized imaging system Lucida 3.0 (Kinetic Imaging, Ltd.) and Ca2+ images were analyzed as described previously 15. To measure [Ca2+]cyt, synaptosomes were stimulated by 10 s pulses of 30 mM KCl (a bar in each graph) in the presence or absence of pharmacological agonists or Ca2+ channels toxins.
3.1 WIN55,212‐2 reduces the glutamate release components associated with N‐ and P/Q‐type Ca2+ channels
Depolarization of nerve terminals with KCl opens Ca2+ channels and initiates the exocytotic release of glutamate. The Ca2+‐dependent release of glutamate after 5‐min depolarization with KCl was 3.66 ± 0.16 nmol/mg of protein (n = 5; Fig. 1 A). The prior addition of the cannabinoid receptor agonist WIN55,212‐2 reduced the release by 37.4 ± 4.3% (n = 4; Fig. 1A). The inhibition by WIN55,212‐2 of the KCl‐induced release was almost completely abolished (2.4% ± 3.5, n = 3, data not shown) by the cannabinoid receptor antagonist AM281 (1 μM). Treatment of synaptosomes with pertussis toxin abolished (0.56 ± 5.5%, n = 3, data not shown) the inhibition of release by WIN55,212‐2. The blockade of Na+ channels with tetrodotoxin (TTx) did not alter (37.1 ± 2.1%, n = 3, Fig. 1A) the inhibition of release by cannabinoids excluding the modulation of voltage dependent K+ channels associated to action potentials. Furthermore, depolarization of the synaptosomal plasma membrane measured with a membrane potential‐sensitive cationic cyanide dye was not modified by WIN55,212.2. Thus, the increase in fluorescence induced by 30 mM KCl (57.0 ± 2.4 arbitrary units) was not altered by WIN55,212‐2 (55.1 ± 4.3 a.u., n = 5, data not shown). In order to know to what extent this inhibition resulted from a reduction in the activity of Ca2+ channels coupled to glutamate exocytosis, we performed experiments in the presence of Ca2+ channel toxins. In cerebrocortical nerve terminals, glutamate release is primarily coupled to Ca2+ entry through both N‐ and P/Q‐type Ca2+ channels 15, 16, channels that can be selectively blocked by ω‐CgTx‐GVIA 17 and ω‐Aga‐IVA 18, respectively. Blocking N‐type channels with ω‐CgTx‐GVIA reduced the KCl‐evoked release by 30.8 ± 2.6% (n = 3) and partially occluded the inhibition by WIN55,212‐2 (17.2 ± 3.8%, n = 5, Fig. 1A and B). Blocking P/Q‐type Ca2+ channels with ω‐Aga‐IVA reduced glutamate release by 65.5 ± 1.3% (n = 4, Fig. 1A and B) and also partially occluded the inhibition of release by WIN55,212‐2 in this case by 20.9 ± 2.0% (n = 4; Fig. 1B). Thus, these data indicate that cannabinoid receptors inhibit glutamate release coupled to both the P/Q‐ and the N‐type Ca2+ channels.
3.2 WIN55,212‐2 reduces the entry of Ca2+ in nerve terminals containing N‐ or P/Q‐type Ca2+ channels
To further examine whether cannabinoid receptors reduce the activity of Ca2+ channels, we visualized Ca2+ in single nerve terminals. We have previously shown that the two major subpopulations of cerebrocortical nerve terminals exist, containing N‐ or P/Q‐types of Ca2+ channels 19. In these experiments, nerve terminals were first stimulated with KCl in the presence of either ω‐CgTx‐GVIA to block N‐type channels or ω‐Aga‐IVA to block P/Q‐type channels. Subsequently, KCl was added in the presence of the cannabinoid agonist WIN55,212‐2 and finally KCl was added alone. In the experiments with ω‐CgTx‐GVIA and WIN55,212‐2 (Fig. 2 ), a total of 812 nerve terminals from five fields were analyzed. In a subpopulation of nerve terminals (9.3 ± 0.5%), the Ca2+ responses were reduced by ω‐CgTx‐GVIA but not by WIN55,212‐2, indicating the presence of N‐type channels but the absence of WIN55,212‐2‐sensitive receptors modulating Ca2+ entry. In another subpopulation of nerve terminals (22.5 ± 1.4%), Ca2+ responses were insensitive to ω‐CgTx‐GVIA but they were reduced by exposure to WIN55,212‐2, indicating the presence of WIN55,212‐sensitive cannabinoid receptors but not of N‐type Ca2+ channels. In a third subpopulation of terminals (28.8% ± 4.5), Ca2+ responses were reduced both by ω‐CgTx‐GVIA and WIN55,212 revealing the presence of cannabinoid receptors and N‐type Ca2+ channels in individual nerve terminals. Finally, 39.0 ± 2.3% of the synaptic boutons were insensitive to both ω‐CgTx‐GVIA and WIN55,212.
In another set of experiments, nerve terminals were stimulated in the presence of ω‐Aga‐IVA to block P/Q‐type Ca2+ channels. A total of 455 nerve terminals from four fields were analyzed. This identified nerve terminals whose Ca2+ responses were inhibited only by ω‐Aga‐IVA (36.7 ± 10.8%) and another subpopulation of nerve terminals in which the Ca2+ responses were only reduced by WIN55,212‐2 (27.2 ± 4.1%). Terminals in which Ca2+ entry was blocked both by ω‐Aga‐IVA and WIN55,212‐2, and in which both P/Q‐type Ca2+ channels and cannabinoid receptors are therefore present represented 27.0 ± 3.5% of the total population. Finally, in some nerve terminals (8.8 ± 2.2%) Ca2+ responses were insensitive to ω‐Aga‐IVA and to WIN55,212‐2.
In this study, we show that the activation of cannabinoid receptors inhibits glutamate release in the whole population of cerebrocortical nerve terminals and that this effect can be partially occluded by blocking N‐ and P/Q‐types of Ca2+ channels. Ca2+ imaging experiments of individual nerve terminals indicate that cannabinoid receptors reduce the influx of Ca2+ in a subset of nerve terminals that express either N‐ or P/Q‐types of Ca2+ channels. This suggests that the mechanism by which cannabinoid receptors inhibit glutamate release in the cerebral cortex may involve the inhibition of the two major Ca2+ channels that trigger release.
A number of studies have suggested that blocking Ca2+ channels is responsible for the inhibition of synaptic transmission by cannabinoid receptors, but only a few studies have directly measured the activity of presynaptic channels 10, 11. We have found that the inhibition of glutamate release by cannabinoid receptors is partially occluded by blocking N‐ and P/Q‐type Ca2+ channels with ω‐CgTx‐GVIA and ω‐Aga‐IVA, respectively. This suggests that cannabinoid receptors reduce Ca2+ channel activity in some but not in all nerve terminals expressing N‐ or P/Q‐type Ca2+ channels. We corroborated this proposal by imaging Ca2+ in synaptosomes that were blocked by ω‐CgTx‐GVIA or ω‐Aga‐IVA and that were sensitive or insensitive to WIN55,212‐2. These experiments provide evidence that the Ca2+ channels were directly modulated by cannabinoid receptors. Cannabinoid receptors also modulate K+ channels and thus, they can indirectly reduce Ca2+ channels activity and the subsequent release of glutamate 20. Nevertheless, it is unlikely that K+ channels are activated during KCl‐evoked responses in nerve terminals due to the high external K+ concentration. In addition, KCl‐evoked responses are insensitive to the Na+‐channels blocker tetrodotoxin 19, 21 and, therefore, it seems more likely that the inhibitory receptors target release‐coupled Ca2+ channels rather than ionic channels involved in the waveform of action potentials.
The N‐ and P/Q‐types of Ca2+ channels are segregated among different populations of nerve terminals in the cerebral cortex of adult rats 20. The finding that cannabinoid receptors inhibit both types of Ca2+ channels suggests that they influence the activity of those Ca2+ channels with which they are co‐expressed rather than selectively acting on the signaling pathways specific to a subtype of Ca2+ channels. Similar results were obtained in a recent study in the cerebellum where the three types of Ca2+ channels (N, P/Q and R) present in granule cell presynaptic boutons were inhibited by cannabinoid receptors 11. In contrast, our results do not agree with the selective regulation of N‐type Ca2+ channels by cannabinoid receptors in the hippocampus 8, striatal neurons 1 and in trigeminal caudal neurons 9. In these preparations, cannabinoid‐mediated inhibition of excitatory synaptic transmission was eliminated by pre‐treatment with antagonists of N‐type Ca2+ channels.
Cannabinoid agonists inhibit the Ca2+‐dependent release by 37.43 ± 1.3%, whereas Ca2+ imaging experiments show that cannabinoid receptors reduced the influx of Ca2+ in 51–54% of the nerve terminals. If cannabinoid receptors were restricted to glutamatergic nerve terminals these data would indicate that cannabinoid receptors do not exert complete control over glutamate release, in spite of the significant reduction of Ca2+ influx (Figs. 2C and 3 C). However, this possibility is rather unlikely given the high local [Ca2+]c that triggers release 22, since a reduction in Ca2+ entry would prevent the firing of those nerve terminals expressing the receptor. Alternatively, Ca2+ imaging may detect the effects of cannabinoids on GABAergic nerve terminals as well as on glutamatergic nerve terminals explaining the increased sensitivity of this technique. We have previously shown that GABAergic nerve terminals represent a fraction of 24% in the cerebrocortical preparation 20. Furthermore, cannabinoid receptors are known to be expressed in GABAergic nerve terminals where they reduce the release of GABA by a mechanism that involves the inhibition of Ca2+ channel activity 23.
In summary, we show that the two major types of Ca2+ channels coupled to glutamate release in the cerebral cortex are modulated by cannabinoid receptors.
This work was supported by Ministerio de Ciencia y Tecnología (MCyT) Grant BFI2001‐1436 and Dirección General de Investigación de la Comunidad de Madrid Grant 08.5/0008.1/2003. We thank M. Sefton for editorial assistance.
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