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1Department of Cellular Membranology, Bogomoletz Institute of Physiology, Kiev, Ukraine; and 2Department of Experimental Neurophysiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Submitted 22 November 2005; accepted in final form 20 May 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Ohno-Shosaku et al. 2001; Wilson et al. 2001). Endocannabinoids, such as anandamide (N-arachidonoyl-ethanolamine, AEA) and 2-arachidonylglycerol (2-AG) (Devane et al. 1992; Di Marzo et al. 1994; Stella et al. 1997), are released from the postsynaptic neuron. Consequently, they can activate retrogradely presynaptic G-protein-coupled cannabinoid CB1 receptors (CB1R), leading to transient inhibition of excitatory and/or inhibitory synaptic transmission (Brown et al. 2003; Diana et al. 2002; Kreitzer and Regehr 2002; Maejima et al. 2001; Wilson and Nicoll 2001; Yoshida et al. 2002). Cannabinoid receptors (CBR) are distributed widely throughout the brain, and such retrograde signaling has been observed in numerous synapses belonging to different brain regions, including the cerebellum (Brown et al. 2003; Diana et al. 2002; Kreitzer and Regehr 2002; Wilson and Nicoll 2001, 2002; Yoshida et al. 2002).
It has been shown that CB1R-mediated reduction of presynaptic calcium influx is responsible for the decrease in neurotransmitter release in cannabinoid-mediated synaptic modulation. One of the proposed mechanisms of cannabinoid action is a retrograde modulation of presynaptic calcium channels (Alger 2002; Hoffman and Lupica 2000; Kreitzer and Regehr 2001; Sullivan 1999). In hippocampal inhibitory synapses, as well as in excitatory synapses onto striatal neurons (Huang et al. 2001) and trigeminal caudal neurons (Liang et al. 2004), it has been suggested that modulation of solely N-type calcium channels plays a major role in cannabinoid-mediated inhibition. However, by monitoring optically the presynaptic Ca2+ influx and measuring the excitatory postsynaptic current (EPSC) amplitudes, Brown et al. (2004) have shown recently that cannabinoids reduced Ca2+ influx through all the three, N-type, P/Q-type, and R- type of calcium channels in cerebellar granule cell presynaptic boutons in CB1R-dependent manner (Brown et al. 2004). Here using the conventional whole cell patch-clamp technique, we demonstrated that P-type calcium currents in isolated Purkinje neurons are directly modulated by endocannabinoids independent of CB1R activation. Therefore in addition to the well-described activity-dependent retrograde signaling mediated by presynaptic cannabinoid receptors, endocannabinoids can also exert direct modulation of excitability and Ca2+ influx in postsynaptic Purkinje neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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This study was carried out on acutely dispersed cerebellar Purkinje neurons from 11- to 12-day-old Wistar rats that were prepared as described in detail elsewhere (Panchenko et al. 1993). Briefly, the cerebellum was cut into 300- to 400-µm-thick slices and incubated with 2.4 mg/ml of protease XXIII from Aspergillus oryzae (Sigma, Germany) for 35–40 min at 33°C. After rinsing off the enzyme, single cells were isolated by triturating the pieces of tissue through several fire-polished pipettes with openings of 0.5–0.1 mm. The Purkinje neurons were identified by their characteristic shape and partially preserved dendritic arborization.
Whole cell recording from isolated cells
Currents through voltage-activated calcium channels were recorded at room temperature (20–22°C) in a whole cell configuration of the patch-clamp technique (Hamill et al. 1981), using an A-M Systems 2400 patch-clamp amplifier (Bio-Medical Products) connected to a 100-kHz Lab Master DMA board (Scientific Solutions, Solon, OH) in a personal IBM computer. Patch pipettes were pulled from borosilicate glass tubes (Sutter Instrument, Novato, CA) on a P97 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA). Pipettes had the resistances of 2–4 M
, when filled with the intracellular solution containing (in mM) 70 tris(hydroxymethyl)aminomethane (Tris)-phosphate, 40 tetraethylammonium chloride (TEA-Cl), 5 MgCl2, 20 Tris-Cl, 5 ethyleneglycol-bis(
-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA), 5 adenosine 5'-triphosphate, Tris salt (ATP), and 0.5 guanosine 5'-triphosphate, Tris salt (GTP) (adjusted to pH 7.3 with Tris-OH). Liquid junction potentials were compensated. After rupture of the membrane, the series resistance (8–12 M) was compensated (70–90%). Only the cells with negligible leaks (<50 pA) were used, thus current records were not leak-subtracted.
High-voltage-activated (HVA) P-type calcium current was measured at a holding potential of –70 mV to ensure complete inactivation of the low-voltage-activated (LVA) T-type calcium currents in Purkinje neurons (Panchenko et al. 1993; Regan 1991). Under these experimental conditions, the whole cell current was completely blocked by 
-Aga IVA (McDonough et al. 1997; Mintz et al. 1992). To avoid Ca2+-dependent inactivation processes, Ba2+ was chosen as a charge carrier through Ca2+ channels. The external control solution contained (in mM) 20 TEA-Cl, 100 choline-Cl, 2 BaCl2, 2 MgCl2, and 20 Tris-Cl. Drug-containing solutions were applied by a "concentration clamp" technique using a "jumping table" set-up (Pharma Robot, Kiev, Ukraine). The currents were filtered at 3 kHz and digitized at sampling intervals of 140, 200, 260, or 2,400 µs. Data were analyzed using jumping table software (Pharma Robot) running on an IBM-PC computer.
All curve fitting and statistics were done with Microcal Origin software (Microcal, Northampton, MA). The amplitude of currents was measured from the baseline to the peak value. An inhibitory action of the substance was measured as the mean ratio (I0 – I)/I0, where I is the amplitude of the current under the action of the substance and I0 is the amplitude of the current in the control saline at a test stimulus corresponding to the maximum of current-voltage (I-V) curves. Dose-dependence smooth curves were fitted to the data points according to the Hill equation I/I0 = 1/[1 + ([S]/IC50)n], where [S] is the concentration of the substance, IC50 is the half-inhibition concentration of the substance, and n is the Hill coefficient. The inactivation kinetics of the current were fitted by a double-exponential function, I = If*exp(–t/
f) + Is*exp(–t/s), where If, Is are the fast and slow amplitudes of the current inactivation, and f, f are the fast and slow inactivation time constants. The effect of the substance on the current under study was averaged at least for three cells for each concentration of the tested agent. Modulation of the inactivation kinetics by the drug was obtained as the mean ratio drug/con where drug is the inactivation time constant of the current under the action of the drug and con is the inactivation time constant of the current in the control saline at a test stimulus corresponding to the maximum of I-V curves. Cumulative data were calculated as means ± SD throughout the study.
Cerebellar slice preparation and recording conditions
Sprague-Dawley rats at 14–20 days of age were killed by decapitation, and the cerebellum was removed. Sagittal cerebellar slices (300-µm-thick) were sectioned using a vibratome in ice-cold solution containing (in mM) 125 NaCl, 3 KCl, 10 glucose, 26 NaHC03, 1.2 NaH2PO4, 1 CaCl2, and 3 MgCl2 (carboxygenated with 5% CO2-95% O2). Slices were maintained at room temperature (20–22°) in the recording solution until use (1–8 h).
Slices were transferred to a recording a chamber which was continuously superfused with recording solution containing (in mM) 125 NaCl, 3 KCl, 10 glucose, 26 NaHC03, 1.2 NaH2PO4, 2 CaCl2, and 2 MgCl2 (carboxygenated with 5% CO2-95% O2). Neurons were visualized via a x40 water-immersion objective using infrared differential interference contrast (IR-DIC) video microscopy. To block rapid synaptic transmission and CB1R activation, the recording solution also contained the AMPA receptor antagonist NBQX, the GABAA receptor antagonist picrotoxin and the CB1R antagonist SR141716A. Whole cell current-clamp recordings were made with borosilicate glass pipettes (3–5 M) filled with (in mM) 130 K-gluconate, 10 Na-gluconate, 4 NaCl, 4 MgATP, 0.3 NaGTP, 4 phosphocreatine, and 10 HEPES, pH 7.3. Whole cell current-clamp recordings were performed using an EPC-9 amplifier with PULSE software (HEKA Electronic, Lambrecht, Germany).
Materials
Synthetic and endogenous cannabinoids were obtained from Tocris (Bioscience, Avonmouth, UK). SR141716A was a gift from Solvay Pharmaceuticals (Weesp, the Netherlands).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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External application of AEA (2 µM) induced inhibition of P-current peak amplitude [(I0 – I)/I0 = 84 ± 5%, n = 6, P < 0.02] accompanied by acceleration of the inactivation kinetics (Fig. 1). The development of the effects induced by AEA was slow (minutes) and only slightly reversible on wash-out of the drug. In experiments when depolarization pulses were applied only after 5-min-long preincubation with AEA, saturating modulation of P currents was observed with the first test pulse, indicating that the action of AEA on P-currents apparently was not use dependent.
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Inactivation of P currents (measured at the maximum of the I-V curve) under control conditions developed in a biexponential manner, with the mean values for the time constants of fast (f) and slow (s) inactivation being f = 82 ± 8 ms and s = 1428 ± 175 ms (n = 3). The acceleration of the inactivation kinetics of P currents in the presence of AEA was manifested as a concentration-dependent decrease of the values of s, while the values of f were not significantly altered (Fig. 1F).
Both the activation and deactivation kinetics of P currents were not affected by AEA. The influence of AEA on the steady-state inactivation of P currents was not evaluated due to significant overlapping of the activation and inactivation dependencies (Regan 1991).
To reveal a possible involvement of cannabinoid receptors in the modulation of P current by AEA, we conducted experiments in the presence of the selective CB1R antagonists SR141716A and AM 251. Preapplication of the antagonists themselves produced partial inhibition of P current (in case of SR141716A inhibition of peak amplitude was 35 ± 11%, n = 5). However, a subsequent application of AEA in the presence of antagonist produced the above-described effects on P current. For 10 µM of AEA inhibition of the P-current peak amplitude in the presence of SR141716A was 84 ± 6% (n = 4) compared with 92 ± 5% (n = 5) in control (Fig. 2C).
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, 2000). However, the TRPV1 receptor antagonist capsazepine, did not influence AEA-induced inhibition of P current or acceleration of its inactivation kinetics, demonstrating that these receptors were also not involved in the observed modulation of P current by AEA. Capsazepine itself produced partial inhibition of P current (33 ± 7%, n = 9). Subsequent application of AEA in the presence of the antagonist inhibited P-current amplitude and accelerated the inactivation kinetics in the same manner as under control conditions. On application of 10 µM of AEA, the inhibition of the P-current peak amplitude in the presence of capsazepine was 85 ± 4% (n = 5), as compared with 92 ± 5% (n = 5) in control (Fig. 2C).
It is known that activated G proteins can directly interact with voltage-activated calcium channels to cause inhibition in a voltage-dependent or -independent manner. Strong depolarization can induce relief from voltage-dependent inhibition. To check whether cannabinoid modulation of P current is mediated by G proteins in a voltage-dependent manner, we used a double-pulse protocol (Bean 1989; Elmslie et al. 1990; Grassi and Lux 1989). In these experiments, modulation of P-type channels by AEA was not abolished by strong depolarizing prepulses to +50 mV (Fig. 2, A and B). Taken together these data suggest that the effects produced by AEA on P-currents are not mediated by G-protein-coupled CBRs or other G-protein-coupled receptors.
Another endogenous cannabinoid, 2-AG, induced qualitatively similar effects on P current as AEA; these were inhibition of the peak amplitude by 78 ± 5% (n = 3, at 10 µM) and speed-up of the inactivation kinetics. As in the case of AEA, the effects of 2-AG were use and voltage independent (data not shown).
Modulation of P currents is not mediated by endocannabinoid metabolites
The endogenous cannabinoids AEA and 2-AG are rapidly hydrolyzed in the brain to arachidonic acid (AA) (Bisogno et al. 1997; Hillard et al. 1995). This polyunsaturated fatty acid (structurally related to endocannabinoids) also appears to play an important role in normal intracellular and/or intercellular signal transduction in the nervous system. In particular, AA demonstrates the capacity to modulate different types of neuronal Ca2+ channel activity (Keyser and Alger 1990; Liu et al. 2001; Schmitt and Meves 1995; Zhang et al. 2000). However, modulation of P-type calcium channels by AA has not been reported so far. Therefore we investigated the possibility that accumulation of AA as a result of AEA hydrolysis is responsible for AEA-induced modulation of P-current. To avoid metabolism of AEA, and subsequent AA accumulation, we used the AEA analogue (R)-(+)-methanandamide [(R)-N-(2-hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide]. (R)-(+)-methanandamide to a great extent reproduced the effects of AEA: the mean inhibition induced by 2 µM of (R)-(+)-methanandamide was 60 ± 13% (n = 3, P < 0.05). Similar to AEA, (R)-(+)-methanandamide produced considerable acceleration of the inactivation kinetics of P current (Fig. 3, A and C). Thus these data suggest that AEA itself, and not an AEA metabolite, directly modulates P currents.
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In a separate set of the experiments, we studied modulation of P current by arachidonic acid and compared this with the effects of cannabinoids. AA itself affected P current in a qualitatively similar manner to AEA and (R)-(+)-methanandamide, but with a significantly lower efficacy (Fig. 3 B, D, and F). At a concentration of 10 µM, AA inhibited P currents by 70.6 ± 2% (n = 3, P < 0.01) and slightly accelerated the P-current inactivation kinetics. These data are consistent with earlier data on AA-induced inhibition of L- and N-type calcium channels in rat sympathetic neurons as well as of native and recombinant T-type calcium channels (Liu et al. 2001; Zhang et al. 2000). However, it should be noted that, in contrast to the biphasic effect of AA (transient enhancement followed by inhibition) reported for N-type channels (Barrett et al. 2001; Liu et al. 2001), in our experiments AA induced only inhibition of P current.
Taken together, our results demonstrate that AA mimics the effects of endocannabinoids on P current, but displays a significantly lower affinity compared with that of endocannabinoids. Therefore taking into account the structural similarity between AA and cannabinoids, and the similar effects they have on P currents, one can suggest that both compounds interact with the same site(s) on the P-type channel. However, it remains to be elucidated whether this action is exerted by binding to the channel protein itself or by an indirect modification of the lipid environment.
Effects of the synthetic selective CB1R agonist WIN 55,212–2 on P currents
A selective CB1R agonist, aminoalkylindole WIN 55,212–2, in concentrations of 1–50 µM also produced inhibition of the peak P current (however, less prominent than AEA). The mean inhibition induced by 10 µM of WIN 55,212–2 was 68 ± 10% (n = 7). Similarly to AEA, WIN 55,212–2 induced acceleration of the inactivation kinetics of P current (Fig. 4A). Inhibition induced by WIN 55,212–2 was not use dependent, and it was incomplete even when a concentration of 50 µM was applied (Fig. 4B).
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S, was used. Loading of the cell with 0.5 mM GDPS did not affect inhibition induced by 10 µM of WIN 55,212–2. As in the case of AEA, application of strong depolarizing prepulses failed to reveal the involvement of G proteins in the effects induced by WIN 55,212–2 (Fig. 5, A and B). The effects of WIN 55,212–2 were also not abolished by both CB1 cannabinoid and vanniloid receptor antagonists (Fig. 5C).
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Action of cannabinoids on P currents is not mediated by intracellular calcium or intracellular protein kinases
To test whether Ca2+ influx through P-type channels is involved in the modulation of P-currents by cannabinoids, we performed a set of experiments using Ca2+ as P-current carrier, instead of Ba2+ (used in the majority of experiments). The effects produced by WIN 55,212–2 in these conditions were the same as in the experiments with Ba2+ as the P-current carrier [inhibition of the peak amplitude was, on average, 61.8 ± 2% (n = 3) in the presence of extracellular Ca2+ compared with 68 ± 10% in the presence of Ba2+]. These data indicate that the entry of Ca2+ into the cell via P-type calcium channels is not important for cannabinoid-induced modulation of P current.
It is known that cannabinoid agonists can stimulate numerous signaling pathways through both receptor- and nonreceptor-mediated intracellular cascades (for review, see Howlett et al. 2002). In particular, it was shown that cannabinoid agonists can modulate ion channels both through G-protein-mediated inhibition of cAMP production and through a CBR-independent increase in the concentration of intracellular calcium (Felder et al. 1993). Intracellular signaling pathways activated by cannabinoids in the CNS include also MAP kinase (Bouaboula et al. 1995) and probably, c-Jun N-terminal kinase via a phosphoinositide 3'-kinase-dependent process (Rueda et al. 2000). Additionally, Netzeband et al. (1999) showed that CB1Rs can interact with phospholipase C, leading to the mobilization of intracellular Ca2+ (Netzeband et al. 1999). To explore possible involvement of intracellular pathways initiated by cannabinoid application, we conducted experiments with a nonspecific protein kinase inhibitor, H89 (1 µM); it is active against PKA, PKC, and CaMKII. However, this treatment failed to alter the effects induced by WIN 55,212–2 on P current (inhibition of the peak amplitude was, on average, 78 ± 5% (n = 3)). Furthermore, to test the involvement of Ca2+ released from intracellular stores, we performed experiments after a preexposure of neurons with caffeine (20 mM), which induces a massive rise of the intracellular Ca2+ level. Under these conditions, cannabinoid–induced effects on P-currents also remained unaltered [inhibition of the peak amplitude was 72 ± 3% (n = 3)].
WIN 55,212–2 increases the firing rate of spontaneous firing in Purkinje neurons in the presence of CB1R antagonist.
To address the functional implication of the direct action of cannabinoids on P-type calcium currents in cerebellum, we tested the effects of WIN 55,212–2 on spontaneous activity of Purkinje neurons using whole cell current-clamp recordings in cerebellar slices. Purkinje neurons display spontaneous and rhythmic activity characterized by prolonged periods of bursting followed by periods of quiescence (Llinas and Sugimori 1980). P-type Ca2+ channels have been shown to play an important role in eliciting repetitive firing. According to Womack et al. (2004), blocking only half of the P/Q-type voltage-gated calcium channels (corresponding to removal of 25% of total action potential-evoked calcium entry) caused a significant increase in the rate and decrease in the regularity of Purkinje neuron spontaneous firing via decreased recruitment of calcium-activated potassium channels (Womack et al. 2004). Therefore we expected that inhibition of P current by WIN 55,212–2 should increase the firing rate. We compared the firing rate during the first second of spontaneously occurring firing episodes after a quiescence period. Experiments were performed in the presence of SR141716A to prevent CB1R activation. We have found that WIN 55,212–2 (10 µM) indeed significantly increased the rate of firing compared with control to 192 ± 38% (P < 0.05, n = 4). Thus cannabinoids modulate intrinsic membrane properties involved in Purkinje cell firing in a CB1R-independent manner.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The pharmacological profile of cannabinoid-induced effects on P-type calcium current was different from that of classical cannabinoid receptors. The order of potency of different cannabinoids for inhibition of P current differs from that observed for CB1R activation. The synthetic cannabinoid agonist WIN55,212–2 is much more potent at activating the CB1R compared with endogenous cannabinoids such as AEA (Breivogel et al. 2001), whereas AEA was more potent at modulating P currents. It should be noted, that the effective concentration range for AEA effects on P current, as estimated from our experiments (IC50 = 1.04 ± 0.04 µM) is comparable to that for activation of the CB1R (EC50 = 1.4 ± 0.3 µM) (Breivogel et al. 2001). These data indicate that CB1R-mediated and direct effects of cannabinoids on P channels could occur on release of the same amount of endogenous cannabinoids.
Although our results clearly indicate that cannabinoids can directly modulate P-type calcium current without activation of CB1Rs or TRPV1 receptors, the mechanism underlying this modulation remains unclear. It could be that cannabinoids interact with modulatory sites within the P-type channel. However, because of the highly lipophilic properties of cannabinoids, we cannot exclude the possibility that they alter P-type calcium channel gating properties by an interaction with the plasma membrane lipid bilayer. In this respect it is important to note that the endogenous cannabinoid AEA has dynamic properties similar to those of membrane phospholipids, producing no perturbation to the bilayer (Tian et al. 2005). Nevertheless, irrespective to the exact mechanism, it is clear that modulation of P current functioning by cannabinoids could occur under physiological conditions.
Activation of CB1Rs accounts for most of the behavioral effects of cannabimimetic drugs. However, although most of these effects are absent in CB1R-deficient mice, the endogenous cannabinoid AEA still induce catalepsy, analgesia, and decreasing spontaneous activity in these mice (Baskfield et al. 2004; Di Marzo et al. 2000). Furthermore, in mice, the typical cannabimimetic effects of AEA on spontaneous activity, body temperature, and pain perception are not reversed by treatment with the selective CB1R antagonist SR141716A (Adams et al. 1998). These data indicate the existence of functionally important non-CB1R targets for brain cannabinoid signaling. Indeed, numerous studies have shown that cannabinoids may exhibit pharmacological action on different voltage- and ligand-gated ion channels and receptors independently of CB1/2 Rs (for review, see Di Marzo et al. 2002; Oz 2006). For example, AEA at low micromolar range of concentrations inhibits T-type calcium channels, Shaker-related KV 1.2 K+ channels (Chemin et al. 2001; Poling et al. 1996). Moreover, it was shown, that AEA can directly interact with serotonin receptor 5-HT3A (Godlewski et al. 2003), NMDA receptor (Hampson et al. 1998), nicotinic acetylholine receptor (nACh 
7) (Oz et al. 2003), and inhibitory glycine receptors GlyR (Hejazi et al. 2006; Lozovaya et al. 2005). Here we demonstrate that P-type Ca2+ channels in Purkinje neurons can also be an important target for direct, non-CBR mediated endocannabinoid signaling.
Activation of P/Q-type voltage-gated calcium channels strongly contributes to neurotransmitter release in a majority of mammalian central synapses (Regehr and Mintz 1994; Wheeler et al. 1994). Even subtle differences in the gating properties of these channels may strongly influence the efficacy of synaptic transmission. The activity of P/Q-type calcium channels can be modulated in various ways, including inhibition by G proteins (Currie and Fox 1997; Dolphin 1995; Regehr and Mintz 1994; Sun and Dale 1999) and feedback regulation by Ca2+/calmodulin (Lee et al. 1999). The present data imply that activity of both pre- and postsynaptic P-type channels in Purkinje neurons can also be directly modulated by cannabinoids. These channels can be located close to the points of release of endocannabinoids in the postsynaptic cell, where the concentration of endocannabinoids can transiently reach quite high levels. Indeed it has been shown recently that in the cerebellum, a major 2-AG biosynthetic enzyme, diacylglycerol lipase- (DAGL) was predominantly expressed in Purkinje cells. DAGL distribution was selective to somatodendritic elements of Purkinje neurons with the highest level in spiny branchlets (Yoshida et al. 2006). On the other hand, P-type calcium channels are also found to be expressed intensely on Purkinje cell dendrites, spiny branchlets, and spines with weaker reactivity present in the soma (Hillman et al. 1991). Thus endocannabinoids could play a role of "local" messenger that mediates feedback inhibition of P currents on depolarization.
Purkinje neurons occupy a central position in the cerebellar circuitry: they integrate excitatory imputs from climbing and parallel fibers into, respectively, "complex" or "simple" spikes (Eccles 1967). Signals for motor coordination and balance are coded in the rate and pattern of firing of cerebellar Purkinje neurons, the only neurons that project out of the cerebellar cortex (Ito 1984). The activity of a Purkinje neuron is controlled both by synaptic input and by intrinsic conductances that cause it to fire spontaneously (Nam and Hockberger 1997; Womack and Khodakhah 2002).
In cerebellar Purkinje neurons under physiological conditions, Ca2+ entry via P-type calcium channels during action potentials is coupled to the activation of Ca2+-activated potassium current so that the net current attributable to Ca2+ entry is outward (Raman and Bean 1999). This implies an important role of P-type Ca2+ channels in eliciting repetitive firing. Partial block of P/Q-type calcium channels in spontaneously firing Purkinje neurons results in a significant increase in firing rate and bursting (Womack and Khodakhah 2002). By current-clamp whole cell recordings, we demonstrate that WIN 55,212–2 also induces increasing of firing rate in Purkinje neurons in cerebellar slices in a manner similar to that of P-type calcium channel-selective toxin (Womack et al. 2004). Therefore it can be suggested that inhibition of P/Q channels by endocannabinoids, released by strong depolarization, can occur under conditions of high-frequency activity. Consequently this could result in a time-dependent alteration of firing frequency.
Modulation of P-type current inactivation by synthetic and endogenous cannabinoids is of particular interest. We suggest that in addition to well-known calmodulin-mediated modulation (Lee et al. 1999), this endocannabinoid-mediated acceleration of inactivation could contribute to the mechanisms of Ca2+-dependent inactivation of P-type channels. Accumulation of intracellular calcium during the train of stimuli would lead to the liberation of the endocannabinoids and subsequent enhancement of P-type channels inactivation. These relatively slow modulatory effects of cannabinoids can be especially pronounced in rounds of repeating activity.
Abnormal Purkinje cell activation, which results in inhibition of the deep cerebellar nuclei, induces symptoms of cerebellar dysfunction. For example, in several human and animal models, mutations that decrease P/Q-current density cause cerebellar ataxia (Pietrobon 2002). Interestingly, cannabinoids can also cause static and gait ataxia in dogs and mice, and this is attributed to cerebellar dysfunction (Dar 2000; Patel and Hillard 2001). Therefore it could be that direct modulation of P current by cannabinoids is involved in cannabinoid induced ataxia symptoms.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Lozovaya, Dept. of Cellular Membranology, Bogomoletz Institute of Physiology, 4 Bogomoletz St., Kyiv 01024, Ukraine (E-mail: n_lozovaya{at}yahoo.com); N. Burnashev, Dept. of Experimental Neurophysiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands (E-mail: nail.burnashev{at}falw.vu.nl)
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