Cannabidiol-induced intracellular Ca2+ elevations in hippocampal cells

Neuropharmacology 50 (2006) 621e631 www.elsevier.com/locate/neuropharm

Cannabidiol-induced intracellular Ca2þ elevations in hippocampal cells Alison J. Drysdale, Duncan Ryan, Roger G. Pertwee, Bettina Platt* School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK Received 7 October 2005; received in revised form 9 November 2005; accepted 15 November 2005

Abstract The phytocannabinoid cannabidiol (CBD) is at the forefront of therapeutic cannabinoid research due to its non-psychotropic properties. Research supports its use in a variety of disorders, yet the cellular mechanisms of its action remain unclear. In this study, the effect of CBD upon Ca2þ homeostasis in hippocampal cells was characterised. CBD (1 mM) elevated intracellular Ca2þ ([Ca2þ]i) by wþ45% of basal Ca2þ levels in both glia (77% responders) and neurones (51% responders). Responses to CBD were reduced in high excitability HEPES buffered solution (HBS), but not affected in low excitability/low Ca2þ HBS. CBD responses were also significantly reduced (by 50%) by the universal Ca2þ channel blocker cadmium (50 mM) and the L-type specific Ca2þ channel blocker nifedipine (20 mM). Interestingly, intracellular store depletion with thapsigargin (2 mM) had the most dramatic effect on CBD responses, leading on average to a full block of the response. Elevated CBD-induced [Ca2þ]i responses (>þ100%) were observed in the presence of the CB1 receptor antagonist, AM281 (1 mM), and the vanilloid receptor antagonist, capsazepine (CPZ, 1 mM). Overall, our data suggest that CBD modulates hippocampal [Ca2þ]i homeostasis via intracellular Ca2þ stores and L-type VGCC-mediated Ca2þ entry, with tonic cannabinoid and vanilloid receptor signalling being negatively coupled to this pathway. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cannabidiol; Calcium; CB1; Vanilloid; Voltage-gated calcium channels; Intracellular calcium stores; IP3 receptor; Ryanodine receptor

1. Introduction Cannabidiol (CBD) is the major non-psychotropic constituent of Cannabis sativa (marijuana) (for a comprehensive review, see Pertwee, 2004), and has been suggested to be of potential therapeutic benefit in a number of applications e.g. epilepsy, neurodegenerative disease, inflammation, cancer and glaucoma (Drysdale and Platt, 2003; Platt and Drysdale, 2004). CBD-containing compounds have been tested in clinical trials (e.g. Whittle et al., 2001; Wade et al., 2004), and approved for symptom relief of neuropathic pain in some countries. On the cellular level, CBD is reported to bind to the two cannabinoid receptors, CB1 and CB2, with low affinity (Showalter et al., 1996; Bisogno et al., 2001; Pertwee, 2004), but able to antagonise other cannabinoids such as WIN-55,212 and CP55940, via CB1/2 independent mechanisms (Pertwee et al., 2002, 2005). Additionally, stimulation of vanilloid receptors * Corresponding author. Tel.: þ44 1224 555741; fax: þ44 1224 555719. E-mail address: [email protected] (B. Platt). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.11.008

(VR1) and inhibition of hydrolysis and uptake of the endocannabinoid anandemide (AEA) has been reported in the micromolar range, potentially linking CBD to a stimulation of the endocannabinoid system (Bisogno et al., 2001; Costa et al., 2004). Thus, although neuroprotective and neuromodulatory effects of CBD and other cannabinoids have been elucidated, the exact mechanisms by which they occur remain elusive. Whilst only the two afore-mentioned cannabinoid receptors have been cloned and characterised to date, many cannabinoids, including CBD, have been shown to exert their actions via independent pathways, providing strong evidence for further, as yet uncharacterised, cannabinoid receptors (Pertwee et al., 2002; Pertwee et al., 2005). Moreover, the CBD analogue, abnormal-CBD operates via a G protein coupled receptor (GPCR) distinct from both CB1 and CB2 receptors in rat isolated mesenteric artery models (Begg et al., 2003; Offertaler et al., 2003). Accordingly, studies in cannabinoid receptor knockout mice have provided further evidence for novel receptor types, with one particular novel receptor type being localised in the hippocampus (e.g. Breivogel et al., 2001).

622

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

With regards to receptor distribution in the CNS, until recently only neuronal cells were thought to express cannabinoid receptors, but cannabinoid receptors on microglial cells have now been proposed (Waksman et al., 1999; Nunez et al., 2004). Interestingly, recent research has shown that both endogenous cannabinoids and phytocannabinoids are potentially able to prevent microglial recruitment during neuroinflammation thereby limiting their exacerbation of cell damage (Walter et al., 2002; Walter et al., 2003). A putative link between neuroprotection and cannabinoids may be via the modulation of [Ca2þ]i homeostasis, vital to the maintenance of healthy physiological function, and disturbed in diverse CNS disorders ranging form acute trauma to neurodegenerative diseases. CBD’s effects upon [Ca2þ]i levels have been studied in leukocytes (Kaplan et al., 2003) and other cannabinoids have been proven to modulate Ca2þ homeostasis in the hippocampus. Mechanisms by which cannabinoids may affect Ca2þ homeostasis include the modulation of NMDA receptor stimulation (Shohami et al., 1993; Eshhar et al., 1995; Nadler et al., 1995; Shohami et al., 1997; Shen and Thayer, 1998; Netzeband et al., 1999); inhibition of voltage-gated calcium channels (VGCCs) (Caulfield and Brown, 1992; Mackie and Hille, 1992; Twitchell et al., 1997) and Kþ channels (Hampson et al., 2000; Robbe et al., 2001), and via gap junction modulation (Venance et al., 1995). Since most in vitro results attained to date were in studies of synthetic and endogenous cannabinoids (eCBs) such as WIN55,212-2, CP55,940 and AEA, it was therefore of interest to characterise responses to CBD in hippocampal culture. Data presented here identify CBD as a potent modulator of [Ca2þ]i, and a pharmacological characterisation of this action is provided for both neurones and glia. Some aspects of this work have been presented in abstract form (Drysdale et al., 2005). 2. Methods 2.1. Hippocampal cultures All procedures were performed according to institutional guidelines in compliance with the UK Home Office Animals (Scientific Procedures) Act, 1986. Hippocampal tissue was dissected from 1e3 day old neonatal Sprague-Dawley rats and placed in ice-cold HEPES buffered solution (HBS; composition: 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 25 mM D-glucose (all compounds from VWR International, Poole, UK, excluding HEPES from SigmaeAldrich, Poole, UK; pH 7.4). Tissue was then chopped into fine pieces and transferred to protease solution (type X and type XIV, each 1 mg/ml HBS; SigmaeAldrich) where it was enzymatically dissociated for 35e40 min at room temperature. Afterwards, tissue was washed twice in 2-ml volumes of HBS and then repeatedly triturated before double centrifugation. The resulting cell pellet was resuspended in 1.5 ml minimal essential medium (MEM) supplemented with 10% foetal bovine serum (FBS) (Invitrogen Ltd., Paisley, UK) and 1% 2 mM L-glutamine (SigmaeAldrich). This suspension was then plated onto poly-L-lysine-coated (0.02 mg/ml, SigmaeAldrich) culture dishes and incubated for 45e60 min (37
C in a humidified atmosphere of 5% CO2 in air). Previously incubated and supplemented MEM was then added to establish a final volume of 2 ml per culture dish. At 2 days in vitro (DIV), medium was changed to Neurobasal-AÔ medium supplemented with 2% B27 (Invitrogen) to promote neurones and reduce glia proliferation.

2.2. Fura-2 AM Ca2þ imaging Experiments were typically conducted using hippocampal cultures at 5e 10 days in vitro (DIV). Cultures were washed in HBS and incubated for 60 min in the dark at room temperature, with 10 mM cell-permeable fluorescent Ca2þ indicator, Fura-2-AM (Cambridge Bioscience, Cambridge, UK). Cells were then washed again with HBS and all experiments performed at room temperature. Tetrodotoxin (TTX, 0.5 mM; Alomone Labs, Jerusalem, Israel), a sodium channel blocker, was added to all perfusion media to inhibit spontaneous cell firing and transmitter release. Cultures were perfused at a rate of w2 ml/min. Under the microscope (Olympus, 40 objective), a suitable field of cells was selected and captured in greyscale using the UltraView software (PerkineElmer, Beaconsfield, UK). Ratiometric imaging was conducted using alternating excitation wavelengths of 340 and 380 nm, from a Xenon lamp regulated by a monochromator (Spectromaster I, PerkineElmer) and an emission filter (wavelength 510 nm). Background levels of fluorescence were calculated and subtracted prior to the commencement of experiments. Image acquisition rate was set to 3 s and ratio values plotted against time for multiple regions of interest (ROIs, neurones and glia, determined by morphological analysis and by fast neuronal responses to application of KCl or NMDA.

2.3. Drug application HBS was the standard perfusion solution used (see above). To achieve low excitability (low ex.) and high excitability (high ex.) HBS, constituents were altered to 2 mM KCl, 1 mM CaCl2, 3 mM MgCl2 and 7 mM KCl, 3 mM CaCl2, 0.5 mM MgCl2, respectively. Nifedipine and cadmium (SigmaeAldrich) were applied at 20 and 50 mM respectively, for a period of 20 min prior to 1 mM CBD application. AM281 (Tocris Cookson Ltd., Avonmouth, UK, stock: 1 mM in DMSO) and CPZ (SigmaeAldrich, stock: 100 mM in DMSO) were applied at 1 mM for 10 min prior to 1 mM CBD application. Thapsigargin (2 mM; Alomone Labs, Jerusalem, Israel, stock 1 mM in DMSO) was applied for five minutes previous to 1 mM CBD application.

2.4. Statistical analysis Experimental values obtained were ratio units, since ratio unit values were found to show highest reproducibility between experiments whilst quantitative calibration produced variable and unreliable data, in agreement with previous reports (Xiong et al., 2004). Graphical illustration and statistical analyses (other than baseline comparisons) were conducted after calculation of change in fluorescence (DF ) as a percentage of the average resting fluorescence (F ) i.e. as % DF/F (e.g. Clodfelter et al., 2002). Statistical analyses were performed using GraphPad PrismÒ statistics package with a KruskaleWallis test due to the non-parametric distribution of data, followed by a Dunn’s multiple comparison post hoc test unless stated otherwise. To directly compare peak responses to drugs within and between experiments, non-parametric paired (Wilcoxon matched pairs test) and unpaired (ManneWhitney U-test) t-tests were used, respectively. In experiments to determine response magnitude of first versus second CBD applications, Wilcoxon Signed Rank statistical analyses were used, where values were calculated as a percentage of the first CBD response and compared to a theoretical median of 100%. The response rate was calculated as a percentage of viable cells (i.e. those responding to 50 mM NMDA or 25 mM KCl probes).

3. Results 3.1. CBD raises Ca2þ in both neurones and glia In the first set of experiments, the effect of a 5-min application of 1 mM CBD was investigated after establishment of a steady baseline for 10 min (Fig. 1A). The CBD application was found to increase [Ca2þ]i by a similar degree in both neurones and glia (glia: 43  3% of baseline fluorescence, n ¼ 78,

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

A

170

170

160

160

150

150

140

140

∆F/F

∆F/F

A

130 120

623

130 120

110

110

100 100 90 90

80

1 µM CBD

1 µM CBD

70 0

5

10

15

20

25

0

30

5

10

B

50

25

30

35

40

45

90

70

30 20 77

51

0

Glia

Neurones

Fig. 1. (A) Time course of CBD-induced [Ca2þ]i responses. Sample time course of n ¼ 9 ROIs (5 glia in grey, 4 neurones in black) depicting their response to a 5-min application of 1 mM CBD (grey bar) as % DF/F versus time. (B) Comparison of CBD induced [Ca2þ]i response by cell type (glia: n ¼ 78; neurones: n ¼ 73). Responder rates are shown within each bar (in %).

neurones: 454%, n ¼ 73), as summarised in Fig. 1B. Interestingly, fewer neurones (51%) responded to the CBD application than glia (77%), while control experiments conducted with 0.1% DMSO (vehicle concentration for CBD application) showed no significant effects upon [Ca2þ]i levels (data not shown). To assess whether the observed transient rise in [Ca2þ]i leads to delayed cell death, 48-h incubations with 1 and 10 mM CBD were conducted and cytochemical analysis with propidium iodide showed no significant cell death (data not shown). Importantly, the majority of cells showed a sustained Ca2þ level, not only in the presence of CBD but also after washout (see Fig. 1A). Following the CBD application, only <10% of glia and <20% of neurones fully recovered to within 0.1 ratio units of baseline [Ca2þ]i (n ¼ 4 glia, 8 neurones), in agreement with previous reports on poor recovery from cannabinoid application in other preparations (e.g. Pertwee et al., 1996) The remainder of cells either showed partial or no recovery (see Fig. 1B). In a second set of experiments the reproducibility of the CBD responses was determined, with a second dose of 1 mM CBD applied after a 15-min recovery period. Of those cells initially responding to 1 mM CBD, approximately 40% of glia and 60% of neurones could elicit a second response, albeit a smaller one calculated relative to the new, non-recovered baseline. A sample time course is shown for the most common response pattern (Fig. 2A), i.e. a significantly decreased second response (P < 0.05) in cells that did not recover from the initial application. When data were grouped based on the

of initial response

∆F/F

20

80

40

10

15

Time (minutes)

Time (minutes)

B

1 µM CBD

80

60 50 40 30 20 10

G 69 N 56

G 26 N 26

G5 N 19

Partial recovery

Full recovery

0 No recovery

Combined

Fig. 2. (A) Sample time course depicting response to two 5-min applications of 1 mM CBD (n ¼ 5 ROIs, all glia) with a 15-min recovery period between applications, as % DF/F versus time. (B) Magnitude of response to the second 1 mM CBD application as a percentage of the first response categorised according to their ability to recover from the initial CBD application. Values shown in boxes depict % responders, grouped according to recovery, i.e. no (n ¼ 54 glia, 24 neurones), partial (n ¼ 20 glia, 11 neurones) or full (n ¼ 4 glia, 8 neurones) recovery (G, glia; N, neurones). The fourth bar shows the combined mean response amplitude of all responding cells.

criterion of recovery, the magnitude of the second response was found to be smallest in cells that showed full recovery (Fig. 2B). Based on these observations, it was concluded that no fully reproducible CBD responses could be obtained within one experiment, thus requiring independent experiments for pharmacological characterisation. 3.2. Contribution of intra- and extracellular Ca2þ sources to the CBD response In order to investigate the source of Ca2þ for the CBDinduced [Ca2þ]i rise, the influence of altered extracellular solution composition upon the CBD-induced Ca2þ response was investigated. Experiments were conducted in each of three solutions - control (as above, in standard HBS), high excitability (high ex.) and low excitability/low Ca2þ (low ex.) solutions (see Section 2). Nominally Ca2þ-free experiments were not conducted since this was found to cause leakage and partial depletion of Ca2þ from intracellular stores, thus leading to

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

A

Glia

160 High Ex HBS Low Ex HBS Standard HBS

150 140 130

∆F/F

diverse and unspecific alterations of hippocampal Ca2þ signalling (for data on the role of intracellular stores, see below). Initial basal [Ca2þ]i levels were compared and found to differ significantly between the different HBS solutions (P < 0.0001), with significantly increased basal [Ca2þ]i in both glia and neurones in high excitability HBS compared to control (P < 0.05 glia, n ¼ 49; P < 0.001 neurones, n ¼ 67; see Fig. 3). Also, neuronal basal [Ca2þ]i levels were significantly higher than glial levels in both high and low excitability solutions (P < 0.05 high ex. HBS, n ¼ 67; P < 0.01 low ex. HBS, n ¼ 54). Mean time courses for three sample experiments in each of the three perfusion solution types for both glia and neurones are shown in Fig. 4A and B. Interestingly, the CBD-induced Ca2þ rise was significantly smaller in neurones perfused with high excitability HBS (P < 0.001, n ¼ 64), as summarised in Fig. 5A and B, providing evidence against the influx of Ca2þ from extracellular sources as the main source. However, the percentage of responding cells in both high and low excitability HBS showed increased rates compared to controls in both modified HBS solutions (>90% in each category). This is indicative of the crucial role of Ca2þ gradients and intracellular Ca2þ levels in CBD signalling. To further elucidate the potential role VGCCs may play in the Ca2þ rise evoked by CBD, a series of experiments were conducted whereby a 10-min application of the general Ca2þ channel blocker cadmium (50 mM) preceded CBD application (Fig. 6A and B). In addition, experiments with the selective Ltype Ca2þ channel blocker nifedipine (20 mM) were performed

120 110 100 90

1 µM CBD

80 0

5

10

15

20

25

20

25

30

Time (minutes)

B

Neurones 160 Standard HBS Low Ex HBS High Ex HBS

150 140 130

∆F/F

624

120 110 100 90

1 µM CBD

** 80

*

1.0

0

***

[Ca2+]i (ratio units)

0.9

0.7

0.6

0.5

(N )

) (N

.H BS

BS .H

H BS

w Lo

h ig

Ex

Ex

d. St

H

Lo

w

Ex

Ex h ig H

(N )

(G )

.H

.H BS

BS

(G

(G )

)

0.4 H BS

15

30

Fig. 4. Sample mean time courses of three sample experiments as % DF/F versus time, in extracellular solutions differentially affecting excitability. In (A) glia, standard HBS (n ¼ 22); high excitability HBS (n ¼ 7); low excitability HBS (n ¼ 21) and in (B) neurones, standard HBS (n ¼ 13); high excitability HBS (n ¼ 26); low excitability HBS (n ¼ 21).

0.8

d.

10

Time (minutes)

*

St

5

Fig. 3. Comparison of basal [Ca2þ]i values (presented as ratio units) in experiments where cells were perfused with extracellular solutions differentially affecting excitability: standard HBS (5.4 mM KCl, 1.8 mM CaCl2 and 1 mM MgCl2), high excitability HBS (7 mM KCl, 3 mM CaCl2 and 0.5 mM MgCl2), n ¼ 49 glia, 67 neurones; or low excitability HBS (2 mM KCl, 1 mM CaCl2 and 3 mM MgCl2), n ¼ 78 glia, 54 neurones. Significances are indicated for comparison with the respective control in standard HBS (std. HBS), and for comparison between cell types. *P < 0.05; **P < 0.01; ***P < 0.001. G: glia; N: neurones.

(Fig. 6C and D). As summarised in Fig. 7, the amplitude of the CBD-induced [Ca2þ]i rise was significantly decreased compared to controls (P < 0.001 for both glia and neurones, n ¼ 78 and 35 respectively in cadmium and n ¼ 38 and 13 respectively in nifedipine). No significant difference was observed between cell types for either channel blocker (P > 0.05). In addition, the percentage of responsive glial cells was markedly increased to 93% in cadmium and 100% in nifedipine from 77% in control, and in neuronal cells this increased from 51% in control to 88% in cadmium but remained at 50% in nifedipine. Since our data suggested that interference with extracellular sources of Ca2þ cannot be the main route that causes a Ca2þ rise by CBD, potential Ca2þ release from intracellular stores was subsequently investigated. The cell-permeable intracellular store depleting agent thapsigargin, which potently and irreversibly inhibits ER Ca2þ-ATPases, was used. This compound

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

A

in the majority of cells, and responsible for the sustained nature of Ca2þ responses in all hippocampal cells.

Glia

60 50

3.3. Are cannabinoid or vanilloid receptors involved in the CBD response?

∆F/F

40 30 20 10 99

96

77

BS H Ex

H

ig h

Lo w

Ex

St an da rd

H

H

BS

BS

0

B

Neurones

60 50

***

40

∆F/F

625

30 20

The next set of experiments addressed the involvement of CB1 and VR1 receptors in the CBD response using the selective CB1 receptor antagonist AM281, and the VR1 antagonist CPZ, a synthetic analogue of capsaicin. When CBD was applied in the presence of AM281 (1 mM) (Fig. 9A) the resulting [Ca2þ]i was significantly increased (glia 113  17%, n ¼ 31, neurones 107  16%, n ¼ 25) compared to na€ıve CBD applications (P < 0.001). In order to investigate the possibility that CBD may exert its effect via vanilloid receptors, CPZ was administered for ten min previous to the 1 mM CBD application (see Fig. 9B). In CPZ-treated cells, the CBD-induced [Ca2þ]i rise was also found to be significantly increased (186  22% in glia, n ¼ 20, 154  15% in neurones, n ¼ 25; P < 0.001). Only in glia was the response significantly higher in CPZ compared to AM281 (P < 0.05; see Fig. 9C). The percentage of responsive cells was lower in the presence of AM281 than in CPZ (49% glia, 36% neurones in AM281; 74% for both cell types in CPZ). 4. Discussion

10 51

86

92

BS H Ex w Lo

h ig H

St

an

da

rd

Ex

H

H

BS

BS

0

Fig. 5. Comparison of CBD induced [Ca2þ]i response in extracellular solutions differentially affecting excitability (A) in glia (standard HBS, n ¼ 75; high excitability HBS, n ¼ 48; low excitability HBS, n ¼ 78) and (B) in neurones (standard HBS, n ¼ 74; high excitability HBS, n ¼ 64; low excitability HBS, n ¼ 50). Values within each column represent the percentage of all viable cells responding to the CBD application. Significance is indicated for comparison with controls in standard HBS (***P < 0.001).

stimulates initial release of Ca2þ from intracellular stores thence preventing any subsequent store-dependent responses from occurring, and eventually leads to apoptosis. After application of thapsigargin, mean responses to CBD were found to be fully blocked in glia and neurones (17  4% and 7.5  7%, respectively). Further analysis revealed that CBD responses in the presence of thapsigargin can be categorised into two different subsets of responses: (i) a proportion of cells (15% of glia, 20% of neurones) showed a CBD response, with the peak response comparable in size to na€ıve conditions (P > 0.05), but with a non-sustained profile (see Fig. 8A and C), or (ii) responses to CBD led to a decrease in [Ca2þ]i from pre-CBD baseline [Ca2þ]i levels (66% of glia, 26% of neurones, Fig. 8B and D, P < 0.001). Fig. 8E and F summarise response size and responder rates by cell type. These data indicate that intracellular stores are the main source of the Ca2þ rise caused by CBD

4.1. The effect of CBD upon Ca2þ homeostasis Although CB agonist-stimulated Ca2þ release has been observed in other studies (Netzeband et al., 1999; Chou et al., 2001), this study is the first to demonstrate the modulation of intracellular [Ca2þ]i in primary hippocampal culture by CBD. Both neurones and glia responded, with the latter having a higher responder rate, providing first evidence that this CBD effect is unlikely to involve CB1 receptors, which are reportedly absent in glia (e.g. Howlett et al., 2002). Since differential distribution of cannabinoid receptors and related differential cellular responses have been found in many preparations, this observation in itself points towards a common, potentially intracellular, side of action. Nevertheless, the rise in Ca2þ caused by CBD may be able to induce the production of endocannabinoids (Brenowitz and Regehr, 2003; Van der Stel et al., 2005), thus potentially linking CBD’s action to CB1/x receptormediated pathways (see Fig. 10). Also involved here may be the suggested inhibition of AEA hydrolysis and uptake by CBD, thus potentially raising the AEA tone, though higher CBD concentrations (>20 mM) may be required for such an action (Bisogno et al., 2001). Since higher eCBs levels are more likely to reduce rather than raise Ca2þ (as also indicated by our results obtained with cannabinoid antagonists, see below), this potential connection cannot explain the rise in Ca2þ caused by CBD. Ca2þ responses induced by CBD were found to be sustained under control conditions, with recovery observed in less than 10% of glia and less than 20% of neurones. The remainder showed either partial recovery or no recovery at all.

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

626

Glia

135

B

130

125

125

120

120

115

115

110

110

105

105

100

100

95

95

1 µM CBD 50 µM Cadmium

90

Neurones

135

130

∆F/F

∆F/F

A

1 µM CBD 50 µM Cadmium

90

85

85 0

5

10

15

20

25

0

30

5

10

Glia

145 140 135 130 125 120 115 110 105 100 95 90 85 80

D

∆F/F

∆F/F

C

1 µM CBD 20 µM Nifedipine

0

5

10

15

20

15

20

25

30

Time (minutes)

Time (minutes)

25

30

35

Time (minutes)

Neurones

145 140 135 130 125 120 115 110 105 100 95 90 85 80

1 µM CBD 20 µM Nifedipine

0

5

10

15

20

25

30

35

Time (minutes)

Fig. 6. Sample time courses of CBD induced [Ca2þ]i responses in experiments following a 10-min application of 50 mM cadmium in (A) glia (5 representative sample ROIs), (B) neurones (5 representative sample ROIs); or following application of 20 mM nifedipine (C) glia (5 representative sample ROIs) in 10 mM, (D) neurones (5 representative sample ROIs) in 20 mM.

Overall, these data indicate a lack of a desensitising response in the continuous presence of CBD. Of the cells that showed an initial response to CBD, w40% of glia and w60% of neurones could elicit a second, albeit smaller response. Intriguingly, where a cell recovered most fully from the initial CBD application, their response to the second application was smaller compared to those that showed partial or no recovery. Thus, sustained responses did not occlude subsequent signalling. The lack of recovery could potentially be due to the lipophilic nature of CBs, as they are not efficiently washed in most experimental conditions. However, different rates of recovery observed in subsets of cells argue against a mere chemical explanation for incomplete recovery, and also support the hypothesis of a possible intracellular site of action (see also Van der Stel et al., 2005), leading to sustained Ca2þ release of from intracellular stores, as supported by data obtained with thapsigargin (see below and Fig. 10). 4.2. Determination of the Ca2þ source for the CBD-induced Ca2þ response There are disadvantages to absolutely excluding Ca2þ from the extracellular surroundings since all intracellular and

transmembrane gradients are affected and intracellular stores depleted. Instead, by modifying the components of the perfusion solution, one can alter the cell’s electrochemical gradient and thus affect Ca2þ influx in a more physiological range. High excitability HBS perfusion solution was found to increase basal [Ca2þ]i levels in both glia and neurones, with neuronal basal [Ca2þ]i levels being higher compared to glia, in line with the excitable nature of the former. The CBD-induced [Ca2þ]i elevation was reduced in neurones perfused with high excitability HBS solution compared to standard HBS, indicating that Ca2þ influx from the extracellular space is not the major route. No differences were observed in low excitability HBS, further supporting this hypothesis, since a decrease in the CBD-induced [Ca2þ]i response would be expected under conditions of a reduced Ca2þ gradient. Accordingly, in neuroblastoma cells, reduction of extracellular Kþ was found to abolish desacetyllevonantrodol (DALN)-stimulated Ca2þ uptake (Rubovitch et al., 2002), and previous studies on WIN55,212-2 in rat striatal neurones showed that this compound causes Ca2þ influx from extracellular space into intracellular presynaptic terminals upon depolarisation (Huang et al., 2001). Nevertheless, for our data, the percentage of responsive cells was increased in both perfusion solutions and

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

A

Glia

50

∆F/F

40

30

* ***

20

10 77

100

93

if N in CB

N

aï

ve

CB

D

D

CB

D

in

ct

Cd

rl

0

B

Neurones

50

∆F/F

40

30

A large proportion of the CBD-induced [Ca2þ]i response was still apparent in the presence of these channel blockers, again suggesting that VGCC entry does not represent the principal mode of Ca2þ entry. Overall, it appears that interference with routes of Ca2þ entry from extracellular sources may facilitate the responsiveness of release from intracellular stores upon CBD application, while at the same time reducing the size of the CBD responses. This may be based on the interaction between extracellular Ca2þ sources and filling of intracellular stores, presumably involving L-type VGCCs (Verkhratsky and Toescu, 2003; Rubovitch et al., 2004; Power and Sah, 2005). The central role of intracellular Ca2þ stores for the CBD response was confirmed by the data obtained with thapsigargin, and found to be responsible for the maintenance phase of the CBD response, since the remaining responders showed a transient rather than a sustained response pattern. The observation that CBD was able to reduce Ca2þ levels in a subset of cells (after the initial Ca2þ rise caused by thapsigargin) also points to a potential link between the suggest protective action of CBD and intracellular Ca2þ regulation. A thapsigargininduced release of eCBs may also have negatively affected the action of CBD (see Fig. 10 and Van der Stel et al., 2005).

***

***

20

627

4.3. Involvement of CB1 and VR1 receptors in the CBD responses

10 51

50

88

N if in CB D

in D CB

N

aï

ve

CB

D

ct

Cd

rl

0

Fig. 7. Comparison of CBD-induced [Ca2þ]i response following Ca2þ channel blocker application (A) in glia (n ¼ 78 in cadmium; n ¼ 38 in nifedipine) and (B) in neurones (n ¼ 35 in cadmium; n ¼ 13 in nifedipine). Responder rates are given in % within each column. Significances are indicated for comparison with na€ıve CBD controls (*P < 0.05; ***P < 0.001).

cell types. Therefore, the pattern observed suggests that excitability change and modification of the Ca2þ gradient has a modulatory impact on the pathway responsible for the CBD response. Interactions between CB signalling and VGCCs has been described in a large variety of studies, though usually looking into effects of CB1 agonists on VGCC-mediated currents (Caulfield and Brown, 1992; Mackie and Hille, 1992; Sullivan, 1999; Nogueron et al., 2001; Brown et al., 2004). This inhibitory pathway is of direct relevance for transmitter release regulation. Here, we found that the CBD-induced [Ca2þ]i rise was reduced in the presence of both nifedipine and cadmium in neurones and glia. The rate of inhibition did not differ between the two blockers, suggesting that the CBD-induced Ca2þ influx regulated via VGCCs is L-type mediated. Similar observations have been made in neuroblastoma cells where DALN induced Ca2þ uptake was found to act via L-type Ca2þ channels (Rubovitch et al., 2002).

Although CBD binds to CB1 receptors, its affinity for the receptor relative to other CBs is very low (Mukhopadhyay et al., 2002), and neither psychoactive effects nor coupling to cAMP signalling pathways have been observed (Pertwee, 2004). It has been suggested that CBD may bind to the recently proposed ‘‘VR1-like’’ or CBx receptor or other, as yet unidentified receptors. On the other hand, there may be interactions between CBD and CB1-signalling pathways, as suggested above. Therefore, our experiments tested whether CBD (a) is acting on CB1 receptors or on a VR1-like pathway, and/or (b) modulated by an endogenous CB1 tone. In agreement with CBD response observed in glia, the CB1selective antagonist AM281 did not block but rather dramatically enhanced the CBD-induced [Ca2þ]i rise, negating the possibility that the effect is directly CB1 receptor mediated. On the other hand, responder rates were reduced by w30% in AM281, potentially suggesting that there may be subsets of cells where CBD acts via a CB1-receptor dependent pathway, while the majority of cells’ responses were facilitated in the presence of AM281. The latter may be tonically regulated via a CB1 pathway, suppressing the CBD mediated Ca2þ rise for instance via the modulation of VGCCs (Fig. 10). In these cells, the blockade of tonically active CB1 receptors could unmask a facilitated CBD response. Pre-treatment with CPZ also disproved the theory that the CBD-induced Ca2þ signalling may be VR1-mediated in hippocampal cells, though CPZ’s specificity has been questioned with non-specific actions having been reported in addition to VR1 antagonism (e.g. Docherty et al., 1997). In any case, the CBD-induced [Ca2þ]i rise in CPZ was again increased

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

628

Glia (↑[Ca2+]i)

350

D

350

250

300

200

250

150

200

100

150

50

2 µM Thapsigargin

Neurones (↓[Ca2+]i)

400

300

∆F/F

∆F/F

A

100

1 µM CBD

0

5

10

15

20

25

30

0

35

5

10

15

Time (minutes)

B

1 µM CBD

2 µM Thapsigargin

50

0

Glia (↓[Ca2+]i)

300

20

25

30

35

Time (minutes)

E

Glia

70 60 50

250

∆F/F

200

20 10

150

ns

30 70

15

***

-10 100 1 µM CBD

20

25

30

35

↑

Neurones (↑[Ca2+]i)

F

350

Neurones

70 60

300

po st Th ap

15

Time (minutes)

M ea n

10

po st Th ap

5

↓

0

po st Th ap

Co nt ro l

-30

50

ns

50 40

∆F/F

250 200

30

10 150

***

20 52 52

20

***

0 -10

15

20

25

30

35

Time (minutes)

po st Th ap

10

↑

-30 5

↓

1 µM CBD

50 0

46

26

-20

po st Th ap

2 µM Thapsigargin

Co nt ro l

100

po st Th ap

∆F/F

81

66

-20 2 µM Thapsigargin

C

***

0

M ea n

∆F/F

40

Fig. 8. Sample time courses of CBD-induced [Ca2þ]i responses in experiments following a 5-min application of 2 mM thapsigargin. (A) Increased, transient [Ca2þ]i glial responses (5 representative sample ROIs). (B) Decreased [Ca2þ]i responses in glia. (C) Increased [Ca2þ]i responses in neurones (5 representative sample ROIs). (D) Decreased neuronal [Ca2þ]i responses (3 ROIs), with trendlines to illustrate the typical [Ca2þ]i maintained level in thapsigargin. (E,F) Summary of CBD-induced [Ca2þ]i responses in glia (E) (n ¼ 71 control, n ¼ 8 increase, n ¼ 35 decrease) and in neurones (F) (n ¼ 57 control, n ¼ 16 increase, n ¼ 20 decrease). Percent values within each column represent the responder rates, significances are indicated for comparison with respective na€ıve CBD controls (***P < 0.001).

compared to na€ıve CBD responses, without effects on the responder rate. VR1 receptors, originally localised to the peripheral nervous system, has later been unveiled in brain and especially in hippocampal CA1 and CA3 regions (Mezey et al., 2000). The discovery that AEA is an agonist at VR1

(Zygmunt et al., 1999; Smart et al., 2000) and that capsaicin analogues interact with CB1 receptors (Di Marzo et al., 1998), suggested an overlap between the CB and vanilloid systems. Some of CBD’s pharmacological effects are reported to be mediated by VR1 receptors e.g. CBD desensitised VR1 to

A

B

225

400 350

200

300

175

∆F/F

∆F/F

250 150

200

125 150 100

100

75

1 µM CBD

1 µM CBD

50

1 µM AM281

50

1 µM CPZ

0 0

5

10

15

20

25

0

5

Time

C

15

20

25

Time (minutes)

Glia

250

10

D

*

Neurones

250

*** ***

150

∆F/F

∆F/F

***

200

200

150

100

100

50

50 77

49

74

***

51

36

74

0

0 Control

+ AM281

+ CPZ

Control

+ AM281

+ CPZ

Fig. 9. (A) Sample time course of CBD-induced [Ca2þ]i response in cultures with 1 mM AM281 applied 10 min previous to the 1 mM CBD application (4 glia in grey; 3 neurones in black). (B) Sample time course of CBD induced [Ca2þ]i response in cultures with 1 mM CPZ applied 10 min previous to the 1 mM CBD application (4 glia, 3 neurones). Comparison of CBD induced [Ca2þ]i response in the presence of 1 mM AM281 and 1 mM CPZ in glia (C) (AM281, n ¼ 31; CPZ, n ¼ 20) and in neurones (D) (AM281, n ¼ 25; CPZ, n ¼ 20). Values within each column represent the percentage of all viable cells responding to the CBD application. Significances indicated above columns are for comparison with na€ıve CBD controls (*P < 0.05; ***P < 0.001).

Ca2+[mM] Ca2+[nM]

CBD RyR

Ca2+

Ca2+

VGCC

CB1/x eCB = ATPase

Fig. 10. CBD and the regulation of intracellular Ca2þ. Low intracellular Ca2þ concentrations are maintained via ATPases that pump Ca2þ out of the cell and/ or into intracellular compartments. Influx of Ca2þ, for instance via L-type voltage gated calcium channels (VGCCs), contributes to the regulation of store filling and can cause further release of Ca2þ via Ryanodine receptor (RyR) activation (so-called calcium-induced Ca2þ release, CICR), depending on the spatio-temporal pattern of the Ca2þ rise. A rise in intracellular Ca2þ can trigger the release of endocannabinoids (eCBs), and may provide a negative feedback circuit to suppress further Ca2þ rise, for instance via inhibition of VGCCs. It is proposed that CBD may be acting on intracellular stores, since the CBD-induced Ca2þ rise was abolished by the intracellular ATPase inhibitor thapsigargin and facilitated by CB1 antagonists. For further information, see text.

capsaicin’s actions in brain membranes, leukaemia cells and cells over-expressing VR1 (Bisogno et al., 2001). Moreover, CBD and vanilloid agonists such as capsaicin have many pharmacological effects in common e.g. anti-inflammatory, anticonvulsive and anti-rheumatoid effects (Consroe et al., 1981; Dib and Falchi, 1996; Lorton et al., 2000; Malfait et al., 2000), and CBD’s anti-hyperalgesic effect has been specifically attributed to VR1 receptors (Costa et al., 2004). Here, the CBD-induced rise in Ca2þ does however not appear to be mediated by CB1 or VR1-like receptors, but is apparently modulated by them. In conclusion, the CBD-induced Ca2þ rise in hippocampal cells shows a strong dependence on intracellular store signalling, Ca2þ gradients and VGCCs. Since the CBD response was exacerbated in the presence of CB/VR receptor antagonists, we conclude that an endogenous cannabinoid ‘‘tone’’ may exist via CB1 and VR1 receptors, coupled negatively to the CBD signalling pathway, supporting the hypothesis of cross-talk between different, CB-dependent signalling cascades.

Acknowledgement The authors would like to thank GW Pharmaceuticals for supplying CBD.

630

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631

References Begg, M., Mo, F.M., Offertaler, L., Batkai, S., Pacher, P., Razdan, R.K., et al., 2003. G protein-coupled endothelial receptor for atypical cannabinoid ligands modulates a Ca2þ-dependent Kþ current. J. Biol. Chem. 278, 46188e46194. Bisogno, T., Hanus, L., De Petrocellis, L., Tchilibon, S., Ponde, D.E., Brandi, I., et al., 2001. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br. J. Pharmacol 134 (4), 845e852. Breivogel, C.S., Griffin, G., Di Marzo, V., Martin, B.R., 2001. Evidence for a new G protein-coupled cannabinoid receptor in mouse brain. Mol. Pharmacol. 60, 155e163. Brenowitz, S.D., Regehr, W.G., 2003. Calcium dependence of retrograde inhibition by endocannabinoids at synapses onto Purkinje cells. J. Neurosci. 23, 6373e6384. Brown, S.P., Safo, P.K., Regehr, W.G., 2004. Endocannabinoids inhibit transmission at granule cell to Purkinje cell synapses by modulating three types of presynaptic calcium channels. J. Neurosci. 24, 5623e5631. Caulfield, M.P., Brown, D.A., 1992. Cannabinoid receptor agonists inhibit Ca current in NG108e15 neuroblastoma cells via a pertussis toxin-sensitive mechanism. Br. J. Pharmacol. 106, 231e232. Chou, K.J., Tseng, L.L., Cheng, J.S., Wang, J.L., Fang, H.C., Lee, K.C., et al., 2001. CP55,940 increases intracellular Ca2þ levels in Madin-Darby canine kidney cells. Life Sci. 69, 1541e1548. Clodfelter, G.V., Porter, N.M., Landfield, P.W., Thibault, O., 2002. Sustained Ca2þ-induced Ca2þ release underlies the post-glutamate lethal Ca2þ plateau in older cultured hippocampal neurones. Eur. J. Pharmacol 447, 189e200. Consroe, P., Martin, A., Singh, V., 1981. Antiepileptic potential of cannabidiol analogs. J. Clin. Pharmacol. 21 (Suppl.), 428Se436S. Costa, B., Giagnoni, G., Franke, C., Trovato, A.E., Colleoni, M., 2004. Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid, cannabidiol, in a rat model of acute inflammation. Br. J. Pharmacol. 143, 247e250. Di Marzo, V., Melck, D., Bisogno, T., De Petrocellis, L., 1998. Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action. Trends Neurosci. 21, 521e528. Dib, B., Falchi, M., 1996. Convulsions and death induced in rats by Tween 80 are prevented by capsaicin. Int. J. Tissue React 18, 27e31. Docherty, R.J., Yeats, J.C., Piper, A.S., 1997. Capsazepine block of voltageactivated calcium channels in adult rat dorsal root ganglion neurones in culture. Br. J. Pharm. 121, 1461e1467. Drysdale, A.J., Platt, B., 2003. Cannabinoids: mechanisms and therapeutic applications in the CNS. Curr. Med. Chem. 10, 2719e2732. Drysdale, A.J., Pertwee, R.G., Platt, B., 2005. Regulation of intracellular calcium stores by the plant cannabinoid, cannabidiol. Soc. Neurosci. Abstr., 690.9. Eshhar, N., Striem, S., Kohen, R., Tirosh, O., Biegon, A., 1995. Neuroprotective and antioxidant activities of HU-211, a novel NMDA receptor antagonist. Eur. J. Pharmacol. 283, 19e29. Hampson, R.E., Mu, J., Deadwyler, S.A., 2000. Cannabinoid and kappa opioid receptors reduce potassium K current via activation of G(s) proteins in cultured hippocampal neurons. J. Neurophysiol. 84, 2356e2364. Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A., et al., 2002. Classification of cannabinoid receptors. Pharmacol. Rev. 54, 161e202. Huang, C.C., Lo, S.W., Hsu, K.S., 2001. Presynaptic mechanisms underlying cannabinoid inhibition of excitatory synaptic transmission in rat striatal neurons. J. Physiol. (Lond) 532, 731e748. Kaplan, B.L., Rockwell, C.E., Kaminski, N.E., 2003. Evidence for cannabinoid receptor-dependent and -independent mechanisms of action in leukocytes. J. Pharmacol. Exp. Ther. 306, 1077e1085. Lorton, D., Lubahn, C., Engan, C., Schaller, J., Felten, D.L., Bellinger, D.L., 2000. Local application of capsaicin into the draining lymph nodes attenuates expression of adjuvant-induced arthritis. Neuroimmunomodulators 7, 115e125. Mackie, K., Hille, B., 1992. Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells. Proc. Natl. Acad. Sci. USA 89, 3825e3829.

Malfait, A.M., Gallily, R., Sumariwalla, P.F., Malik, A.S., Andreakos, E., Mechoulam, R., et al., 2000. The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc. Natl. Acad. Sci. USA 97, 9561e9566. Mezey, E., Toth, Z.E., Cortright, D.N., Arzubi, M.K., Krause, J.E., Elde, R., et al., 2000. Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1-like immunoreactivity, in the central nervous system of the rat and human. Proc. Natl. Acad. Sci. USA 97, 3655e3660. Mukhopadhyay, S., Shim, J.Y., Assi, A.A., Norford, D., Howlett, A.C., 2002. CB(1) cannabinoid receptor-G protein association: a possible mechanism for differential signaling. Chem. Phys. Lipids 121, 91e109. Nadler, V., Biegon, A., Beit-Yannai, E., Adamchik, J., Shohami, E., 1995. 45Ca accumulation in rat brain after closed head injury; attenuation by the novel neuroprotective agent HU-211. Brain Res. 685, 1e11. Netzeband, J.G., Conroy, S.M., Parsons, K.L., Gruol, D.L., 1999. Cannabinoids enhance NMDA-elicited Ca2þ signals in cerebellar granule neurons in culture. J. Neurosci. 19, 8765e8777. Nogueron, M.I., Porgilsson, B., Schneider, W.E., Stucky, C.L., Hillard, C.J., 2001. Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons. J. Neurochem. 79, 371e381. Nunez, E., Benito, C., Pazos, M.R., Barbachano, A., Fajardo, O., Gonzalez, S., et al., 2004. Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain: an immunohistochemical study. Synapse 53, 208e213. Offertaler, L., Mo, F.M., Batkai, S., Liu, J., Begg, M., Razdan, R.K., et al., 2003. Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor. Mol. Pharmacol. 63, 699e705. Pertwee, R.G., 2004. The pharmacology and therapeutic potential of cannabidiol. In: Di Marzo, V. (Ed.), Cannabinoids. Kluwer Academic/Plenum, London, pp. 32e83. Pertwee, R.G., Fernando, S.R., Griffin, G., Ryan, W., Razdan, R.K., Compton, D.R., et al., 1996. Agonist-antagonist characterization of 6#-cyanohex-2#-yne-delta 8-tetrahydrocannabinol in two isolated tissue preparations. Eur. J. Pharmacol. 315, 195e201. Pertwee, R.G., Ross, R.A., Craib, S.J., Thomas, A., 2002. ()-Cannabidiol antagonizes cannabinoid receptor agonists and noradrenaline in the mouse vas deferens. Eur. J. Pharmacol. 456, 99e106. Pertwee, R.G., Thomas, A., Stevenson, L.A., Maor, Y., Mechoulam, R., 2005. Evidence that ()-7-hydroxy-4#-dimethylheptyl-cannabidiol activates a non-CB(1), non-CB(2), non-TRPV1 target in the mouse vas deferens. Neuropharmacology 48, 1139e1146. Platt, B., Drysdale, A.J., 2004. Search and rescue: identification of cannabinoid actions relevant for neuronal survival and protection. Curr. Neuropharm. 2, 103e114. Power, J.M., Sah, P., 2005. Intracellular calcium store filling by an L-type calcium current in the basolateral amygdala at subthreshold membrane potentials. J. Physiol. 562, 439e453. Robbe, D., Alonso, G., Duchamp, F., Bockaert, J., Manzoni, O.J., 2001. Localization and mechanisms of action of cannabinoid receptors at the glutamatergic synapses of the mouse nucleus accumbens. J. Neurosci. 21, 109e116. Rubovitch, V., Gafni, M., Sarne, Y., 2002. The cannabinoid agonist DALN positively modulates L-type voltage-dependent calcium-channels in N18TG2 neuroblastoma cells. Brain Res. Mol. Brain Res. 101, 93e102. Rubovitch, V., Gafni, M., Sarne, Y., 2004. The involvement of VEGF receptors and MAPK in the cannabinoid potentiation of Ca2þ flux into N18TG2 neuroblastoma cells. Brain Res. Mol. Brain Res. 120, 138e144. Shen, M., Thayer, S.A., 1998. Cannabinoid receptor agonists protect cultured rat hippocampal neurons from excitotoxicity. Mol. Pharmacol. 54, 459e462. Shohami, E., Novikov, M., Mechoulam, R., 1993. A nonpsychotropic cannabinoid, HU-211, has cerebroprotective effects after closed head injury in the rat. J. Neurotrauma 10, 109e119. Shohami, E., Gallily, R., Mechoulam, R., Bass, R., Ben-Hur, T., 1997. Cytokine production in the brain following closed head injury: dexanabinol (HU-211) is a novel TNF-alpha inhibitor and an effective neuroprotectant. J. Neuroimmunol. 72, 169e177.

A.J. Drysdale et al. / Neuropharmacology 50 (2006) 621e631 Showalter, V.M., Compton, D.R., Martin, B.R., Abood, M.E., 1996. Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands. J. Pharmacol. Exp. Ther. 278, 989e999. Smart, D., Gunthorpe, M.J., Jerman, J.C., Nasir, S., Gray, J., Muir, A.I., et al., 2000. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br. J. Pharmacol. 129, 227e230. Sullivan, J.M., 1999. Mechanisms of cannabinoid-receptor-mediated inhibition of synaptic transmission in cultured hippocampal pyramidal neurons. J. Neurophysiol. 82, 1286e1294. Twitchell, W., Brown, S., Mackie, K., 1997. Cannabinoids inhibit N- and P/Qtype calcium channels in cultured rat hippocampal neurons. J. Neurophysiol. 78, 43e50. Van der Stel, M., Trevisani, M., Vellani, V., De Petrocellis, L., Schiano Moriello, A., Campi, B., et al., 2005. Anandamide acts as an intracellular messenger amplifying Ca2þ influx via TRPV1 channels. EMBO J 24, 3026e3037. Venance, L., Piomelli, D., Glowinski, J., Giaume, C., 1995. Inhibition by anandamide of gap junctions and intercellular calcium signalling in striatal astrocytes. Nature 376, 590e594. Verkhratsky, A., Toescu, E.C., 2003. Endoplasmic reticulum Ca(2þ) homeostasis and neuronal death. J. Cell. Mol. Med. 7, 351e361.

631

Waksman, Y., Olson, J.M., Carlisle, S.J., Cabral, G.A., 1999. The central cannabinoid receptor (CB1) mediates inhibition of nitric oxide production by rat microglial cells. J. Pharmacol. Exp. Ther. 288, 1357e1366. Walter, L., Franklin, A., Witting, A., Moller, T., Stella, N., 2002. Astrocytes in culture produce anandamide and other acylethanolamides. J. Biol. Chem. 277, 20869e20876. Walter, L., Franklin, A., Witting, A., Wade, C., Xie, Y., Kunos, G., et al., 2003. Nonpsychotropic cannabinoid receptors regulate microglial cell migration. J. Neurosci. 23, 1398e1405. Whittle, B.A., Guy, G.W., Robson, P., 2001. Prospect for new cannabis-based prescription medicines. J. Cannabis Ther. 1, 183e205. Wade, D.T., Makela, P., Robson, P., House, H., Bateman, C., 2004. Do cannabis-based medicinal extracts have general or specific effects on symptoms in multiple sclerosis? A double-blind, randomized, placebo-controlled study on 160 patients. Mult. Scler. 10, 434e441. Xiong, J., Camello, P.J., Verkhratsky, A., Toescu, E.C., 2004. Mitochondrial polarisation status and [Ca2þ]i signalling in rat cerebellar granule neurones aged in vitro. Neurobiol. Aging 25, 349e359. Zygmunt, P.M., Petersson, J., Andersson, D.A., Chuang, H., Sorgard, M., Di Marzo, V., et al., 1999. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400, 452e457.