The effects of the cannabinoid receptor agonist WIN 55,212-2 on endogenous extracellular glutamate levels in the prefrontal cortex of the awake rat and in primary cultures of rat cerebral cortex neurons were investigated. In the prefrontal cortex WIN 55,212-2 (0.1 and 1 mg/kg i.p.) increased dialysate glutamate levels from of the awake rat, while the lower (0.01 mg/kg) and the higher (2 mg/kg) doses were ineffective. Furthermore, the WIN 55,212-2 (0.1 mg/kg)- induced increase of dialysate glutamate levels was counteracted by pretreatment with the selective CB1 receptor antagonist SR141716A (0.1 mg/kg i.p.) and by the local perfusion with a low-calcium Ringer solution (Ca2+ 0.2 mM). In primary cultures of rat cerebral cortex neurons, WIN 55,212-2 (0.01–100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration–response curve. The facilitatory effect of WIN 55,212-2 (1 nM) was fully counteracted by SR141716A (10 nM), by the replacement of the normal Krebs Ringer-bicarbonate buffer with a low Ca2+ medium (0.2 mM) and by the IP3 receptor antagonist xestospongin C (1 μM). These in vivo and in vitro findings suggest an increase in cortical glutamatergic transmission by CB1 receptors, an effect that may underlie some of the psychoactive and behavioural actions of acute exposure to marijuana.
Among the recognized responses of the central nervous system to marijuana inhalation are deficits in cognitive functions and in memory process (Ameri, 1999; Hampson and Deadwyler, 1999; Sullivan, 2000). These effects are presumably mediated via the activation of a CB1 cannabinoid-receptor subtype probably located in the brain regions that mediate higher cognitive functions. High density of CB1 receptors has been detected in the cerebral cortex, hippocampus, basal ganglia and cerebellum (Herkenham et al., 1990). Morphological and biochemical evidence have demonstrated that in the hippocampus cannabinoid receptors are present at high density on the presynaptic terminals of glutamatergic synapses (Twitchell et al., 1997) and their activation is associated with a reduction of glutamate release (Piomelli et al., 2000). The cerebral cortex, and in particular the prefrontal cortex, is also a major site subserving cognition and memory processing due in part to complex interactions between local cortical neurons (Frith and Dolan, 1996; Courtney et al., 1998; Fuster, 2000). In view of the effect of marijuana on memory and of the presence of cannabinoid receptors in the prefrontal cortex, it has been postulated that the impairment of cognition associated with cannabis inhalation may reflect neurochemical changes in this brain region (Jentsch et al., 1997; Diana et al., 1998; Braida and Sala, 2000). Since glutamate is the major excitatory neurotransmitter involved in cortical transmission, and little is known about the effects of cannabinoids on cortical glutamatergic transmission (Auclair et al., 2000), the aim of the present study was to investigate the effects of WIN 55,212-2 and SR141716A, cannabinoid receptor agonist and antagonist respectively (D'Ambra et al., 1992; Rinaldi-Carmona et al., 1994), on cortical endogenous glutamate levels. Towards this aim, we combined the in vivo microdialysis technique with an in vitro preparation such as the primary cultures of rat cerebral cortex neurons.
Materials and Methods
WIN 55,212-2 mesylate ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone) (Tocris Cookson Ltd, Bristol, UK) and SR141716A (N-piperidino-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) (Sanofi-Synthelabo, Montpellier, France) were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was <0.01% and this concentration of DMSO did not change glutamate levels when it was perfused in the control rats or cell cultures. Xestospongin C was purchased from Calbiochem (San Diego, CA, USA) while ω-conotoxin MVIIC and l-trans-pyrrolidine-2,4-dicarboxylic acid were purchased from Tocris Cookson. The culture dishes were purchased from NUNC A/S, Denmark. Fetal calf serum and basal Eagle's medium were obtained from GIBCO (Grand Island, NY, USA). Poly-l-lysine, trypsin, soybean trypsin inhibitor, DNase, cytosine arabinoside, gentamycine sulphate and glutamine were obtained from Sigma Chemical Co. (St Louis, MO, USA).
In Vivo Microdialysis
Male adult Sprague–Dawley rats with a body weight of 300–320 g were housed in cages in groups of five animals at a constant room temperature (20°C) and exposed to a 12:12 h light–dark cycle (lights on at 06.00 a.m.). Food and water were provided ad libitum. Following delivery, the animals were allowed to adapt to the environment for at least 1 week before the experiment started.
Briefly, the animals were anaesthetized with halothane/air (1.5% mixture), mounted in a David Kopf stereotaxic frame and one microdialysis probe (2 mm dialysing membrane length) was implanted into the right or the left frontal cortex using the following stereotaxic coordinates (A, +3.5; L ±2.8; V, –3.5) (Paxinos and Watson, 1986). After the implantation, the probe was secured to the skull with methacrylic cement and 36 h later microdialysis was performed.
On the day of the microdialysis experiment, the probe was perfused with Ringer solution (in mM: Na+ 147; K+ 4; Ca2+ 1.4; Cl– 156; glucose 2.7) at a constant flow rate of 2 μl/min. In order to achieve stable dialysate glutamate levels, collection of samples started 300 min after the onset of the perfusion and perfusates were collected every 20 min. WIN 55,212-2 (0.01–2 mg/kg) was administered i.p. after three stable baseline levels of glutamate had been achieved, and perfusate samples were collected for a further 120 min. When required, the CB1 receptor antagonist SR141716A (0.1 mg/kg) was injected i.p. either alone or 10 min before WIN 55,212-2 (0.1 mg/kg). Alternatively, a low calcium medium (Ca2+ 0.2 mM) was locally perfused 40 min before WIN 55,212-2 (0.1 mg/kg) and remained until the end of the experiment.
At the end of each experiment, the brain was removed from the skull and the location of the probe was carefully verified in 30 μm thick coronal cryostat sections. Only those animals in which the probe was correctly located were included in this study.
The in vivo experimental procedures were approved by the local Ethics Committee and by the Italian Ministero della Sanita' (licence no. 111/94-B).
In Vitro Cortical Cell Cultures
Cerebral cortical cells were prepared from 1 day old Sprague–Dawley rats (Alho et al., 1988). After the resuspension in the plating medium, the cells were counted and then plated on poly-l-lysine (5 μg/ml)-coated dishes at a density of 2.5 × 106 cells/dish. The plating medium consisted of Eagle's Basal Medium supplemented with inactivated fetal calf serum, 25 mM KCl, 2 mM glutamine and 100 μg/ml gentamycine. Cultures were grown at 37°C in a humidified atmosphere, 5% CO2/95% air. Cytosine arabinoside (10 μM) was added within 24 h of plating to prevent glial cell replication. The cultures were used in experiments after 8 days in vitro.
On the day of the release experiment, the cells were rinsed twice by replacing the culture medium with Krebs Ringer-bicarbonate buffer (37°C). Thereafter, five consecutive fractions were collected, renewing this solution (400 μl) every 30 min. The first two samples were used to assess basal glutamate levels, while pharmacological treatments were carried out during the third fraction. WIN 55,212-2 was applied 15 min before the end of the third fraction. When required, the CB1 receptor antagonist SR141716A, the IP3 receptor antagonist xestospongin C (Gafni et al., 1997; Netzeband et al., 1999) and the N, P and Q type Ca2+ channel blocker ω-conotoxin MVIIC (Hillyard et al., 1992) were added 10 min before WIN 55,212-2. The effect of WIN 55,212-2 in low calcium medium was evaluated by replacing the culture medium with Krebs Ringer-bicarbonate buffer containing 0.2 mM Ca2+ at the onset of the experiment.
In a further set of experiments, the effect of WIN 55,212-2 on the l-[3H]glutamate uptake in cortical cell cultures was analysed and compared with the effect of the specific glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (Bridges et al., 1991). To this purpose, the cells were incubated for 15 min at 37°C in Krebs Ringer-bicarbonate buffer containing l-[3H]glutamate (0.3 μCi ) in the absence or in the presence of WIN55,212-2 (1 and 10 nM) or PDC (0.1 mM). Thereafter, the uptake was halted by replacing the incubation medium with ice-cold Krebs Ringer-bicarbonate buffer. The radioactivity accumulated in the cells was extracted by a 30 min incubation period (37°C) with 0.5 ml of acidic ethanol (95% ethanol/5% 0.1 M HCl) and quantified by liquid scintillation spectrometry. Experiments were carried out in duplicate. Non-specific uptake and/or absorption were determined by performing parallel experiments at 0°C.
Endogenous Glutamate Assay
Endogenous glutamate was quantified by using a high-performance liquid chromatography/fluorimetric detection system, including precolumn derivatization o-phthaldialdehyde reagent and a Chromsep 5 (C18) column. The mobile phase consisted of 0.1 M sodium acetate, 10% methanol and 2.5% tetrahydrofuran, pH 6.5. The limit of detection for glutamate was 30 fmol/sample.
The microdialysis data from individual time points have been reported as percentages of the mean of the three basal samples prior to the treatments. The data were calculated as mean ± SEM. In addition, the area under the curve, which reflects the duration of the effect over the experimental time period (120 min), was calculated for each animal. The area values (overall effects) were calculated as percentage changes in basal value over time (Δ basal % × time) by using the trapezoidal rule.
In the in vitro study, the effects of the treatments on the endogenous extracellular glutamate levels during the third fraction were reported and expressed as percentage changes of basal values, as calculated by the means of the two fractions collected prior treatment.
The statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by the Newman–Keuls test for multiple comparisons.
In Vivo Microdialysis
Effect of WIN 55,212-2
Basal dialysate glutamate levels from the prefrontal cortex of the awake rat were 0.226 ± 0.061 μM (n= 18) and remained stable over the duration of the experiment (180 min). As shown in Figure 1, i.p. injection of WIN 55,212-2 (0.1 and 1 mg/kg) increased dialysate glutamate levels, with the maximal effect observed at the 0.1 mg/kg dose (145 ± 7% and 128 ± 6% of basal values respectively) while the lower (0.01 mg/kg) and the higher (2 mg/kg) doses were ineffective.
SR141716A and Low-calcium Medium Alone and Together with WIN 55,212-2
In a further set of experiments, the effect of WIN 55,212-2 (0.1 mg/kg) in the presence of the selective CB1 receptor antagonist SR141716A or of a low-calcium perfusate medium was studied. As shown in Figure 2A, i.p. injection of SR141716A (0.1 mg/kg), which by itself had no effect on cortical dialysate glutamate levels, completely counteracted the WIN 55,212-2 (0.1 mg/kg)-induced increase in dialysate glutamate levels.
Local perfusion with low-calcium (0.2 mM) Ringer solution alone did not affect basal dialysate cortical glutamate levels. However, the facilitatory effect of WIN 55,212-2 (0.1 mg/kg) was completely counteracted when the low-calcium Ringer solution was perfused into the prefrontal cortex 40 min prior to administration of the cannabinoid receptor agonist (Fig. 2B).
In Vitro Cortical Cell Cultures
Effect of WIN 55,212-2 on Extracellular Endogenous Glutamate Levels
Extracellular endogenous glutamate levels in control cortical cell cultures were 0.137 ± 0.017 nM (n = 14) and remained essentially stable over the duration of the experiment (five collected fractions; 150 min). The addition of WIN 55,212-2 (0.01–100 nM) to the medium during the third fraction was associated with a bell-shaped concentration-dependent increase in extracellular glutamate levels (Fig. 3). The maximal effect was found at 1 nM concentration (314 ± 34%, n = 23) with a less pronounced increase at the 10 nM concentration (226 ± 24%, n = 19). On the contrary, the lower (0.01 and 0.1 nM) and the higher (100 nM) concentrations of the cannabinoid agonist were ineffective (103 ± 8%, n = 15, 135 ± 11%, n = 16 and 139 ± 19%, n = 15 respectively).
Extracellular glutamate levels in the fourth and fifth fractions were similar in control and treated cells (data not shown).
Effect of WIN 55,212-2 on l-[3H]Glutamate Uptake
In view of the above and in order to evaluate further the biochemical mechanisms underlying the modulation of extracellular glutamate levels by WIN 55,212-2, the effect of the cannabinoid receptor agonist on l-[3H]glutamate uptake in cortical cell cultures was analysed and compared with the effect of the specific glutamate uptake inhibitor PDC. As expected, the inclusion into the medium of PDC (0.1 mM) markedly reduced l-[3H]glutamate uptake down to 45 ± 4% of control values (n = 11). On the contrary, WIN 55,212-2 (1 and 10 nM) failed to affect the l-[3H]glutamate uptake (102 ± 3% and 104 ± 5% of control values respectively; n = 12).
SR141716A and Low-calcium Medium Alone and Together with WIN 55,212-2
The effect of WIN 55,212-2 (1 nM) in the presence of the selective CB1 receptor antagonist SR141716A was also tested. As shown in Figure 4, SR141716A (10 nM), which by itself was ineffective, completely prevented the WIN 55,212-2-induced increase in glutamate levels (109 ± 5%, n = 7). On the contrary, the lower 0.1 mM concentration of SR141716A failed to significantly affect the WIN 55,212-2-induced increase in glutamate levels.
Interestingly, when WIN 55,212-2 (1 nM) was added to cell cultures maintained from the onset of the experiment in a low-calcium medium (Ca2+ 0.2 mM), no increase in local glutamate levels was observed (125 ± 9%, n = 8; Figure 4).
Involvement of IP3-controlled Ca2+ Stores and Voltage-sensitive Ca2+ Channels (VSCCs) on WIN 55,212-2-induced Increase of Extracellular Glutamate Levels
These experiments were performed to investigate whether the Ca2+ release from intracellular stores controlled by IP3 or the stimulation of VSCCs contributed to the WIN 55,212-2-induced (1 nM) increase of extracellular glutamate levels. As shown in Figure 5, when the IP3 receptor antagonist xestospongin C (1 μM) was added to the medium 10 min before WIN 55,212-2 (1 nM), it counteracted the enhancement of extracellular glutamate levels induced by the cannabinoid receptor agonist (144 ± 17%, n = 7). However, under the same experimental conditions, the N, P and Q type Ca++ channel blocker ω-conotoxin MVIIC (2 μM) was ineffective (260 ± 20%, n = 7). When applied alone, the toxins by themselves did not affect basal glutamate levels (data not shown).
The present results provide the first in vivo and in vitro evidence for a CB1 receptor-mediated increase on endogenous glutamate levels in the cerebral cortex. In fact, the cannabinoid receptor agonist WIN 55,212-2, at low mg/kg doses, enhances dialysate glutamate levels in the prefrontal cortex of the awake rat and increases, in a nanomolar concentration range, extracellular glutamate levels in primary cultures of rat cerebral cortex neurons. In view of the observation that WIN 55,212-2 failed to affect l-[3H]glutamate uptake in cortical cell cultures, while the specific glutamate uptake inhibitor PDC markedly reduced it under the same experimental conditions, it seems unlikely that, at least under the present in vitro conditions, a blockade of glutamate uptake contributes to the increase on extracellular glutamate levels produced by the cannabinoid receptor agonist. Furthermore, the finding that, in both the methodological approaches, the selective CB1 receptor antagonist SR141716A counteracts the effect of WIN 55,212-2 suggests that CB1 receptors play a key role in regulating cortical glutamatergic transmission. In this context, it is worth noting that other psychoactive drugs such as amphetamine and phencyclidine also increase glutamate levels in the prefrontal cortex (Adams and Moghaddam, 1998; Del Arco et al., 1998). Thus, it might be speculated that a potentiation of cortical glutamate neurotransmission could be a common mechanism underlying some of the shared behavioural effects of these compounds.
The observation that, under the present in vivo experimental procedure, basal dialysate cortical glutamate levels are not affected by local perfusion with a low-calcium Ringer solution, is in line with previous findings that show also a TTX-insensitivity of basal glutamate levels (Moghaddam, 1993; Rocher et al., 1999; Timmerman et al., 1999; Antonelli et al., 2000). Taken together, these results support the view that basal dialysate glutamate levels do not mainly originate from an exocytotically releasable pool (Timmerman et al., 1999), despite the fact that we allowed for an extended 6 h wash-out period prior to sample collection. However, the observation that the WIN 55,212-2-induced increase in glutamate levels was largely reduced in a low-calcium medium suggests the involvement of a calcium-dependent mechanism in this effect. Similarly, the in vitro results show that the replacement of the normal Krebs Ringer-bicarbonate buffer with a low-calcium medium (0.2 mM) also abolished the WIN 55,212-2 (1 nM)-induced increase in extracellular glutamate levels from cortical cells, whereas the spontaneous glutamate levels were calcium-independent. Although this finding could lead to questions about the physiological relevance of the modulation of spontaneous glutamate levels, the possibility that under basal conditions the calcium omission could mask the expected calcium-dependent component by the simultaneous efflux of cytoplasmatic (i.e. non-vesicular) pools cannot be ruled out. On the other hand, it seems likely that the activation of cortical glutamate transmission observed following the CB1 receptor stimulation might involve a calcium-dependent neurosecretory process both in vivo and in vitro.
The observed increase in cortical glutamate levels is unexpected in light of the fact that the CB1 receptor transduction mechanism is thought to mediate an inhibitory neuromodulatory action. Thus, cannabinoids acting via CB1 receptor activation have been shown to decrease local glutamate release from cerebellar granule cells, periacqueductal grey and hippocampal neurones (Shen et al., 1996; Di Marzo et al., 1998; Levenes et al., 1998; Piomelli et al., 2000). Inhibitory effects on other neurotransmitter systems and brain regions, such as the cholinergic system in rat hippocampal slices (Gifford et al., 1997) and noradrenergic transmission in human, guinea pig and rat hippocampus (Schlicker et al., 1997) have also been reported. Furthermore, a recent study (Auclair et al., 2000) reported an inhibitory effect of cannabinoid agonists on glutamatergic excitatory postsynaptic currents in slices of rat prefrontal cortex, a finding which is in contrast with the present results. However, it is worth noting that those authors used a different concentration of the agonist (1 μM). Unfortunately, under the present experimental conditions higher concentrations of WIN 55,212-2 could not be tested because of a primary effect of DMSO vehicle itself at a concentration >0.1% on cortical glutamate levels. In addition, the discrepancy may be due, at least in part, to the different approaches used to investigate the effect of WIN 55,212-2 on glutamate transmission and in this context further studies will be necessary to elucidate this point.
Despite the apparent discrepancies with some previous findings, the CB1-mediated facilitatory effect of WIN 55,212-2 on extracellular glutamate levels is consistent with recent excitatory actions of the drug. In particular, it has been recently reported that at low doses WIN 55,212-2 and HU210 stimulate cortical and hippocampal acetylcholine release (Acquas et al., 2000), while at higher doses WIN 55,212-2 decreases hippocampal acetylcholine release in a similar experimental approach (Gessa et al., 1998). Furthermore, Δ9-tetrahydrocannabinol (Δ9-THC) induces Ca2+ release from thapsigargin-sensitive Ca2+ stores in DDT1MF-2 smooth muscle cells (Filipeanu et al., 1997). Furthermore, WIN 55,212-2- and Δ9-THC increase intracellular Ca2+ levels in neuroblastoma (NG108-15) cells (Sugiura et al., 1996, 1997) and both cannabinoid receptor agonists WIN 55,212-2- and HU210 enhance intracellular Ca2+ levels elicited by NMDA and KCl stimulation in cultured cerebellar granule neurons (Netzeband et al., 1999). Taken together, these findings provide evidence that cannabinoids may trigger a CB1 receptor-mediated Ca2+ release from intracellular Ca2+ stores. To explain these findings it has been hypothesized that βγ subunits of Gi/Go proteins coupled to CB1 receptors may activate phospholipase C and increase IP3 and Ca2+ release from IP3-gated Ca2+ stores (Netzeband et al., 1999). Since increased intracellular Ca2+ promotes neurosecretion, it could be suggested that under the present experimental conditions cannabinoids enhance intracellular calcium levels via a preferential mobilization of calcium from the intracellular stores. This mechanism could underlie the Ca2+-dependent facilitatory effect of WIN 55,212-2 on extracellular glutamate levels observed in our study. Such a hypothetical mechanism is in line with the observation that, under the present in vitro conditions, the IP3 receptor blockade by xestospongin C abolished the facilitatory effect of WIN 55,212-2 on extracellular basal glutamate levels, whereas the Ca2+-channel blocker ω-conotoxin MVIIC did not prevent the action of the cannabinoid agonist. Finally, in view of the finding (Maneuf and Brotchie, 1997) that in certain conditions CB1 receptor transduction may also involve a Gs protein, the possibility that such a mechanism may also be involved in the facilitation of glutamate transmission observed in our study cannot be excluded.
The lack of effect of the higher doses or concentrations of WIN 55,212-2 observed either in vivo or in vitro may be referred to several causes. Namely, it can be speculated that WIN 55,212-2 could affect other neurotransmitters, which may in turn reduce cortical glutamate levels thus balancing the WIN 55,212-2-induced excitatory effect. On the other hand, it cannot be excluded that cannabinoid receptor activation by the higher doses/concentrations of WIN 55,212-2 could activate several cellular mechanisms coupled through different G-protein-mediated events (i.e. excitatory and inhibitory), leading to a lack of effect as net final result.
In summary, our findings indicate that acute administration of cannabinoids at low doses/concentrations enhances cortical glutamate transmission, an effect that disappears at higher doses/concentrations. In view of this, both in vitro and in vivo studies evaluating the effects of chronic cannabinoid administration should be undertaken, in order to evaluate the relevance of cortical cannabinoid–glutamate interactions in the central actions of marijuana.
The authors thank Dr M. Mossé (Sanofi-Synthelabo, Montpellier, France) for supplying SR141716A and Dr W. T. O'Connor (Department of Human Anatomy and Physiology, Conway Institute of Biomolecular and Biomedical Research, University College, Dublin, Ireland) for his valuable suggestions.This study was supported by an Italian MURST ‘Cofin 1999’ grant.
Address correspondence to Dr Sergio Tanganelli, Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Via Fossato di Mortara 17–19, 44100 Ferrara, Italy. Email: email@example.com.