Animals and treatments

All animal care and experimental procedures complied with Italian (D.L. 116/92) and EECabO.J. of E.C. L358/1 (18/12/86) regulations on the protection of laboratory animals and were approved by the Animal Ethics Committee of the Second University of Naples. All efforts were made to minimize animal suffering and to reduce the number of animals used. A total of 260 Wistar male rats (250–300 g) were used (Harlan, Milan, Italy). Rats were housed three per cage under controlled illumination (12:12 h light : dark cycle; light on 06:00 h) and environmental conditions (ambient temperature 20–22°C, humidity 55–60%) for at least 1 week before the commencement of experiments. Rat chow and tap water were available ad libitum.

Groups of 12–16 animals per treatment were used in order to have at least 6–8 ON and OFF cell recordings with each animal being used for a single cell recording.

Rats receiving intra-vl-PAG microinjections of vehicle or different doses of CBD and CBC, alone or in combination with antagonists were grouped as follows:

• A group of rats received an intra-vl-PAG microinjection of 200 nL of vehicle (0.2% dimethyl sulfoxide, DMSO, in rtificial CSF).
• Groups of rats received intra-vl-PAG microinjections of same volume of CBD (1.5, 3 and 6 nmol) alone or CBD (3 nmol) in combination with the selective TRPV1 antagonist, 5′-iodo-resiniferatoxin (I-RTX, 1 nmol), or the selective CB₁ receptor antagonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251, 0.5 nmol), or the selective adenosine A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.05 nmol), or the selective TRPA1 antagonist (Z)-4-(4-chlorophenyl)-3-methylbut-3-en-2-oxime (AP18, 6 nmol), or the selective 5-HT_1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY100635, 0.34 nmol).
• Groups of rats received intra-vl-PAG microinjections of CBC (3 and 6 nmol) alone or CBC (6 nmol) in combination with I-RTX (1 nmol), AM251 (0.5 nmol), DPCPX (0.05 nmol) or AP18 (6 nmol).
• A group of rats received intra-vl-PAG microinjections of OMDM-2 (1.5 and 3 nmol), a selective inhibitor of endocannabinoid cellular reuptake (Ortar et al., 2003).
• A group of rats received intra-vl-PAG microinjections of mustard oil (3 and 6 nmol), a classical agonist at TRPA1 channels.

As, to our knowledge, no other study has been published describing the effects of the drugs using a similar administration route in the rat, we performed preliminary experiments (not shown) with several doses of all drugs in order to find the lowest doses able to change RVM cell activities and/or tail-flick latencies or, in the case of the antagonists, the highest doses inactive per se.

Electrophysiological analyses and tail flick test

In order to perform direct intra-vl-PAG administrations of drugs or respective vehicle, rats were anaesthetized with pentobarbital (50 mg kg⁻¹, i.p.) and a 26-gauge, 12 mm-long stainless steel guide cannula was stereotaxically lowered until its tip was 1.5 mm above the vl-PAG by applying coordinates (A: −7.8 mm and L: 0.5 mm from bregma, V: 4.3 mm below the dura) from the atlas of Paxinos and Watson (1986). The cannula was anchored with dental cement to a stainless steel screw in the skull. We used a David Kopf stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) with the animal positioned on a homeothermic temperature control blanket (Harvard Apparatus Limited, Edenbridge, Kent, UK). Only those rats whose microinjected site was located within the vl-PAG, recognized by histological methods, were used for data computation (Figure 2).

Figure 2

Schematic illustration of the location of periaqueductal gray (PAG) microinjection sites (A) and rostral ventromedial medulla (RVM) ON or OFF cell recording sites (B). Vehicle or drug microinjections were performed in the ventrolateral (vl)-PAG (filled …

Anaesthesia was maintained with a continuous infusion of propofol (5–10 mg kg⁻¹ h⁻¹, i.v.), through a non-sterile catheter (polyethylene tubing, ID 0.58 mm, OD 0.96 mm, Becton Dickinson & Co., Franklin Lakes, NJ, USA) inserted into the left jugular vein, and adjusted so that tail flicks were elicited with a constant latency of 4–5 s. A thermal stimulus was elicited by a radiant heat source of a tail flick unit (Ugo Basile, Varese, Italy), focused on the rat tail approximately 3–5 cm from the tip. The intensity of the radiant heat source was adjusted to 50 mW (corresponding to 50 mJ per second) at the beginning of each experiment in order to elicit a constant tail-flick latency. A glass-insulated tungsten filament electrode (3–5 MW) (FHC Frederick Haer & Co., Bowdoin, ME, USA) was lowered into the RVM using the following stereotaxic coordinates: 2.8–3.3 mm caudal to lambda, 0.4–0.9 mm lateral and 8.9–10.7 mm depth from the surface of the brain (Paxinos and Watson, 1986) (Figure 2). RVM noxious stimuli-responding neurons were identified by the characteristic OFF cell pause and ON cell burst immediately prior to tail flick responses (Fields et al., 1991). The recorded signals were amplified and displayed on both analogue and a digital storage oscilloscope to ensure that the unit under study was unambiguously discriminated throughout the experiment. Signals were also fed into a window discriminator, whose output was processed by an interface (CED 1401) (Cambridge Electronic Design Ltd, Cambridge, UK) connected to a Pentium III PC. Spike2 software (CED, version 4) was then used to create peristimulus rate histograms online and to store and analyse digital records of single-unit activity offline. The configuration, shape and height of the recorded action potentials were monitored and recorded continuously using a window discriminator and Spike2 software for online and offline analyses. Once an ON or OFF cell was identified from its background activity, we optimized spike size before all treatments. This study only included neurons whose spike configuration remained constant and could clearly be discriminated from the background activity throughout the entire experiment. For each neuron the ongoing activity was obtained by averaging the firing rate (spikes s⁻¹) for 50 s before the tail flick trials (carried out every 5 min). Moreover, the peak height of the tail-flick-related burst (spikes s⁻¹) of the ON cells and the duration of the tail-flick-related pause (the time elapsing between the pause onset and the first action potential following tail flick) of OFF cells were also quantified. Recording sites were recognized with an electrolytic lesion at the conclusion of the experiment. The locations of all the studied neurons were reconstructed and plotted on standardized sections. Cells located outside the RVM were excluded from the study (Figure 2).

Tail flick latencies and extracellular recordings were considered before and after microinjecting drugs, or respective vehicle, into the vl-PAG. When CBD or CBC was administered in combination with I-RTX, AM251, DPCPX, AP18 or WAY100635, the two drugs were always co-injected. Tail flick latencies were monitored in the same rats undergoing RVM ON and OFF cell recordings.

Analysis of endocannabinoid levels in the PAG

In order to perform the endocannabinoid analysis, a different cohort of rats was used. Rats were decapitated 20 min after intra-PAG drug/vehicle microinjections, brains were rapidly removed and immersed in oxygenated ice-cold artificial cerebrospinal fluid. A brainstem slice of 1.30–1.35 mm was cut throughout the rostral-caudal PAG using a vibrotome (Vibratome 1500, Warner Instruments, CT, USA) (interaural from + 1.9 mm to + 0.7 mm,Paxinos and Watson, 1986). The slice of tissue containing the PAG/dorsal raphe was then further dissected under a optical microscope for microsurgery to isolate the vl-PAG (M650, Wild Heerbrugg, Glattbrugg, Switzerland) to be homogenized accordingly to the following protocol. In brief, tissues were homogenized in five volumes of chloroform/methanol/Tris HCl 50 mM (2:1:1) containing 20 pmol of d⁸-arachidonoyl ethanolamide (AEA) or d⁵-2-arachidonoyl glycerol (2-AG). Deuterated standards were synthesized from commercially available deuterated arachidonic acid and ethanolamine or glycerol, as described, respectively, in Devane et al. (1992) and Bisogno et al. (1997). Homogenates were centrifuged at 13 000×g for 16 min (4°C), the aqueous phase plus debris was collected and extracted again twice with one volume of chloroform. The organic phases from the three extractions were pooled and the organic solvents evaporated in a rotating evaporator. Lyophilized extracts were resuspended in chloroform/methanol (99:1, v v⁻¹). The solutions were then purified by open bed chromatography on silica as described in Bisogno et al. (1997). Fractions eluted with chloroform/methanol 9:1 by volume (containing AEA and 2-AG) were collected, the excess solvent was evaporated with a rotating evaporator, and aliquots were analysed by isotope dilution-liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC-APCI-MS) carried out under conditions described previously (Marsicano et al., 2002) and allowing the separation of the four compounds. Mass spectrometric (MS) detection was carried out in the selected ion monitoring mode using m/z-values of 356 and 348 (molecular ions + 1 for deuterated and undeuterated AEA) and 384.35 and 379.35 (molecular ions + 1 for deuterated and undeuterated 2-AG).The area ratios between the signals of the deuterated and undeuterated compounds varied linearly with varying amounts of undeuterated compounds (30 fmol–100 pmol). AEA and 2-AG levels in unknown samples were therefore calculated on the basis of their area ratios with the internal deuterated standard signal areas. For 2-AG, the areas of the peaks corresponding to 1(3)-and 2-isomers were added together. The amounts of endocannabinoids were expressed as pmol g⁻¹ of wet tissue weight.

Data analysis

Results were expressed as means ± SEM of latency time to the tail withdrawal reflex or spikes s⁻¹ obtained by averaging the ongoing cell firing recorded in 50 s before tail flick trials (which were carried out every 5 min). Tail-flick-related ON cell burst was calculated as means ± SEM of the number of spikes in the 10 s interval starting from the beginning of the increase in the cell frequency. The duration of the cell pause was expressed as means ± SEM of the time elapsing between the pause onset and the 1st spike after the tail flick. Comparisons between pretreatment and post-treatment ongoing and tail-flick-related cell activity changes were performed by anova for repeated measures. Comparisons between different treated groups of rats were performed by using Wilcoxon signed-ranks test. P < 0.05 was considered statistically significant.

Materials

5′-iodo-resiniferatoxin, DPCPX, AM 251, OMDM-2, and propofol were supplied by Tocris (Bristol, UK) and AP-18, WAY100635, pentobarbital and mustard oil were from Sigma-Aldrich (Milano, Italy). CBD and CBC were obtained from GW Pharmaceuticals, Salisbury, UK. Receptor and channel nomenclature followsAlexander et al. (2009)

Effect of intra-vl-PAG CBD and CBC on ongoing ON and OFF cell activity in anaesthetized rats

The results were based on RVM neurons (one cell recorded from each animal per treatment) at a depth of 9900–10 955 µm from the surface of the brain. All recorded neurons, identified as OFF cells by the characteristic pause induced by the tail flick trail, were spontaneously active and discharged with a mean frequency of 7.58 ± 0.6 spikes s⁻¹ (Figure 3B).

Figure 3

Effect of vehicle, cannabidiol (CBD) (1.5, 3 and 6 nmol) alone or CBD (3 nmol) in combination with AM251 (0.5 nmol), I-RTX (1 nmol), DPCPX (0.05 nmol), AP18 (6 nmol) or WAY100635 (0.34 nmol) on the spontaneous firing of rostral ventromedial medulla ON …

Neurons identified as ON cells by a burst of activity just before tail flick responses were spontaneously active in 33.2% of the cases and inactive in the remaining cases. ON cells with spontaneous activity were chosen in order to characterize drug-related changes on both ongoing and tail-flick-related activity. The population of ON cells with spontaneous activity had a mean frequency of 7.12 ± 0.4 spikes s⁻¹ (Figure 3A).

Microinjection of vehicle did not change the spontaneous activity of either ON (F_(1,14) = 0; P > 0.05) or OFF cells (F_(1,14) = 0.02; P > 0.05). Microinjections of CBD (1.5, 3 and 6 nmol) into the vl-PAG caused a decrease on the firing activity of the ON cells. CBD (3 nmol) induced a greater effect with a maximal decrease observed 35 min after microinjection (F_(1,14) = 28.64; P < 0.01) (Figure 3A). The same treatment produced a decrease in the firing activity of the OFF cells, which was maximal 30 min after the microinjection of CBD (3 nmol) (F_(1,14) = 88.65; P < 0.01) (Figure 3B). CBD (3 nmol) also reduced tail-flick-related ON cell burst (F_(1,14) = 8.71; P < 0.05) (Figure 4A) but was devoid of activity on the OFF cell pause. The effects induced by CBD (3 nmol) on the ON and OFF cell were prevented when AM251 (0.5 nmol) (Figures 3C and D, ​,4B),4B), DPCPX (0.05 nmol), AP18 (6 nmol) (Figures 3E and F, ​,4C)4C) or WAY-100635 (0.34 nmol) (Figures 3C and D, ​,4B)4B) were co-injected. I-RTX (1 nmol) only antagonized the effect of CBD (3 nmol) on the OFF cells at 5 and 10 min after drug microinjection (Figure 3D).

Figure 4

Effects of intra-ventrolateral periaqueductal gray microinjections of vehicle, cannabidiol (CBD) (3 nmol), cannabichromene (CBC) (6 nmol), OMDM-2 (3 nmol) and mustard oil (6 mol) on the tail flick induced RVM ON burst of firing (A). B shows the effect …

Cannabichromene (3 and 6 nmol) induced effects similar to those elicited by CBD, that is, a decrease on the ongoing activity of the ON and OFF cells. In particular, the decrease in the ON cell ongoing activity was maximal 30 min after the injection of CBC (6 nmol) (F_(1,14) = 22.65; P < 0.01) (Figure 5A). CBC (6 nmol) also reduced tail-flick-related ON cell burst (F_(1,14) = 9.15; P < 0.01) (Figure 4A) without changing the OFF cell pause. The decrease in the OFF cell ongoing activity started 5 min after CBC (6 nmol) microinjection and was maximal 35 min after its administration (F_(1,14) = 49.97; P < 0.01) (Figure 5D). Similarly to CBD, the effect of CBC (6 nmol) was antagonized by AM251 (0.5 nmol) (Figures 4C, 5C and D), DPCPX (0.05 nmol) and AP18 (6 nmol) (Figures 4C, 5E and F), although not by I-RTX (1 nmol) (Figures 4C, 5C and D).

Figure 5

Effect of vehicle, cannabichromene (CBC) (3 and 6 nmol) alone or CBC (6 nmol) in combination with AM251 (0.5 nmol), I-RTX (1 nmol), DPCPX (0.05 nmol) and AP18 (6 nmol) on the spontaneous firing of RVM ON (A, C and E) or OFF (B, D and F) cells. A and B …

Effect of intra-vl-PAG CBD and CBC on tail-flick-related nociception in anaesthetized rats

Tail flicks were elicited every 5 min for at least 10 min prior to microinjecting drugs or respective vehicle into the vl-PAG. Data related to pretreatment intervals were considered as basal tail flick latencies (4.5 ± 0.3 s). Intra-vl PAG microinjection of vehicle did not change the tail-flick latency as compared with basal values. Tail-flick latency proved to be significantly increased by CBD (1.5 and 3 nmol). The dose of 3 nmol CBD produced the highest antinociceptive effect (F_(1,30) = 38.12; P < 0.01) while the dose of 6 nmol was devoid of antinociceptive effect (Figure 6A). The antinociceptive effect of CBD (3 nmol) was blocked by AM251 (0.5 nmol) or WAY100635 (0.34 nmol) (Figure 6C), DPCPX (0.05 nmol) or AP18 (6 nmol). I-RTX (1 nmol) was instead able to block CBD (3 nmol) antinociceptive effect only at 15 min after drug microinjection (Figure 6C).

Figure 6

Effect of vehicle, cannabidiol (CBD) (1.5, 3 and 6 nmol) and cannabichromene (CBC) (3 and 6 nmol) alone or CBD (3 nmol) or CBC (6 nmol) in combination with AM251 (0.5 nmol), I-RTX (1 nmol), DPCPX (0.05 nmol) and AP18 (6 nmol) on the tail-flick latency. …

Tail-flick latency was also increased by CBC (3 and 6 nmol) with a maximal effect at the higher dose used (F_(1,30) = 40.70; P < 0.01) (Figure 6B) and at 15 min after its administration. Such an antinociceptive effect was prevented by AM251 (0.5 nmol) (Figure 6D), DPCPX (0.05 nmol) or AP18 (6 nmol) (Figure 6F) but not by I-RTX (1 nmol) (Figure 6D).

Effect of intra-vl-PAG injection of mustard oil and OMDM-2 on ongoing ON and OFF cell activity and tail-flick-related nociception in anaesthetized rats

In order to substantiate the involvement of TRPA1 channels and endocannabinoid cellular uptake in the effects of the phytocannabinoids, we next tested pharmacological tools specific for these two targets. Both OMDM-2 (1.5 and 3 nmol), a selective and potent inhibitor of endocannabinoid cellular uptake (Ortar et al., 2003), and mustard oil isothiocyanate (3 and 6 nmol), the prototypical activator of TRPA1 (Jordt et al., 2004) injected into the vl-PAG, reduced ON and OFF cell ongoing activity (Figure 7A–D). In particular, OMDM-2 (1.5 nmol) decreased the ON cell ongoing activity with a noticeable delay (35 min). The highest dose of OMDM-2 (3 nmol) decreased the ON cell ongoing activity as early as 15 min after its administration and showed a maximal effect 35 min after its administration (F_(1,14) = 116.46; P < 0.01) (Figure 7A). OMDM-2 also decreased the OFF cell ongoing activity but only at the higher dose used (3 nmol). This effect was evident after 15 min and maximal 35 min after its administration (F_(1,14) = 135.98; P < 0.01) (Figure 7B). The lowest dose of mustard oil (3 nmol) did not change the ON cell ongoing activity. The highest dose (6 nmol) decreased the ON cell ongoing activity after 20 min showing a maximal effect after 35 min from its administration (F_(1,14) = 23.82; P < 0.01) (Figure 7C). The highest dose of mustard oil (6 nmol) also decreased the OFF cell ongoing activity already after 5 min from its administration showing a maximal effect 35–40 min after its administration (F_(1,14) = 28.55;P < 0.01) (Figure 7D). OMDM-2 (3 nmol) and mustard oil (6 nmol) decreased also the tail-flick-induced ON cell burst (Figure 4A) but were devoid of activity on the OFF cell pause. OMDM-2 (3 nmol) and mustard oil (6 nmol) increased the tail-flick latency (F_(1,30) = 22.49 and F_(1,30) = 8.98 respectively; both P < 0.01) (Figure 7E and F).

Figure 7

Effect of vehicle, OMDM-2 (1.5 and 3 nmol) or mustard oil (3 and 6 nmol) on the spontaneous firing of rostral ventromedial medulla ON (A and C) and OFF (B and D) cells and on nocifensive behaviour through tail flick test (E and F). Each point represents …

Effect of intra-vl-PAG CBD and CBC on endocannabinoid levels in the PAG

The effect of the intra vl-PAG injection of maximally active doses of CBD (3 nmol) and CBC (6 nmol) on PAG endocannabinoid levels of lipid extracts of the PAG were determined by LC-MS (Table 1). CBD significantly elevated 2-AG by 2.6-fold, but not anandamide levels. CBC, instead, significantly elevated both 2-AG levels, by almost 3.9-fold and anandamide levels, by almost 1.7-fold.

Table 1

Amounts of anandamide and 2-arachidonoylglycerol (2-AG) in the rat periaqueductal grey (PAG) after injection of cannabidiol (CBD) or cannabichromene (CBC)

This study was partly supported by a research grant from GW Pharmaceuticals.

Abbreviations

2-AG

2-arachidonoyl glycerol

AEA

arachidonoyl ethanolamide (anandamide)

CB₁

cannabinoid receptor type 1

CBC

cannabichromene

CBD

cannabidiol

FAAH

fatty acid amide hydrolase

RVM

rostral ventromedial medulla

THC

Δ⁹-tetrahydrocannabinol

TRPA1

transient receptor potential channels of ankyrin type-1

TRPV1

transient receptor potential channels of vanilloid type-1

vl-PAG

ventrolateral periaqueductal grey

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