Effects of intra-ventrolateral periaqueductal grey palmitoylethanolamide on thermoceptive threshold and rostral ventromedial medulla cell activity

European Journal of Pharmacology 676 (2012) 41–50

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Neuropharmacology and Analgesia

Effects of intra-ventrolateral periaqueductal grey palmitoylethanolamide on thermoceptive threshold and rostral ventromedial medulla cell activity Vito de Novellis a, 1, Livio Luongo a, 1, Francesca Guida a, Luigia Cristino c, Enza Palazzo a, Roberto Russo b, Ida Marabese a, Giuseppe D'Agostino b, Antonio Calignano b, Francesca Rossi d, Vincenzo Di Marzo e,⁎, Sabatino Maione a,⁎⁎ a

Endocannabinoid Research Group at the Department of Experimental Medicine, Division of Pharmacology “L. Donatelli”, The Second University of Naples, 80138 Naples, Italy Department of Experimental Pharmacology, University of Naples Federico II, Naples, Italy c Endocannabinoid Research Group at the Institute of Cybernetics, Consiglio Nazionale delle Ricerche, Pozzuoli (NA), Italy d Department of Pediatrics, Second University of Naples, via De Crecchio 4, 80138 Naples, Italy e Endocannabinoid Research Group at the Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli (NA), Italy b

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Article history: Received 29 August 2011 Received in revised form 22 November 2011 Accepted 24 November 2011 Available online 3 December 2011 Keywords: PEA PPARα receptor Periaqueductal grey Rostral ventromedial medulla Pain (Rat)

a b s t r a c t Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α (PPAR-α) ligand, exerts antinociceptive and anti-inflammatory effects. PEA (3 and 6 nmol) was microinjected in the ventrolateral periaqueductal grey (VL PAG) of male rats and effects on nociceptive responses and ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) ON and OFF cells were recorded. Intra-PAG microinjection of PEA reduced the ongoing activity of ON and OFF cells and produced an increase in the latency of the nociceptive reaction. These effects were prevented by a selective PPAR-α antagonist, GW6471 and by a largeconductance Ca2 +-activated K + channel inhibitor, charybdotoxin. Cannabinoid 1 (CB1) receptor blockade by AM251 increased the PEA-induced effect both on the ongoing activity of the ON cell and on the latency to tail flick without affecting the effect of PEA on the OFF cell. Conversely, a transient receptor potential vanilloid type 1 (TRPV1) blocker, I-RTX, had no effect on the ON cell activity and tail flick latency, whereas it blocked the PEA-induced decrease in ongoing activity of the OFF cell. PEA decreased the burst and increased the latency of tail flick-evoked onset of ON cell activity in a manner antagonised by GW6471 and charybdotoxin. AM251 and I-RTX, instead, enhanced these latter effects. In conclusion, intra-VL PAG PEA induces antinociceptive effects associated with a decrease in RVM ON and OFF cell activities. PPAR-α receptors mediate, and CB1 and TRPV1 receptors antagonise, PEA-induced effects within the PAG-RVM circuitry. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Peroxisome proliferator-activated receptors (PPARs) are members of proteins termed nuclear receptors (Mangelsdorf et al., 1995) which fatty acids or fatty acid derivatives activate. PPAR stimulation leads to the regulation of promoters of gene encoding for proteins involved in lipid metabolism, inflammation (Tontonoz et al., 1994) and nociception (Kostadinova et al., 2005; Lo Verme et al., 2005, 2006). Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts as PPAR-α ligand (Lo Verme et al., 2005). PEA reduces the pain behaviour elicited by formalin (Calignano et al., 1998), carrageenan (Conti et al., 2002), NGF (Farquhar-Smith and Rice, 2003), sciatic nerve ligation (Helyes et al., 2003) or subcutaneous granuloma (De

⁎ Corresponding author. Tel.: + 39 0818675093. ⁎⁎ Corresponding author. Tel.: + 39 081 5667650. E-mail addresses: [email protected] (V. Di Marzo), [email protected] (S. Maione). 1 These authors contributed equally to this work. 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.11.034

Filippis et al., 2011). Its brain concentrations are higher than anandamide (Cadas et al., 1997; Di Marzo et al., 1994), whereas the expression and distribution of PPAR-α within the central nervous system (CNS) have been described (Benani et al., 2004; Moreno et al., 2004). More recently, rapid broad-spectrum analgesia by PEA has been documented to be mediated by non-genomic effects such as activation of largeconductance Ca2 +-activated K + channels (Lo Verme et al., 2006). PEA does not bind cannabinoid CB1 or CB2 receptors (Lambert and Di Marzo, 1999; Lambert et al., 2002). However, PEA might inhibit anandamide metabolic degradation, due to its competition for fatty acid amide hydrolase (FAAH) (Ueda et al., 2000), thus leading to increased anandamide levels and analgesic actions through the stimulation of CB1 receptor and desensitisation of TRPV1 (Costa et al., 2008; Di Marzo et al., 2001). The PAG-RVM pathway is a key circuitry in pain processing. PAGmediated antinociception involves the recruitment of pain modulating RVM neurons. Three classes of neurons in RVM have been shown to respond differently to pain-stimuli (Fields et al., 1995; Vanegas et al., 1984): ON cells are activated, OFF cells are inhibited and Neutral cells

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are unaffected (Fields et al., 1991, 2006). Intra-PAG microinjections of CB1 and TRPV1 agonists change ON and OFF cell activity and produce analgesia (de Novellis et al., 2005). Moreover, we have shown that URB597, a FAAH inhibitor, changed the nociceptive threshold, the ON and OFF cells activity and increased the anandamide levels within the PAG (Maione et al., 2006). It has been shown that FAAH inhibition elevates PEA levels (Jhaveri et al., 2008; Kathuria et al., 2003), which can also produce analgesia by activating PPAR-α (Lo Verme et al., 2006) and other mechanisms (Costa et al., 2008). Based on PEA-induced antinociception through multiple mechanisms we hypothesised that PEA microinjection within the VL PAG may affect thermoceptive responses and the electrophysiological activity of RVM ON and OFF cells. Furthermore, owing to the fact that PEA competes with anandamide for hydrolysis by FAAH, thus elevating anandamide levels, the effects of intraVL PAG PEA microinjection on thermoceptive response to tail flick and ongoing and tail flick-related activity of RVM ON and OFF cell could be affected not only by PPAR-α blockers but also by CB1 or TRPV1 receptors. 2. Material and methods 2.1. Animals A total of 132 Wistar male rats (250–300 g) were used. Rats were housed 3 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. The experimental procedures were approved by the Animal Ethics Committee of the Second University of Naples. Animal care was in compliance with Italian Legislative Decree D.L. 116/92 and European Commission Directive L358/1, 18/12/86 regulations on the protection of laboratory animals. For all experimental procedures n = 6–8 animals were used. All efforts were made to minimise animal suffering and to reduce the number of animals used. 2.2. Surgical preparation for intra-PAG microinjections In order to perform intra-VL PAG administrations of drugs or respective vehicle, 0.5% DMSO in artificial cerebrospinal fluid (aCSF, composition in mM: KCl 2.5; NaCl 125; MgCl2 1.18; CaCl2 1.26), rats were anaesthetised with pentobarbital (50 mg/kg, i.p.) and a 26gauge, 12 mm-long stainless steel guide cannula was stereotaxically lowered until its tip was 1.5 mm above the VL PAG by applying coordinates from the atlas of Paxinos and Watson (1986) (A: −7.8 mm and L: 0.5 mm from bregma, V: 4.3 mm below the dura). VL PAG was considered in this study, since previous studies have shown the presence of excitatory output neurons projecting to RVM in that area (Moreau and Fields, 1986; Sandkühler and Gebhart, 1984). 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). Direct intra-VL PAG administration of drugs, or respective vehicle, was conducted with a stainless steel cannula connected by a polyethylene tube to a SGE 1-μl syringe, inserted through the guide cannula and extended 1.5 mm beyond the tip of the guide cannula to reach the VL PAG. Volumes of 200 nl drug solutions, or vehicle, were injected into the VL PAG over a period of 60 s and the injection cannula gently removed 2 min later. 2.3. RVM extracellular recordings coupled to tail flick test After implantation of the guide cannula into the VL PAG (described in: Surgical preparation for intra-PAG microinjections), a

tungsten microelectrode was stereotaxically lowered through a small craniotomy into the RVM (AP: 11.5; L: 0.3; V: 9.9–10.9) (Paxinos and Watson, 1986) to record the activity of ON and OFF cells. The OFF cells were identified by the characteristic pause when tail flick stimulus was applied (Fields et al., 1983; Heinricher et al., 1989). As far as the ON cells are concerned, these were identified by a sudden firing increase (spikes/s) beginning just prior to the occurrence of the tail flick reflex. Moreover, only ON cells with spontaneous activity have been included in the analysis of data. Anaesthesia was maintained with a constant, continuous infusion of propofol (5–10 mg/kg/h, i.v.) and was adjusted so that tail flicks were elicited with a constant latency of 4–5 s. The tail flick test was performed during the electrophysiological recording with the animals anaesthetized and immobilised on the stereotaxis frame during the duration of the experiment. A thermal stimulus was elicited by a radiant heat source of a tail flick unit (Ugo Basile, model 7360, Varese, Italy) focused on the rat tail approximately 3–5 cm from the tip. The rat tail was placed over the surface of a slightly projecting window receiving the I.R. energy. The I.R. intensity in our experiments has been set to 50 mW corresponding to 50 mJ per s. Cutoff was set at 20 s in order to prevent tissue damage. The intensity of the radiant heat emission was adjusted at the beginning of each experiment in order to elicit a constant tail flick latency. Tail flick latency in seconds was determined by a timer connected to a photoelectric cell which stopped the timer (and switch off the lamp) at the movement of the tail which was withdrawal. Tail flicks were elicited every 5 min for at least 10 min prior to microinjecting drugs, or respective vehicle, into the VL PAG. Tail flick latencies were monitored in the same rats undergoing RVM ON and OFF cell recordings. Extracellular single-unit recordings were made in the RVM with glass insulated tungsten filament electrodes (3–5 MΩ) (FHC Frederick Haer & Co., ME, USA). The recorded signals were amplified and displayed on 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 El electronic Design Ltd., UK) connected to a Pentium III PC. Spike2 software (CED, version 4) was used to create peristimulus rate histograms on-line and to store and analyse digital records of single-unit activity off-line. The configuration, shape, and height of the recorded action potentials were monitored and recorded continuously using a window discriminator and Spike2 software for on-line and off-line analysis. Once an ON or OFF cell was identified, we optimised spike size before treatments. This study only included neurons whose spike configuration remained constant and could clearly be discriminated from activity in the background throughout the experiment, indicating that the activity from only one neuron was measured. Only one neuron was recorded from each rat. 2.4. Experimental design Groups of animals (n = 12–14 in order to have at least 6 ON and 6 OFF cell recordings) were treated with an intra-VL PAG microinjection of vehicle or PEA (3 or 6 nmol) either alone or PEA (6 nmol) in combination with GW6471 (3 nmol), a PPAR-α antagonist, charybdotoxin (CBTX, 0.15 and 0.3 nmol), a blocker of large-conductance Ca2 +activated K + channel, AM251 (0.5 nmol), a CB1 receptor antagonist, or I-RTX (1 nmol), a TRPV1 channel antagonist. The antagonists were either administered alone or co-injected with PEA. Electrophysiological recordings were carried out simultaneously to the tail flick latency (s) measurements. 2.5. Data analysis and statistical procedures The neuron responses were measured and expressed as spikes/s (Hz) before and after intra-VL PAG vehicle or drug microinjections. Background activity of neurons was measured between tail flicks. In particular, basal values were obtained by averaging the activities

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recorded in 50 s before the application of two consecutive thermal stimulations (each stimulation trial was performed every 5 min). Changes in neuron response were expressed as mean ± S.E.M. (spikes/s, extracellular recordings), while changes in withdrawal latencies were expressed as a percentage of the maximum possible effect (%MPE), using the following formula:

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%MPE ¼ ðtest latencyÞ−ðcontrol latencyÞ  100 ðcutoff timeÞ−ðcontrol latencyÞ: Tail flick-related ON cell burst of firing was calculated as the number of spikes in the 2-s interval beginning 0.5 s before the tail flick, before and after drug treatments. The onset of the ON cell burst was also calculated as the time elapsing between the onset of the applied noxious radiant heat and the beginning of the tail flick-related increase in the cell frequency which was at least double than that of baseline spontaneous activity. We also performed analysis of tail flick-related OFF cell activities before and after drug treatments. The duration (the interval between the pause onset and the 1st spike after the tail flick) and the onset of the cell pause (the interval between the stimulus application and last spike) were determined. Comparisons between pre- and post-treatment ongoing and tail flick-related cell activity changes were performed using ANOVA for repeated measures. Comparisons between differently treated groups of rats were made by using 2-way ANOVA for repeated measures followed by Newman–Keuls post hoc test for multiple comparisons. P b 0.05 was considered statistically significant.

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2.6. Histology At the end of the experiment, a volume of 200 nl of neutral red (0.1%) was also injected into the VL PAG 30 min before killing the rat. Rats were then intracardially perfused with 200 ml phosphate buffer solution (PBS) followed by 200 ml 10% formalin solution in PBS. The brains were removed and immersed in a saturated formalin solution for 2 days. The injection site was ascertained by using 2 consecutive sections (40 μm), one stained with cresyl violet to identify nuclei and the other unstained to determine dye spreading. Only those rats whose microinjected site was located within the VL PAG were used for data computation (Fig. 1A). Recording sites were recognised with an electrolytic lesion at the conclusion of the experiment. The rats were killed with an overdose of pentobarbital, and perfused with 10% formalin. The locations of all the studied neurons were reconstructed and plotted on standardised sections (Fig. 1B). Cells located intentionally or accidentally outside the RVM were excluded from the study. 2.7. Immunohistochemical detection of PPAR-α within PAG In order to perform immunohistochemical staining of PPAR-α receptors in the PAG, rats (n = 4) were deeply anaesthetized (pentobarbital, 60 mg/kg, i.p.) and perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Each brain was removed from the skull, postfixated for 2 h in the same fixative as above and then washed and soaked for cryoprotection in 30% sucrose in PB at 4 °C until it sank. Coronal frozen sections containing PAG were cut through the brain at 14 μm-thick and collected in alternate series onto gelatine-coated slides (Mezel, Germany) to be processed for anti-PPAR-α immunohistochemistry. For this purpose the sections were reacted for 10 min in 0.1% H2O2 to inactivate endogenous peroxidase activity and preincubated for 1 h at room temperature in 10% normal rabbit serum (NRS; Vector Laboratories, Burlingame, CA) in 0.1 M Tris-buffered saline, pH 7.6 (TBS), containing 0.3% Triton X-100 and 0.05% sodium azide (Sigma, St. Louis, MO). The sections were then

- 2.00 Fig. 1. Schematic illustration of the location of periaqueductal grey (PAG) microinjection sites (A) and RVM ON or OFF cell recording sites (B). Vehicle or drug microinjections were performed in the VL PAG (filled circles) (A). Open circles indicate the microinjection sites performed outside the VL-PAG, which were neither associated with change in RVM cell activity nor with tail flick latency and were not included in the study. Moreover, cell recordings were performed by lowering a tungsten electrode into the RVM and ON cells (filled triangles) or OFF cell (open triangles) sites (B) are shown. Many sites are not shown to avoid symbol overlapping. Distances (mm) from the interaural line are indicated.

incubated overnight at 4 °C with NRS diluted (range 1:200–1:400) goat polyclonal anti-PPAR-α (Santa Cruz Biotechnology, Santa Cruz, CA USA). After several rinses, the sections were incubated at room temperature for 2 h in biotinylated rabbit anti-goat IgGs (Vector Laboratories, Burlingame, CA) followed by incubation for 1 h in the avidin– biotin complex (ABC Kit; Vectastain, Vector) diluted in TBS according to the supplier indications, and then in 0.05‰ 3-3′diaminobenzidine for 10 min (DAB Sigma Fast, Sigma-Aldrich, Louis, MO U.S.A.). Controls for specificity of immunoreactivity included: 1) preabsorption of diluted goat polyclonal anti-PPAR-α with its respective immunising peptide (blocking peptide sc-1985P from Santa Cruz Biotechnology, Santa Cruz, CA USA); 2) omission of either the primary or the secondary antibodies. The sections processed for immunohistochemistry were studied at the microscope under bright-field illumination equipped with the appropriate filters (Zeiss Axioscop). In all controls no immunostaining was detected. Images were acquired by using the digital camera Leica DFC 320 connected to the microscope and the image analysis software Leica IM500 for Windows (Leica; Germany). Digital images were processed in Adobe Photoshop, with contrast and brightness being the only adjustments made.

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2.8. Drugs PEA, N-((2S)-2-(((1Z)-1-Methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl) ethoxy)phenyl)propyl)propanamide (GW6471), iodoresiniferatoxin (IRTX) and N-(Piperidin-1-yl)-5-(4-iodophenyl) -1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) were purchased from Tocris Bioscience Ltd, Bristol, UK. All the drugs were dissolved in 0.5% DMSO in aCSF (v/v).

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The tail flick latency, expressed as percentage of the maximum possible effect (% MPE), was elicited every 5 min for at least 10 min before microinjecting drugs or respective vehicle into the VL PAG. Intra-VL PAG microinjection of the lowest dose of PEA (3 nmol) did not significantly change the tail flick latency. The higher dose of PEA (6 nmol) produced a significant (P b 0.001) increase in the latency of the nociceptive reaction of 29.95% ± 2, with a maximal effect 30 min after its administration (Fig. 2A). The antinociceptive effect of PEA (6 nmol) was prevented by both co-injection with GW6471 (3 nmol) (Fig. 2B), but not by co-injection with I-RTX (1 nmol) (Fig. 2C). AM251 (0.5 nmol) enhanced PEA-induced antinociception (37.45% ± 3.2) (Fig. 2C). All antagonists, including GW6471 at the highest tested (12 nmol) (data not shown), were inactive at the doses used when administered per se (Starowicz et al., 2007). For the effects of higher doses I-RTX and AM251 through the same administration route (see de Novellis et al., 2008).

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3. Results 3.1. Effect of PEA on tail flick latency

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3.2. Effect of PEA on the ongoing activity of RVM ON and OFF cells 0

Results are based on RVM neurons (group size = 12–14; one cell recorded from each animal per treatment) at a depth of 8875 to 10,730 μm from the surface of the brain, the location of the neurons being in nucleus raphe magnus, nucleus reticularis gigantocellularis pars α, and nucleus reticularis paragigantocellularis (Fig. 1B). Neurons identified as ON cells by a burst of activity just before tail flick responses were spontaneously active in 32.2% of the cases and inactive in the remaining cases. ON cells with spontaneous activity were chosen to better characterise the activity of this ON cell subgroup and to consider post-drug changes in their spontaneous activity. Thus only the ongoing and tail-flick related activity of the ON cell with spontaneous activity has been included in the analysis of data. The population of ON cells with spontaneous activity had a mean frequency of 7.4 ± 0.6 (ON cells). OFF cells had a spontaneous activity of 8.1 ± 0.7 spikes/s. Injection of vehicle or the lower dose of PEA (3 nmol) into the VL PAG did not significantly change the firing activity of the pronociceptive ON or the antinociceptive OFF cells of RVM (Fig. 3A and B). The higher doses of PEA (6 nmol) caused an inhibition of the ongoing activity of the ON cells, which was significant 15 min after its administration and maximal at 40 min (3.3 ±0.4 and 2.2±0.6 spikes/s, respectively) (F(3–23) = 24.98, P b 0.001) (Fig. 3A, see also Fig. 4A). PEA (6 nmol) reduced the ongoing activity of the OFF cells, an effect which was significant after 20 min and maximal 45 min after its administration (2.9± 0.6 and 0.9± 0.7 spikes/s, respectively) (F(3–23) = 28.01, P b 0.001) (Fig. 3B, see also Fig. 4B). 3.3. Effects of PPAR-α blockade and Ca 2 +-activated K + channel blockade on PEA-induced changes in the ON and OFF cell activity In this set of experiments we used the dose of PEA (6 nmol) able to modify tail flick latency, coinjected with the ineffective dose of GW6471 (3 nmol) or CBTX (0.3 nmol) in order to unmask the contribution of PPAR-α or Ca 2 +-activated K + channels on PEA-induced

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Fig. 2. Tail flick latencies after microinjections into the ventrolateral PAG of vehicle (0.5% DMSO in aCSF), PEA (3 and 6 nmol) alone or PEA (6 nmol) in combination with GW6471 (3 nmol), AM251 (0.5 nmol) and I-RTX (1 nmol). “A” shows the effects of vehicle and PEA at 3 or 6 nmol. “B” shows the effects of PEA (6 nmol) in combination with GW6471 (3 nmol). “C” shows the lack of effect of I-RTX (1 mol) and the enhancement of AM251 (0.5 nmol) on PEA-induced antinociception. Each point represent the mean ± standard error of the mean (S.E.M) of 12–14 observations. ○ indicates significant differences vs vehicle and * vs PEA (6 nmol). P valuesb 0.05 were considered statistically significant.

effect. The same rationale was used for CB1 and TRPV1 antagonists. The effect of PEA (6 nmol) on both ON and OFF cell activities was significantly prevented by its coinjection with GW6471 (3 nmol) (Fig. 5A and B), which was inactive per se at the same and at the highest dose tested (12 nmol) (data not shown). The effect of PEA (6 nmol) on the ongoing activity of the ON and OFF cells was also antagonised by coinjection with the higher dose of CBTX (0.3 nmol) (Fig. 5B and D), but not by the lower dose used (0.15 nmol, not shown). Microinjection of CBTX alone (0.3 nmol, not shown) was per se inactive. 3.4. Effect of CB1 and TRPV1 receptor blockade on PEA-induced changes in the ON and OFF cell activity The effect of PEA (6 nmol) on the ON cell spontaneous activity was modified significantly by AM251 (0.5 nmol) (Fig. 6A and B), which was inactive per se at the same dose (data not shown). In particular, AM251 significantly enhanced the PEA-induced decrease in ON cell ongoing activity. AM251 did not modify PEA-induced decrease of the OFF cell ongoing activity. I-RTX (1 nmol) coinjection with PEA,

V. de Novellis et al. / European Journal of Pharmacology 676 (2012) 41–50

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5.0 ± 0.6 spikes/s (Fig. 7A). PEA (6 nmol) increased the onset of the ON cell burst (from 2.55 ± 0.4 to 4 ± 0.5 s) (F(4–25) = 494.5, P b 0.001) (Fig. 7B). The effect of PEA (6 nmol) on the tail flickinduced burst and onset of burst was significantly prevented by coinjection with GW6471 (3 nmol) or the higher dose of CBTX (0.3 nmol), but not by the lower dose used (0.15 nmol, not shown) (Fig. 7A and B). I-RTX (1 nmol) and AM251 (0.5 nmol) increased the effect of PEA (6 nmol) by further decreasing the burst and increasing the onset of the ON cell burst (Fig. 8A and B). Neither PEA (6 nmol) alone nor PEA in combination with GW6471 (3 nmol), AM251 (0.5 nmol) or I-RTX (1 nmol) affected the tail flick-induced onset or duration of the OFF cell pause (not shown).

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Fig. 3. Effect of intra-VL PAG microinjections of vehicle (0.5% DMSO in aCSF) or PEA (3 or 6 nmol) on the spontaneous firing of RVM ON (A) or OFF (B) cells. The arrows indicate drug microinjections. Each point represents the mean ± standard error of the mean (S.E.M) of 6–8 neurons. ○ indicates significant differences vs vehicle. P values b 0.05 were considered statistically significant.

instead, did not modify PEA-induced decrease in ON cell ongoing activity (Fig. 6A), although it prevented the PEA-induced decrease in OFF cell activity (Fig. 6B).

PPAR-α immunoreactivity was widely present in several cell nuclei throughout the ventrolateral periaqueductal grey (VL PAG, boxed area in Fig. 9A and its high magnification in Fig. 9B). Although the subcellular/nuclear localization of PPAR-α receptor was in accordance with its role as a transcription factor, it was also detected in the cytoplasm and neurites of many cells in the VL PAG (boxed area in Fig. 9B and its high magnification in Fig. 9C) and through the different sections studied, suggesting its role in additional signalling processes as proposed for many other transcription factors. A strong immunoreactivity was also found in the cytoplasm of neurons of the mesencephalic trigeminal nucleus (Fig. 9A and B, arrows). Moreover, a weak immunoreactivity was found in the dorsal and lateral PAG (Fig. 9A). No PPAR-α immunostaining was detected when VL PAG sections adjacent to those that were PPAR-α-ir (shown in Fig. 9D) were made to react with preabsorbed PPAR-α antibody blocking peptide (Fig. 9E). 4. Discussion

3.5. Effect of PEA on the tail flick-related RVM ON and OFF cell activity PEA (6 nmol) modified the tail flick-related ON cell activity. It significantly reduced the ON cell burst from 14.1 ± 0.5 spikes/s to

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In this study we evaluated whether PEA intra-VL PAG microinjections might be able to modify thermoceptive responses and the ongoing and tail flick-evoked activity of ON and OFF cells of the RVM, in

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Fig. 4. Single representative ratemater records showing the effect of tail flick stimulation (the black arrows indicate when the heat stimulus was applied) on the ON cell activity or OFF cell pause before and after PEA (6 nmol). The red arrows indicate when the tail flick reflex occurred. Scale bars indicate 2 s intervals.

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Fig. 5. Effects of intra-VL PAG microinjections of vehicle, PEA (6 nmol) alone or in combination with GW6471 (3 nmol) or CBTX (0.3 nmol) on the spontaneous firing of RVM ON (A) or OFF (B) neurons. Each point represents the mean ± standard error of the mean (S.E.M) of 6–8 neurons. ○ indicates significant differences vs vehicle and * vs PEA. P values b 0.05 were considered statistically significant.

order to gain evidence for its possible role in the descending antinociceptive pathway (D'Agostino et al., 2007). In fact, although the presence of PEA (Cadas et al., 1997) and PPAR-α expression in the CNS (Kainu et al., 1994; Moreno et al., 2004) have been reported, the role of this pathway in descending antinociception is poorly understood. We

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observed that PEA intra-VL-PAG microinjection produces a rapid antinociceptive effect, indicating that also when locally acting in the brain, PEA may operate via a non-genomic mechanism. We provided pharmacological evidence that such mechanism, as suggested for some PPARα-mediated peripheral effects of PEA (Lo Verme et al., 2006), might

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Fig. 6. Effect of vehicle, PEA (6 nmol) alone or in combination with I-RTX (1 nmol) or AM251 (0.5 nmol) on the spontaneous firing of RVM ON (A) or OFF (B) neurons. Each point represents the mean± standard error of the mean (S.E.M) of 6–8 neurons. ○ indicates significant differences vs vehicle and * vs PEA. P valuesb 0.05 were considered statistically significant.

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Fig. 7. Tail flick related burst (A) and onset of burst (B) of RVM ON cells before and after VL-PAG microinjections of vehicle (0.5% DMSO in aCSF), PEA (6 nmol) alone or in combination with GW6471 (3 nmol) or CBTX (0.3 nmol). Each point represents the mean ± S.E.M. of 6–8 recorded neurons. ○ indicates significant differences vs vehicle and * vs PEA. P valuesb 0.05 were considered statistically significant.

V. de Novellis et al. / European Journal of Pharmacology 676 (2012) 41–50

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Fig. 8. Tail flick related burst (A) and onset of burst (B) of RVM ON cells before and after VL-PAG microinjections of vehicle (0.5% DMSO in aCSF), PEA (6 nmol) alone or in combination with I-RTX (1 nmol) or AM251 (0.5 nmol). Each point represents the mean± S.E.M. of 6–8 recorded neurons. ○ indicates significant differences vs vehicle and * vs PEA. P values b 0.05 were considered statistically significant.

47

involve modulation of large-conductance Ca2 +-activated K + channels, expressed in the brain, and reported to be controlled by PPAR-α in the nucleus tractus solitarius (Kaneko et al., 2009). The antinociceptive role of PEA in midbrain seems consistent with previous data showing that PEA levels decrease in the dorsal raphe and RVM of neuropathic rats (Petrosino et al., 2007). Thus, PEA down-regulation might play a role in the onset or maintenance of chronic pain, and exogenous administration of PEA might restore its physiological levels, inducing pain relief. Similar to previous studies (Lo Verme et al., 2005, 2006), we show here that the PEA antinociceptive effect in the PAG-RVM pathway was prevented by GW6471, a PPAR-α selective antagonist, thus suggesting for the first time a role of central PPAR-α in the control of antinociception. However, also other mechanisms of action have been suggested for PEA (Re et al., 2007). For example, PEA enhances the cannabinoid receptor-mediated antiinflammatory effects of anandamide due to its competition with the enzyme FAAH (Fowler et al., 2001; Lambert and Di Marzo, 1999). Moreover, PEA is able to strengthen anandamide activation of TRPV1 channels (De Petrocellis et al., 2001; Smart et al., 2002), thus facilitating TRPV1 activation/desensitization and leading to analgesia (Knotkova et al., 2008; Palazzo et al., 2002). Our data, however, do not support the participation of TRPV1 in the antinociceptive effects of intra-VL PAG PEA. On the other hand, our immunohistochemical data showing expression of PPAR-α receptor in several VL PAG neurons, reported here for the first time, lend anatomical bases to our data with the PPAR-α antagonist GW6471. Indeed, the potential intervention of PPAR-α in the effects of the putative endogenously elevated anandamide levels that might arise from the local administration of PEA in the VL PAG, might also be envisaged, although the existing data on the

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Fig. 9. (A) Immunohistochemical detection of PPAR-α in the rat periaqueductal grey (PAG). (B) High magnification of the ventrolateral (VL)-PAG boxed area in A, showing also labelling in the neuronal cell nuclei and the cytoplasm of neurons of the mesencephal trigeminal nucleus (arrows). (C) High magnification of boxed area in B, showing cytoplasm and neurites of many labelled VL-PAG neurons. (D,E) Control of the selectivity of PPAR-α immunoreactivity. Note in E the absence of signal, after pre-absorption with a specific blocking peptide, in the VL-PAG slice adjacent to the one shown in D.

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increased the effect of PEA on tail flick-related ON cell burst duration and onset, and PEA anti-nociceptive activity. However, the time course of the effects on ON cells was not consistent with that of the effect on tail flick latency, since AM251 caused a rapid enhancement (within 10–15 min) of PEA-induced antinociceptive activity, although this latter effect was still present 45 min after the injection of the antagonist. In any case, CB1 receptor blockade was unable to modify PEA-induced inhibition of OFF cell firing. Thus, it seems possible that PAG output neurons innervating OFF cells are preferentially excitatory neurons directly modulated by somatic TRPV1 channels (Palazzo et al., 2002; Starowicz et al., 2007). Conversely, the PAGRVM pathway regulating ON cell functioning would appear to be mainly under the trans-synaptic control of at least two types of CB1sensitive GABAergic neurons (Rea et al., 2007). According to this scenario, CB1 receptor blockade would enhance the GABA release from interneurons in the PAG, which in turn would inhibit the firing of the GABAergic output neurons, with subsequent disinhibition of RVM inhibitory interneurons and, hence, inhibition of ON cell firing and stronger analgesic effects in the tail-flick test Fig. 10. The fact that a per se inactive dose of a CB1 receptor antagonist becomes active in the presence of PEA, suggests that under these conditions endocannabinoid tone at CB1 is elevated, possibly due to the increased anandamide levels, which might take some time to develop. This would explain why the tested dose of AM251 reduced ON cell activity in the presence of PEA only after some time, thus prolonging the antinociceptive effect of this compound. As to the more rapid effect of PEA plus AM251 on anti-nociception as compared to ON cell inhibition, this may be due to other factors, such as: i) the involvement of other antinociceptive output areas, ii) the fact that behavioural antinociceptive responses are the result of highly integrated functional responses than just the inhibition of the ON cell. Finally, the possibility that AM251 might have acted via a direct activation of PPAR-α, suggested by a recent study (Zhao et al., 2010), appears unlikely here, as this compound was administered at a dose selective for CB1 and it only enhanced PEA-induced inhibition of ON cell firing after some time. The present observations emphasise the emerging opinion that there is independency between ON and OFF cell functioning

capability of anandamide to activate PPAR-α are controversial (Artmann et al., 2008; Lo Verme et al., 2005). The antinociceptive effect of PEA was associated with a decrease, of the ongoing activity of the “pronociceptive” RVM ON cells. There is growing evidence that ON cell activity decrease may be considered “an electrophysiological marker” of antinociception (Heinricher et al., 2009). However, microinjection of PEA also exerted an inhibitory effect on the OFF cells ongoing activity. Being analgesic, PEA should have increased the ongoing activity of the OFF cells, like opioids or cannabinoids, normally do. The inhibitory effect of PEA on OFF cell firing is probably a consequence of its multi-target mechanism (Costa et al., 2008). In fact, I-RTX, a TRPV1 antagonist, at a dose inactive per se, blocked PEA-induced decrease in ongoing activity of the OFF cell. One possible explanation could be that PEA may have enhanced both anandamide levels and action at TRPV1 (De Petrocellis et al., 2001), thus causing TRPV1 desensitisation. This, in turn, might have caused a local depression of VL-PAG glutamatergic output neurons projecting to RVM OFF cells, similar to that previously observed with a per se active concentration of I-RTX (Fig. 10). However, in this previous study, TRPV1 antagonism in the VL-PAG reduced both ongoing and tail flick-evoked OFF cell activity (Starowicz et al., 2007), whereas here PEA only produced the former effect. Furthermore, TRPV1 antagonism stimulated (Starowicz et al., 2007), whereas here PEA inhibited, ON cell activity. This difference may depend from the fact that here TRPV1 channels might have been desensitised by a compound (PEA i.e.) that, unlike I-RTX, also activates other targets, such as the PPAR-α receptor, potentially causing ON cell inhibition. Furthermore, it must be pointed out that TRPV1 antagonism and TRPV1 activation leading to TRPV1 desensitisation are not equivalent. The effects exerted by TRPV1 desensitisation might be the consequence of secondary changes in cell physiology (Cortright and Szallasi, 2009), which might affect cell behaviour more persistently than rapid TRPV1 blockade. Another present finding pointing to the complexity of PEA actions in the VL-PAG is the observation that a per se inactive dose of a CB1 receptor antagonist enhanced PEA-induced inhibition of the ON cell ongoing activity, although with a delay of 40 min. AM251 also

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(Heinricher et al., 2009), and that, in order to obtain analgesia, it is critical to decrease ON cell activity (Bee and Dickenson, 2007). Indeed, reduced ON and OFF cell excitability, with concomitant hypoalgesia, is also produced through, for example, T-type calcium channel blockade (Barbara et al., 2009), or by sodium channel blockers (Bee and Dickenson, 2007). Our data provide further support to this hypothesis, since we found that only those pharmacological treatments, or combinations thereof, which resulted in the modulation of ON cell activity also modulated in the same way tail flick analgesia, even when inactive on OFF cell activity. Instead, I-RTX antagonised PEA inhibition of ongoing OFF cell activity but did not enhance PEA antinociceptive effect. Moreover, when PEA-induced analgesia ended, the OFF cell remained inhibited, whereas the ON cell, commenced to recover (about 45% recovery at 60 min following PEA). However, the observation that PEA did not affect ON/OFF cell activity as rapidly as it affected tail flick latency may be also consistent with the recruitment of VL PAG projection neurons to other “antinociceptive” output pathways that may be important in generating antinociception, as previously demonstrated (de Novellis et al., 2008). Such pathways are also sensitive to TRPV1 desensitisation in PAG output neurons and can act in synergy with CB1-modulated excitatory terminals from descending cortical neurons innervating PAG neurons afferent to other brainstem areas involved in antinociception (Maione et al., 2006; Palazzo et al., 2008; Rea et al., 2007). It is interesting to note that the hybrid TRPV1 antagonist and FAAH blocker, AA-5-HT, also decreased both ON and OFF cell ongoing activity and induced analgesia, when microinjected into the rat VL PAG, just as PEA did in the current study, its effects being accompanied by changes in cell activity not only in the RVM but also in the locus coeruleus (de Novellis et al., 2008).

5. Conclusion In conclusion, we reported here for the first time that after injection into the VL-PAG, PEA exerted analgesic effects and inhibited RVM ON cell activity. PEA-induced analgesia was not associated with an increase but rather with a decrease in ongoing OFF cell activity, and was counteracted by a selective PPAR-α antagonist. Although it is well established that PPAR-α regulates gene expression (Berger and Moller, 2002), the effects of PEA observed in the present study were fairly rapid in their onset (10–15 min), in agreement with other previous studies (D'Agostino et al., 2007; Lo Verme et al., 2006), and consistent with both a partly non-genomic mechanism of action for PPAR-αmediated antinociception and a subsidiary role of other (CB1 receptors or TRPV1 channels) mechanisms. Apart from the appealing perspective of inducing analgesia by intra-PAG PPAR-α stimulation, our data support the hypothesis that multiple molecular targets might intervene in PEA-induced antinociceptive effects.

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