Il Δ9-THC, componente principale della cannabis sativa, attenua la neurotossicità indotta dalla meth

Methamphetamine (METH) is a lipofilic molecule widely abused for its psychostimulant effects. Its long term use can result in neurotoxic and adverse consequences like activation of neuronal nitric oxide synthase (nNOS), production of peroxynitrite, microglia stimulation, astrogliosis and hyperthermia [Maxwell JC and Brecht ML, 2011]. Notably, METH abusers also consume Cannabis [UNODC, 2013]. Several line of research have demonstrated that natural (Δ9-tetrahydrocannabinol, Δ9-THC) and synthetic cannabinoid CB1 receptor agonists exert neuroprotective effects in different models of cerebral damage, including excitotoxic injury [Deng X and Cadet JL, 2009; La Voie MJ, et al. 2004]. In this study we evaluated the neuroprotective effect of Δ9-THC on an animal model of neurotoxicity induced by METH. Rats treated with a binge administation of METH (4 x 10 mg/Kg s.c. 2h apart), were pre-treated or post-treated with 1 mg/Kg or 3 mg/Kg of Δ9-THC (i.p) and sacrificed 3 days after the last METH administration. METH-induced nNOS overexpression and Glial Fibrillary Acidic Protein (GFAP)-immunoreactivity, markers of brain damage, were attenuated by treatment with Δ9-THC. In order to verify the role of CB1 receptor in the mechanism of Δ9-THC-neuroprotection, rats post-treated with Δ9-THC (1 mg/Kg) were pre-treated with the CB1 rceptor antagonist SR141716A (SR). SR partially blocked METH-induced nNOS-overexpression but failed to revert the decreasing effect of Δ9-THC on METH-induced GFAP-immunostaining. These results indicate that Δ9-THC reduces METH-induced brain damage by CB1-dependent and independent mechanisms. In order to identify other neuroprotective mechanisms, we evaluated as markers of neurotoxicity: i) tyrosine hydroxilase (TH), marker of terminal loss; ii) ionized calcium binding adapter molecule 1 (IBA-1), marker of microglial activation; iii) brain derived neurotrophic factor (BDNF) and iiii) CB2 receptor. Post-treatment with Δ9-THC 1mg/Kg increase the METH-induced down-regulation of TH and counteract the IBA-1 over-expression induced by METH. On the contrary, it fails to revert the METH-induced BDNF-increase and CB2-increase. Further studies will be carried out to support the involvement of CB2 receptor on Δ9-THC-mediated mechanism of neuroprotection. References Deng X and Cadet JL (2000) Methamphetamine-induced apoptosis is attenuated in the striata of copper-zinc superoxide dismutase transgenic mice. Brain Res Mol Brain Res. 83: 121-124. LaVoie MJ, Card JP, Hasting TG (2004) Microglial activation precedes dopamine terminal pathology in methamphetamine-induced neurotoxicity. Exp Neurol. 187: 47-57. Maxwell JC and Brecht ML (2011) Methamphetamine: here we go again? Addict Behav. 36: 1168–1173. United Nation Office on Drugs and Crime (UNODC) (2013) World Drug Report 2013 (United Nations publication, Sales No. E.13.XI.6)

Enhanced endocannabinoid-mediated modulation of rostromedial tegmental nucleus drive onto dopamine neurons in sardinian alcohol-preferring rats

The progressive predominance of rewarding effects of addictive drugs over their aversive properties likely contributes to the transition from drug use to drug dependence. By inhibiting the activity of DA neurons in the VTA, GABA projections from the rostromedial tegmental nucleus (RMTg) are well suited to shift the balance between drug-induced reward and aversion. Since cannabinoids suppress RMTg inputs to DA cells and CB1 receptors affect alcohol intake in rodents, we hypothesized that the endocannabinoid system, by modulating this pathway, might contribute to alcohol preference. Here we found that RMTg afferents onto VTA DA neurons express CB1 receptors and display a 2-arachidonoylglycerol (2-AG)-dependent form of short-term plasticity, that is, depolarization-induced suppression of inhibition (DSI). Next, we compared rodents with innate opposite alcohol preference, the Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. We found that DA cells from alcohol-naive sP rats displayed a decreased probability of GABA release and a larger DSI. This difference was due to the rate of 2-AG degradation. In vivo, we found a reduced RMTg-induced inhibition of putative DA neurons in sP rats that negatively correlated with an increased firing. Finally, alcohol failed to enhance RMTg spontaneous activity and to prolong RMTg-induced silencing of putative DA neurons in sP rats. Our results indicate functional modifications of RMTg projections to DA neurons that might impact the reward/aversion balance of alcohol attributes, which may contribute to the innate preference observed in sP rats and to their elevated alcohol intak

α2A adrenergic receptors highly expressed in mesoprefrontal dopamine neurons

α2 adrenoreceptors (α2-ARs) play a key role in the control of noradrenaline and dopamine release in the medial prefrontal cortex (mPFC). Here, using UV-laser microdissection-based quantitative mRNA expression in individual neurons we show that in hTH-GFP rats, a transgenic line exhibiting intense and specific fluorescence in dopaminergic (DA) neurons, α2A adrenoreceptor (α2A-AR) mRNA is expressed at high and low levels in DA cells in the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Confocal microscopy fluorescence immunohistochemistry revealed that α2A-AR immunoreactivity colocalized with tyrosine hydroxylase (TH) in nearly all DA cells in the VTA and SNc, both in hTH-GFP rats and their wild-type Sprague–Dawley (SD) counterparts. α2A-AR immunoreactivity was also found in DA axonal projections to the mPFC and dorsal caudate in the hTH-GFP and in the anterogradely labeled DA axonal projections from VTA to mPFC in SD rats. Importantly, the α2A-AR immunoreactivity localized in the DA cells of VTA and in their fibers in the mPFC was much higher than that in DA cells of SNc and their fibers in dorsal caudate, respectively. The finding that α2A-ARs are highly expressed in the cell bodies and axons of mesoprefrontal dopaminergic neurons provides a morphological basis to the vast functional evidence that somatodendritic and nerve-terminal α2A-AR receptors control dopaminergic activity and dopamine release in the prefrontal cortex. This finding raises the question whether α2A-ARs might function as autoreceptors in the mesoprefrontal dopaminergic neurons, replacing the lack of D2 autoreceptors

Effects of SR on nNOS and GFAP-IR in the CPu.

<p>A. Rats received injections of 1/kg Δ9-THC or VEH at 0.5, 12, 24, 36 and 48 h after the last METH administration (Post-treatment, POST) and were sacrificed 3 days after the last METH injection. SR (1 mg/kg, i.p.) or VEH were administered 15 min before each Δ9-THC or VEH injection. Two-way ANOVA in the CPu (A) showed a significant Δ9-THC x SR interaction (F_(1,40) = 32.45, <i>p</i><0.0001); the administration of SR blunted the effect of Δ9-THC on METH-induced nNOS over-expression. SR alone decreased nNOS labeled neurons compared to that of control. ***p<0.001 vs METH-VEH (VEH pretreated) and <b>^##</b>p<0.01 vs METH-VEH-Δ9-THC (VEH pretreated). B. Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F_(1,35) = 19.86, <i>p</i><0001). Δ9-THC and SR, alone or in combination, attenuated the METH-induced increase of GFAP-IR in the CPu. ***<i>p</i><0.001 vs METH-VEH (VEH pretreated).</p

Effects of SR on nNOS and GFAP-IR in the CPu.

<p>A. Rats received injections of 1/kg Δ9-THC or VEH at 0.5, 12, 24, 36 and 48 h after the last METH administration (Post-treatment, POST) and were sacrificed 3 days after the last METH injection. SR (1 mg/kg, i.p.) or VEH were administered 15 min before each Δ9-THC or VEH injection. Two-way ANOVA in the CPu (A) showed a significant Δ9-THC x SR interaction (F_(1,40) = 32.45, <i>p</i><0.0001); the administration of SR blunted the effect of Δ9-THC on METH-induced nNOS over-expression. SR alone decreased nNOS labeled neurons compared to that of control. ***p<0.001 vs METH-VEH (VEH pretreated) and <b>^##</b>p<0.01 vs METH-VEH-Δ9-THC (VEH pretreated). B. Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F_(1,35) = 19.86, <i>p</i><0001). Δ9-THC and SR, alone or in combination, attenuated the METH-induced increase of GFAP-IR in the CPu. ***<i>p</i><0.001 vs METH-VEH (VEH pretreated).</p

METH increases the number of neuronal nitric oxide synthase (nNOS) neurons and GFAP-immunoreactivity (IR).

<p>Values represent means ± SEM of either number of nNOS positive neurons, expressed per mm² (A) or as percentage of GFAP-IR density (B). **<i>p</i><0.01 and ***<i>p</i><0.001 compared to SAL.</p

Effects of SR on GFAP-IR in the PFC.

<p>Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F_(1,33) = 45.91, <i>p</i><0001). METH-Δ9-THC significantly reduced METH-induced GFAP-IR. Moreover, GFAP-IR was lower in METH-SR-VEH and METH-SR-THC groups as compared to METH-VEH treated rats. ***<i>p</i><0.001 vs METH-VEH (VEH pretreated).</p

Δ9-THC reduces METH-induced increase of nNOS neurons in the CPu.

<p>A. Rats received injections of 1 or 3/kg of Δ9-THC either 0.5 h before each METH injection (PRE) or 0.5, 12, 24, 36, and 48 h after the last METH administration (POST), and were sacrificed 3 days after the last METH injection. Pre- and Post-treatment with both doses of Δ9-THC significantly decreased the number of nNOS positive neurons in the CPu. *<i>p</i><0.05 and **<i>p</i><0.01 vs PRE METH-VEH; <b>^#</b><i>p</i><0.05 and <b>^###</b><i>p</i><0.001 vs POST METH-VEH (Bonferroni's <i>post-hoc</i> test). Horizontal dot lines represent the values of nNOS positive neurons (31±1.03) in SAL-VEH group. B. Representative images of nNOS immunohistochemical staining 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH-Δ9-THC 1 and 3 mg. Scale bar  =  100 µm.</p

Core body temperature: effect of methamphetamine (METH) in the presence and absence of Δ9-THC (1 and 3 mg/kg).

<p>Rats were given SAL (1 mL/kg) or METH (4×10 mg/kg s.c., every 2 h) with and without Δ9-THC (1 and 3 mg/kg) pre-treatment. Body temperature was measured prior to and 1 h after each METH injection. Values are expressed as means ± SEM. Arrows indicate each injection of METH or SAL. No difference in baseline temperature was detected among groups. METH administration resulted in a significant increase in rectal temperature over time in comparison with SAL-treated rats. Both doses of Δ9-THC did not significantly change rectal temperature in METH-administered rats at any time point. METH: *p<0.05, **p<0.01 and ***p<0.001 vs corresponding SAL group at each time point. Δ9-THC1-METH: <b>^##</b>p<0.01 and <b>^###</b>p<0.001 vs corresponding Δ9-THC1-SAL group at each time point; Δ9-THC3-METH: <b>⁺</b>p<0.05 and <b>⁺⁺⁺</b>p<0.001 vs corresponding Δ9-THC 3-SAL group at each time point.</p

Synopsis of the experimental design, including treatment schedule and IHC assays.

<p>A. Pre-treatment: rats received injections of Δ9-THC (1 or 3 mg/kg) or vehicle (VEH) 30 min before each METH or SAL injection, and 3 days (3d) after the last METH or SAL injection were perfused and used for IHC analysis. B. Post-treatment: rats received injections of Δ9-THC (1 or 3 mg/kg) or vehicle (VEH) 0.5, 12, 24, 36 and 48 h after the last METH or SAL administration, and 3 days (3d) after the last METH injection were perfused and used for IHC analysis. C. Post-treatment + SR treatment: rats received injection of SR (1 mg/kg, i.p.) or VEH 15 min prior each Δ9-THC (1 mg/kg) or VEH post-treatment injection, and 3 days (3d) after the last METH or SAL injection were perfused and used for IHC analysis. 0, 2 h, 4 h, 6 h: 1st, 2nd, 3rd and 4th injection of METH (10 mg/kg, s.c.) or SAL; IHC: immunohistochemistry; SR: SR141716A; VEH: vehicle.</p