Alterations in Rat Accumbens Dopamine, Endocannabinoids and GABA Content During WIN55,212-2 Treatment: The Role of Ghrelin

The endocannabinoid/CB1R system as well as the central ghrelin signalling with its growth hormone secretagogoue receptors (GHS-R1A) are importantly involved in food intake and reward/reinforcement processing and show distinct overlaps in distribution within the relevant brain regions including the hypothalamus (food intake), the ventral tegmental area (VTA) and the nucleus accumbens (NAC) (reward/reinforcement). The significant mutual interaction between these systems in food intake has been documented; however, the possible role of ghrelin/GHS-R1A in the cannabinoid reinforcement effects and addiction remain unclear. Therefore, the principal aim of the present study was to investigate whether pretreatment with GHS-R1A antagonist/JMV2959 could reduce the CB1R agonist/WIN55,212-2–induced dopamine efflux in the nucleus accumbens shell (NACSh), which is considered a crucial trigger impulse of the addiction process. The synthetic aminoalklylindol cannabinoid WIN55,212-2 administration into the posterior VTA induced significant accumbens dopamine release, which was significantly reduced by the 3 mg/kg i.p. JMV2959 pretreatment. Simultaneously, the cannabinoid-increased accumbens dopamine metabolic turnover was significantly augmented by the JMV2959 pretreament. The intracerebral WIN55,212-2 administration also increased the endocannabinoid arachidonoylethanolamide/anandamide and the 2-arachidonoylglycerol/2-AG extracellular levels in the NACSh, which was moderately but significantly attenuated by the JMV2959 pretreatment. Moreover, the cannabinoid-induced decrease in accumbens γ-aminobutyric acid/gamma-aminobutyric acid levels was reversed by the JMV2959 pretreatment. The behavioural study in the LABORAS cage showed that 3 mg/kg JMV2959 pretreatment also significantly reduced the systemic WIN55,212-2-induced behavioural stimulation. Our results demonstrate that the ghrelin/GHS-R1A system significantly participates in the rewarding/reinforcing effects of the cannabinoid/CB1 agonist that are involved in cannabinoid addiction processing.

. We were interested in the WIN55,212-2 effects and particularly the JMV2959 pretreatment effects on the cannabinoid-induced accumbens changes. Therefore, the follow groups were chosen for comparison: WIN55,212-2 + saline, WIN55,212-2 + JMV2959, vehicle + saline. Single concentration values from these rat groups and intervals were used for calculation of the particular accumbens metabolite/dopamine metabolic ratios (HVA/DA, 3-MT/DA, DOPAC/DA) (see the main text of article).  Table S2. The average accumbens concentrations (means within rat group) of anandamide/AEA, 2-AG and GABA within the three baseline intervals and the 60 min and 80 min intervals after the WIN55,212-2 administration, once the maximum effects were observed (each sampling interval lasted 20 min). Number of rats in groups: N = 7 (WIN55,212-2 +SALINE), N = 6 (WIN55,212-2 + JMV2959), N = 7 (VEHICLE + SALINE). Table S3. summarises selected results from our previous behavioural study (Jerabek et al. 2017) which support/confirm our present results. The locomotor activity of adult Wistar rats in "open field" within 20 min starting 25 min after intraperitoneal administration of JMV2959 (1, 3 and 6 mg/kg i.p.) did not significantly differ from the control group which was treated with saline (N = 9).  Table S3. Locomotor activity in "open field" within 20 min starting 25 min after intraperitoneal administration of JMV2959 (1, 3 and 6 mg/kg i.p.). Selected data from our previous study (Jerabek et al. 2017).

Discussion
The cannabinoid mechanisms of effect and the endocannabinoid system Cannabinoids including the main cannabis constituent tetrahydrocannabinol/THC and synthetic cannabinoids e.g. WIN55,212-2, most likely mediate their pleasurable, anxiolytic and rewarding/reinforcing effects through the CB1Rs located within the central brain reward circuits, particularly the VTA and the NAC (Herkenham 1991;Matsuda et al. 1990;Parsons and Hurd 2015). Cannabinoids activate these reward pathways in a manner that is consistent with other drugs of abuse, including the trigger dopamine release in the nucleus accumbens The CB1R-dependent effects emerge only during periods of heightened cellular activity.
The GABA inhibition is sufficient to induce the dopamine neuron bursting (Lobb et al. 2010), so, CB1 receptor activation on the VTA GABA terminals facilitates these actions and CB1 agonists generate dopamine cell firing and phasic accumbens dopamine release hence reward seeking (Cheer et al. 2003;Cheer et al. 2004); particularly, involvement of GABA VTA inputs from the rostromedial tegmental nucleus (RMTg) were suggested (Lecca et al. 2012;Melis and Pistis 2012). Bursting VTA dopamine neurons synthesize and release endocannabinoids, which through retrograde CB1 activations depolarisation-induced suppression of inhibition (DSI, e.g. reduced GABA release) enhances/drives dopamine release. The VTA 2-AG seems to play the main role in the dopamine activation, on the contrary, anadamide/AEA impeded the CB1R binding of 2-AG and antagonises its effects in the VTA (Covey et al. 2015;Volkow et al. 2017).
The role of CB2R is still unclear.
It was described in rats that a single exposure to THC can cause a selective remodelling of a population of glutamatergic synaptic inputs to dopamine neurons in the VTA. Specifically, THC rapidly increased the expression of AMPA receptors with the GluA1 subunit selectively at subcortical pedunculopontine nucleus (PNN) glutamatergic inputs to VTA dopamine neurons. This is thought to result in long-term potentiation (LTP) of these synapses and increase the subcortical drive of VTA DA neurons by the PPN and may play a role in long-term modification in mesolimbocortical dopaminergic output. In the contrary, cocaine-induced glutamatergic synapses remodelling in the VTA was not selective, it was observed also on non-PNN inputs (similarly to other abused drugs) (Good and Lupica 2010;Hoffman and Lupica 2013).
The cannabinoid-induced (phasic) dopamine release within the NACSh activates direct GABAergic medium spiny neurons (dMSNs) through excitatory D1Rs and inhibits the indirect MSNs/iMSNs through inhibitory D2Rs. The D1Rs have low affinity for dopamine, thus they are preferentially activated by large surges/phasic release of dopamine and result in the activation of the direct pathway (dMSNs), which project to the midbrain regions including the VTA that in extend increases approach/appetitive behaviour. The D2Rs have a high affinity for dopamine, thus, basal extracellular dopamine concentrations occupy the D2Rs and inhibit the iMSNs output and supress indirect pathway that projects to the ventral pallidum, thus inhibiting the avoidance behaviour. If there is a dopamine concentration decrease (following exposure to aversive stimulus), then it would relieve the D2R-mediated suppression of iMSNs and promote avoidance behaviour. Therefore, the reward-related phasic dopamine release increases the approach behaviour and reduces the avoidance behaviour (Covey et al. 2015;Parsons and Hurd 2015).
The NACSh MSN activity is substantially modulated by glutamatergic inputs from the prefrontal cortex (PFC), basolateral amygdala and ventral hippocampus that express CB1Rs. Activated accumbens MSN cells and probably fast spiking interneurons (FSI) (Schall et al. 2020) produce and release endocannabinoids (2-AG, AEA), which both modulate presynaptic axons within the NACSh. The CB1-mediated suppression of (glutamate) excitatory signalling is preferentially active at iMSN synapses possibly resulting from D2R-mediated endocannabinoid production (2-AG, AEA) from the iMSN cell bodies. Thus, increased endocannabinoid formation preferentially reduces excitatory inputs to iMSNs (in comparison to dMSNs) which results in decreasing avoidance.
Single acute administration of THC in mice caused a CB1-dependent impairment of the long term depression (LTD) of synaptic transmission at excitatory/glutamatergic synapses on the MSNs in the NACSh (Mato et al. 2004), resulting in diminished constraint of glutamatergic release and increased excitation of NACSh cells.
Thus, the THC/cannabinoid single exposure impairs the endocannabinoid-mediated plasticity in the VTA and the NAC, induces an overall enhancement of mesolimbic signalling, and increase in the sensitivity of dopamine neurons to subcortical excitatory drive in specific set of synapses, which may alter motivational processes mediated by the NAC (Covey et al. 2015;Hoffman and Lupica 2013;Panlilio et al. 2015;Parsons and Hurd 2015;Volkow et al. 2017). (The repeated/chronic cannabinoid effects are not discussed in this article). Figure S1. Administration of JMV2959 1 mg/kg i.p. alone also did not induce significant changes in the accumbens dopamine and metabolites (3-MT, DOPAC, HVA) as it is illustrated in the Figure S1. and HVA (dotted line). No significant differences were found among the groups.