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

Abstract

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.

Keywords: 2-arachidonoylglycerol/2-AG; GABA; addiction; anandamide/AEA; dopamine; dopamine metabolism; endocannabinoids; ghrelin/GHS-R1A; nucleus accumbens shell microdialysis; synthetic cannabinoid WIN55,212-2.

Figure 1

Effects of JMV2959 on the WIN55,212-2-induced behavioural changes in rats, in the fully automated behaviour monitoring LABORAS cage. JMV2959 (0, 1 and 3 mg/kg i.p.) was injected immediately before placing the rat into the cage, and after 20 min of habituation a stimulatory WIN55,212-2 dose 0.1 mg/kg or vehicle was administered intraperitoneally. After another 20 min of habituation, behaviour monitoring started and lasted for 20 min (20–40 min after WIN55,212-2 administration). Changes in locomotion duration (A), rear duration (B), immobility duration (C), distance travelled (D) and average speed overall (E) are illustrated as follows: saline + vehicle (open bar; n = 9), saline + WIN55,212-2 (filled bar; n = 7), JMV2959 1 mg/kg + WIN55,212-2 (vertically striped bar; n = 7), JMV2959 3 mg/kg + WIN55,212-2 (diamond bar; n = 8), JMV2959 1 mg/kg + vehicle (horizontally striped bar; n = 4), JMV2959 3 mg/kg + vehicle (little arrows bar; n = 8). The JMV2959 pretreatment effects in comparison to saline + WIN55,212-2 group are expressed as # p < 0.05, ## p < 0.01. Differences among groups in comparison to vehicle + saline group are expressed as * p < 0.05, ** p < 0.01.

Figure 2

Effects of growth hormone secretagogoue receptors (GHS-R1A) antagonist (JMV2959) on WIN55,212-2-induced dopamine and its metabolites’ extracellular changes in the rat nucleus accumbens shell (NACSh). JMV2959 3 mg/kg was given i.p. 20 min before 2.4 mM/0.5 µL WIN55,212-2 or vehicle 0.5 µL was administered into the posterior ventral tegmental area (VTA) injection (n = 6; means ± SEM). Changes in accumbens dopamine are illustrated in graph (A), changes in homovanillic acid (HVA), 3-methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC) are illustrated in the graphs (B–D), respectively. The effects are illustrated as follows: saline + WIN55,212-2 (filled circle; n = 7), 3 mg/kg JMV2959 + WIN55,212-2 (open circle; n = 6), 3 mg/kg JMV2959 + vehicle (open triangle; n = 4), saline + vehicle (dotting; n = 7). Differences between saline + WIN55,212-2 and saline + vehicle groups are expressed as *** p < 0.001, * p < 0.05. Differences JMV2959 + WIN55,212-2 and saline + vehicle groups are expressed as +++ p < 0.001, ++ p < 0.01, + p < 0.05. Differences between groups saline + WIN55,212-2 and 3 mg/kg JMV2959 + WIN55,212-2 are expressed as ### p < 0.001, # p < 0.05.

Figure 3

Extracellular dopamine (DA) metabolic turnover in the NACSh. The graphs show the metabolite/dopamine ratios, the means ± SEMs (n = 6) of concentration values of the metabolite divided by the corresponding values of dopamine concentrations (metabolite/DA). The homovanillic acid/dopamine ratio (HVA/DA) values are illustrated in the graph (A), 3-methoxytyramine/dopamine ratio (3-MT/DA) in the graph (B) and the 3,4-dihydroxyphenylacetic acid/dopamine ratio (DOPAC/DA) in the graph (C). Three baseline interval concentration values before pretreatments (baseline metabolite/DA ratio–white bars) and two intervals with maximal WIN55,212-2 effects (40 min and 60 min intervals; treatment metabolite/DA ratio–black bars) were used for statistical comparison within WIN55,212-2 + saline versus WIN55,212-2 + JMV2959 versus vehicle + saline groups. The effect of JMV2959 pretreatment, thus differences between WIN55,212-2 + saline and WIN55,212-2 + JMV2959, are expressed as ### p < 0.001. Differences between groups relative to the vehicle + saline group are expressed as + p < 0.05 and +++ p < 0.001. Differences between treatment and baseline are expressed as * p < 0.05 and *** p < 0.001.

Figure 4

Effects of GHS-R1A antagonist (JMV2959) on WIN55,212-2-induced endocannabinoid and gamma-aminobutyric acid (GABA) extracellular changes in the rat NACSh. The JMV2959 3 mg/kg was injected i.p. 20 min before 2.4 mM/0.5 µL WIN55,212-2 or vehicle 0.5 µL administered into the posterior VTA injection (n = 6; means ± SEM). Changes in accumbens anandamide/N-arachidonoylethanolamine (AEA) levels are illustrated in graph (A), changes in 2-AG levels in the graph (B) and GABA changes are shown in graph (C). The effects are illustrated as follows: saline + WIN55,212-2 (filled circle; n = 7), 3 mg/kg JMV2959 + WIN55,212-2 (open circle; n = 6), 3 mg/kg JMV2959 + vehicle (open triangle; n = 4) and saline + vehicle (dotting; n = 7). Differences between saline + WIN55,212-2 and saline + vehicle groups are expressed as *** p < 0.001. Differences between JMV2959 + WIN55,212-2 and saline + vehicle groups are expressed as +++ p < 0.001, ++ p < 0.01. Differences between groups saline + WIN55,212-2 and 3 mg/kg JMV2959 + WIN55,212-2 are expressed as ### p < 0.001, # p < 0.05.

Figure 5

Schematic locations of dialysis probes in the nucleus accumbens shell (A); sites of infusions into the ventral tegmental area (B). Schematic locations of probe tips in animals which were involved in statistical analyses of accumbens neurotransmitter concentrations (the bold lines indicate the dialyzing positions in Figure 4A and locations of WIN55,212-2/vehiculum solution administrations into the VTA (dark dots in the lower part of slices in Figure 4B) as described in the atlas of Paxinos and Watson [77]. The distance from bregma (in mm) is indicated on the left of each sectional view.