The effect of self-administered methamphetamine on GABAergic interneuron populations and functional connectivity of the nucleus accumbens and prefrontal cortex

Abstract

Introduction: Methamphetamine (METH, "ice") is a potent and addictive psychostimulant. Abuse of METH perturbs neurotransmitter systems and induces neurotoxicity; however, the neurobiological mechanisms which underlie addiction to METH are not fully understood, limiting the efficacy of available treatments. Here we investigate METH-induced changes to neuronal nitric oxide synthase (nNOS), parvalbumin and calretinin-expressing GABAergic interneuron populations within the nucleus accumbens (NAc), prefrontal cortex (PFC) and orbitofrontal cortex (OFC). We hypothesise that dysfunction or loss of these GABAergic interneuron populations may disrupt the excitatory/inhibitory balance within the brain.

Methods: Male Long Evans rats (N = 32) were trained to lever press for intravenous METH or received yoked saline infusions. Following 14 days of behavioural extinction, animals were given a non-contingent injection of saline or METH (1 mg/kg, IP) to examine drug-primed reinstatement to METH-seeking behaviours. Ninety minutes post-IP injection, animals were culled and brain sections were analysed for Fos, nNOS, parvalbumin and calretinin immunoreactivity in eight distinct subregions of the NAc, PFC and OFC.

Results: METH exposure differentially affected GABAergic populations, with METH self-administration increasing nNOS immunoreactivity at distinct locations in the prelimbic cortex and decreasing parvalbumin immunoreactivity in the NAc. METH self-administration triggered reduced calretinin immunoreactivity, whilst acute METH administration produced a significant increase in calretinin immunoreactivity. As expected, non-contingent METH-priming treatment increased Fos immunoreactivity in subregions of the NAc and PFC.

Conclusion: Here we report that METH exposure in this model may alter the function of GABAergic interneurons in more subtle ways, such as alterations in neuronal firing or synaptic connectivity.

Keywords: Calretinin; Drugs of abuse; GABAergic interneurons; Methamphetamine; Methamphetamine-induced neuroadaptations; Neuronal nitric oxide synthase; Parvalbumin; Relapse to methamphetamine; Self-administration.

Fig. 1

Anatomical coronal diagrams depicting each the brain region of interest and the coordinates (mm from bregma) of each subregion analysed; NAc, nucleus accumbens core; NA Sh, nucleus accumbens shell; Cg1, anterior cingulate gyrus; PrL, prelimbic cortex; IL, infralimbic cortex; MO, medial orbitofrontal cortex; VO, ventral orbitofrontal cortex; LO, lateral orbitofrontal cortex. Adapted from the Rat Brain Atlas (Paxinos and Watson, 2007)

Fig. 2

Mean number of delivered infusions (A), active and inactive lever presses (B) and locomotor activity counts (C) during IVSA and extinction in METH IVSA and yoked controls. Data presented as n = 22 METH IVSA rats, n = 10 yoked controls. D Administration of a non-contingent METH-prime significantly increased active lever pressing in METH/METH animals when compared to Sal/Sal treated animals (**p < 0.001) and the extinction day prior (**p = 0.001). All data presented as mean + SEM. Data presented as n = 5 in Sal/Sal and Sal/METH groups, n = 10 in METH/Sal and METH/METH groups

Fig. 3

Analysis of total nNOS counts within examined brain regions revealed no significant differences across groups (A). Examination of nNOS reactivity in one coordinate of the PrL (+ 3.2 mm from bregma) revealed a significant effect of METH IVSA (B). Correlation analysis revealed a positive correlation between nNOS immunoreactivity in the PrL and METH intake, which trended towards significance (C). All data presented as mean + SEM. Data presented as n = 5 per group or n = 10–20 for correlational analysis

Fig. 4

Analysis of total regional PV immunoreactivity revealed no statistically significant differences across treatment groups (A). However, at one discrete coordinate of the NAc core (+ 1.7 mm from bregma), METH-IVSA yielded a significant reduction in PV immunoreactivity (B). Data presented as mean ∓ SEM, n = 5 per group

Fig. 5

Analysis of total counts of calretinin labelled neurons across brain regions revealed a significant reduction in calretinin immunoreactivity in METH IVSA animals and a significant increase in Sal/METH-exposed animals in the NAc core, NAc shell, Cg1, PrL and IL (A). Examination of one specific coordinate of the brain (+2.7 mm from bregma) also revealed a significant reduction in calretinin immunoreactivity in METH IVSA animals (B). Data presented as mean ± SEM, n = 4 per group. * represents a significant effect of METH IVSA, # represents a significant interaction effect whereby Sal/METH differed to all other groups

Fig. 6

Treatment groups which received a non-contingent injection of METH (1 mg/kg) during relapse testing displayed significantly increased Fos immunoreactivity when compared to groups treated with vehicle (*p < 0.05). NAc core Fos immunoreactivity was also significantly increased in METH/METH animals when compared to Sal/METH animals (#p = 0.020). Data presented as mean  ∓SEM. Data presented as n = 4 in METH/Sal group, n = 5 in all other groups

Fig. 7

Pearson’s correlation coefficients and connectivity diagrams for Sal/Sal (A), Sal/METH (B), METH/Sal (C) and METH/METH (D) treated animals. LHS: Matrices describing the Pearson’s correlation coefficient, p value and n value. The strength of the correlation is depicted by the shade, with weak correlations presented in lighter shades and stronger correlations presented in darker shades. Statistically significant correlations are presented in bold. RHS: Diagrams displaying possible functional connectivity between examined brain regions. Solid lines represent positive correlations which were statistically significant. Bold lines represent significant positive correlations which met q criteria