Gamma aminobutyric acidergic and neuronal structural markers in the nucleus accumbens core underlie trait-like impulsive behavior

Abstract

Background: Pathological forms of impulsivity are manifest in a number of psychiatric disorders listed in DSM-5, including attention-deficit/hyperactivity disorder and substance use disorder. However, the molecular and cellular substrates of impulsivity are poorly understood. Here, we investigated a specific form of motor impulsivity in rats, namely premature responding, on a five-choice serial reaction time task.

Methods: We used in vivo voxel-based magnetic resonance imaging and ex vivo Western blot analyses to investigate putative structural, neuronal, and glial protein markers in low-impulsive (LI) and high-impulsive rats. We also investigated whether messenger RNA interference targeting glutamate decarboxylase 65/67 (GAD65/67) gene expression in the nucleus accumbens core (NAcbC) is sufficient to increase impulsivity in LI rats.

Results: We identified structural and molecular abnormalities in the NAcbC associated with motor impulsivity in rats. We report a reduction in gray matter density in the left NAcbC of high-impulsive rats, with corresponding reductions in this region of glutamate decarboxylase (GAD65/67) and markers of dendritic spines and microtubules. We further demonstrate that the experimental reduction of de novo of GAD65/67 expression bilaterally in the NAcbC is sufficient to increase impulsivity in LI rats.

Conclusions: These results reveal a novel mechanism of impulsivity in rats involving gamma aminobutyric acidergic and structural abnormalities in the NAcbC with potential relevance to the etiology and treatment of attention-deficit/hyperactivity disorder and related disorders.

Keywords: Attention-deficit/hyperactivity disorder; GABA; impulsivity; magnetic resonance imaging; nucleus accumbens; psychostimulants.

Figure 1

High impulsivity in rats is associated with a reduced density of gray matter (GM) in the left nucleus accumbens (NAcb) core. (A) High-impulsive rats (HI, n = 6) make more premature responses on the five-choice serial reaction time task compared with mid-impulsive rats (MI, n = 6) and low-impulsive rats (LI, n = 6) when the prestimulus waiting interval is increased to 7 seconds (long intertrial interval [LITI] = 7 sec) from the intervening 5-second interval (indicated by b). (B) Voxel-based morphometry analysis of orthogonal coronal (i), sagittal (ii), and horizontal (iii) sections superposed on an averaged magnetic resonance imaging template. The results indicate a significant reduction in the density of gray matter in the left NAcb core with a cluster extent of 29 voxels (uncorrected F_1,16 = 116.2, p_{family-wise error} = .003), centered 2.3 mm anterior to bregma, 2.2 mm medial-lateral, 7.4 mm dorsal-ventral . (C) Negative correlation between impulsivity and GM scores, reported for individual subjects, of the most significant voxels in the left NAcb core (r = −.87, p < .001). The insert graph shows the corresponding, nonsignificant relationship between impulsivity and GM scores in the right NAcb core. (D) Three-dimensional composite image of the rat forebrain showing brain areas selected for Western blot analysis. C, cortex; CPu, caudate putamen; L, left; R, right.

Figure 2

High impulsivity in rats is associated with a significant reduction in glutamate decarboxylase (GAD)_65/67, microtubule-associated protein 2 (MAP2), and spinophilin (Spinoph) in the left nucleus accumbens (NAcb) core but not right NAcb core. (A) Representative immunoreactive bands from samples of the left and right NAcb core in low-impulsive (LI) and high-impulsive (HI) rats. (B) Densitometric quantification (relative optical density [R.O.D.] expressed as a % of the mean value of LI rats) of left NAcb core revealed a significant reduction of GAD_65/67 (t₉ = 3.48, **p < .01), MAP2 (t₉ = 2.34, *p < .05), and spinophilin (t₉ = 2.43, *p < .05) in HI rats compared with LI rats. Data are expressed as mean ± SEM. (C) Densitometric analysis of samples from the right NAcb core revealed no significant differences in GAD_65/67, MAP2, and spinophilin (GAD_65/67; t₉ =1.66, p = .13). (D–F) Correlation between impulsivity scores and the relative optical density of GAD_65/67 ([D]r = −.71, p < .01), MAP2 ([E]r = −.66, p < .05), and spinophilin ([F]r = −.47, p = .074) in left NAcb core. Insert graph shows equivalent data for the right NAcb core.

Figure 3

Bilateral reduction in glutamate decarboxylase 65/67 (GAD_65/67) protein in the nucleus accumbens (NAcb) core increases impulsivity in low-impulsive rats on the five-choice serial reaction time task. (A) Individual responses of rats to GAD_65/67 scrambled (Scr) sequence in the NAcb core showing no effect on premature responding compared with vehicle infusions in this region (n = 6). The insert graph shows the intended location of the oligodeoxynucleotide (ODN) microinfusions in the NAcb . (B) Individual responses of rats to GAD_65/67 antisense in the NAcb core showing increased premature responding compared with vehicle infusions (n = 7). (C) Representative immunoblot and related densitometric analysis showing GAD_65/67 antisense-induced decrease of GAD_65/67 protein levels 8 hours after intra-NAcb core microinfusions in selected LI rats. *p < .05. (D) Representative immunoblot and related densitometric analysis of the adjacent NAcb shell showing no differences between GAD_65/67 protein levels 8 hours intra-NAcb core microinfusions in low-impulsive rats. (E) Histograms show difference scores (± SEM) between the effects of vehicle infusions (pre-ODN and post-ODN) and ODN infusions (Scr and antisense oligonucleotide [ASO]). *p < .05 (Scr vs. ASO). (F) Injector tip locations in the NAcb core of rats injected with GAD_65/67 Scr (left) and ASO (right). Anterior-posterior (AP) coordinates are relative to bregma (mm) . DV, dorsal ventral; ML, medial lateral; R.O.D., relative optical density; Vehicle-post/pre, GAD 65/67 ODNs exposure.