Phosphorylation of calcium/calmodulin-dependent protein kinase II in the rat dorsal medial prefrontal cortex is associated with alcohol-induced cognitive inflexibility

Abstract

Repeated cycles of alcohol [ethanol (EtOH)] intoxication and withdrawal dysregulate excitatory glutamatergic systems in the brain and induce neuroadaptations in the medial prefrontal cortex (mPFC) that contribute to cognitive dysfunction. The mPFC is composed of subdivisions that are functionally distinct, with dorsal regions facilitating drug-cue associations and ventral regions modulating new learning in the absence of drug. A key modulator of glutamatergic activity is the holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) that phosphorylates ionotropic glutamate receptors. Here, we examined the hypothesis that abstinence from chronic intermittent EtOH (CIE) exposure dysregulates CaMKII activity in the mPFC to impair cognitive flexibility. We used an operant model of strategy set shifting in male Long-Evans rats demonstrating reduced susceptibility to trial omissions during performance in a visual cue-guided task versus albino strains. Relative to naïve controls, rats experiencing approximately 10 days of abstinence from CIE vapor exposure demonstrated impaired performance during a procedural shift from visual cue to spatial location discrimination. Phosphorylation of CaMKII subtype α was upregulated in the dorsal, but not ventral mPFC of CIE-exposed rats, and was positively correlated with perseverative-like responding during the set shift. The findings suggest that abstinence from CIE exposure induces an undercurrent of kinase activity (e.g. CaMKII), which may promote aberrant glutamatergic responses in select regions of the mPFC. Given the role of the mPFC in modulating executive control of behavior, we propose that increased CaMKII subtype α activity reflects a dysregulated 'top-down' circuit that interferes with adaptive behavioral performance under changing environmental demands.

Keywords: cognition; ethanol; prelimbic.

Figure 1. Cortico-striatal connectivity involved in drug-cue associations and extinction learning

The medial prefrontal cortex (mPFC) can be subdivided into dorsal (dPFC) and ventral (vPFC) regions. The dPFC receives input from the basolateral amygdala, and projects to the dorsal striatum (DS) and nucleus accumbens core (NAcC) to facilitate drug-cue associations. The vPFC integrates input from the basolateral amygdala, hippocampus, and dorsal hypothalamus and primarily projects to the nucleus accumbens shell (NAcS) to facilitate extinction learning. For a more detailed discussion, please see the following reviews that address cortico-striatal connectivity and its importance in modulating drug-induced plasticity (Gass and Chandler, 2013; Gourley and Taylor, 2016; Peters et al., 2009).

Figure 2. Trial omissions in visual cue discrimination are lower in Long-Evans versus Wistar rats

Behavioral performance on an operant model of strategy set-shifting was compared in ethanol-naïve male Wistar (WIS, n=10) and Long-Evans (LE, n=10) rats. The data are separated into two distinct operant procedures: the initial (visual cue) discrimination task in which the rats were required to respond on a lever located on the congruent side of the illuminated cue to receive a food reward, and the set-shift to a spatial discrimination task in which the rats were required to respond on a lever with a set position that was independent of visual cue illumination. Behavioral measurements (mean ± SEM) included total trials (A and D), errors (B and E) and omissions (C and F) until rats achieved a criterion threshold for each task. Generally, we observed no strain differences in cognitive performance across task procedures, except for reduced omissions (C) in LE versus WIS rats during the cue discrimination task. As high omission rates may complicate the assessment of cognitive flexibility, our subsequent studies evaluated the effects of ethanol abstinence in LE rats displaying reduced susceptibility to trial omissions. An asterisk (*) denotes significant group differences (p<0.05).

Figure 3. Abstinence from chronic intermittent ethanol exposure induces deficits in cognitive flexibility that are characterized by increased perseverative-like responding

Behavioral performance on an operant model of strategy set-shifting was compared in male Long-Evans rats serving as ethanol-naïve controls (CON, n=9) or undergoing abstinence from chronic intermittent ethanol vapor exposure (EtOH, n=11). The data are separated into two distinct operant procedures: the initial (visual cue) discrimination task in which the rats were required to respond on a lever located on the congruent side of the illuminated cue to receive a food reward, and the set-shift to a spatial discrimination task in which the rats were required to respond on a lever with a set position that was independent of visual cue illumination. A retest of each operant procedure was administered to distinguish between acquisition and retention of task performance. Behavioral measurements (mean ± SEM) included total trials (A and C) and errors (B and D) until rats achieved a criterion threshold for each task. As compared to CON, EtOH rats undergoing abstinence did not display altered performance on the cue discrimination task (A and B) in either test phase. Alternatively, after approximately 10 days of abstinence, EtOH rats displayed higher trials (C) and errors (D) to criterion on the day of the set-shift, although no group differences emerged during the retest of this association. To gain further insight on impaired cognitive performance, we parsed the errors committed during the set-shift into previously- and never-reinforced (E) responses. As compared to CON, EtOH rats committed more errors that would have been correct under the initial task, although a marginal increase in indiscriminate learning errors was also evident. An asterisk (*) denotes significant group differences (p<0.05).

Figure 4. Abstinence from chronic intermittent ethanol exposure augments the phosphorylation of CaMKII α in the dorsal medial prefrontal cortex, an effect that is associated with inflexible performance

Total and phosphorylated levels of CaMKIIα (threonine 286) were estimated from microdissections of the medial prefrontal cortex (mPFC) in ethanol-naïve controls (CON, n=6) or rats that were re-exposed to ethanol vapor and abstinence procedures (EtOH, n=7) from the behavioral studies. The mPFC wet tissue slice (A) was dissected for regions comprising the dorsal (i.e., primarily prelimbic cortex) or ventral mPFC (i.e., primarily infralimbic cortex). Fluorescence values of immunoreactivity for total and phosphorylated CaMKIIα relative to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analyzed as a percentage change from CON levels (mean ± SEM) across brain regions (B and C). Representative images for each group illustrate the gel band and molecular weight of the proteins quantified in the analysis. Whereas total CaMKIIα levels remained comparable across groups and brain regions, EtOH rats undergoing 10 days of abstinence displayed an increase in phospho-CaMKIIα levels in the dorsal (B) but not ventral (C) mPFC as compared to CON. To gain insight on the role of upregulated phospho-CaMKIIα, we correlated these measures with respective performance during the set-shift (D, E, and F) in CON (open circles) and EtOH (closed circles) rats. Phospho-CaMKIIα in the dorsal mPFC correlated with inflexible performance, showing a positive association with increasing total errors (D), as well as indices of perseverative-like responding via previously-reinforced (E), but not never-reinforced (F) errors. An asterisk (*) denotes significant group differences (p<0.05).