Prefrontal cortical GABAergic signaling and impaired behavioral flexibility in aged F344 rats

Abstract

The prefrontal cortex (PFC) is critical for the ability to flexibly adapt established patterns of behavior in response to a change in environmental contingencies. Impaired behavioral flexibility results in maladaptive strategies such as perseveration on response options that no longer produce a desired outcome. Pharmacological manipulations of prefrontal cortical GABAergic signaling modulate behavioral flexibility in animal models, and prefrontal cortical interneuron dysfunction is implicated in impaired behavioral flexibility that accompanies neuropsychiatric disease. As deficits in behavioral flexibility also emerge during the normal aging process, the goal of this study was to determine the role of GABAergic signaling, specifically via prefrontal cortical GABA(B) receptors, in such age-related deficits. Young and aged rats were trained in a set shifting task performed in operant chambers. First, rats learned to discriminate between two response levers to obtain a food reward on the basis of a cue light illuminated above the correct lever. Upon acquisition of this initial discrimination, the contingencies were shifted such that rats had to ignore the cue light and respond on the levers according to their left/right positions. Both young and aged rats acquired the initial discrimination similarly; however, aged rats were impaired relative to young following the set shift. Among aged rats, GABA(B) receptor expression in the medial prefrontal cortex (mPFC) was strongly correlated with set shifting, such that lower expression was associated with worse performance. Subsequent experiments showed that intra-mPFC administration of the GABA(B) receptor agonist baclofen enhanced set shifting performance in aged rats. These data directly link GABAergic signaling via GABA(B) receptors to impaired behavioral flexibility associated with normal aging.

Keywords: GABA(B) receptors; aging; baclofen; behavioral flexibility; prefrontal cortex; rat.

Fig. 1

Schematic of the set shifting task. (A) Rats were initially required to discriminate between two response levers on the basis of a cue light illuminated above the correct lever (visual cue discrimination). (B) Upon acquisition of the visual cue discrimination, the response strategy was “shifted”, such that the rats had to ignore the cue light and respond on the basis of a particular lever location (e.g., always press the left lever).

Fig. 2

Performance of young and aged rats on the set shifting task. (A) Bar graph shows that young and aged rats’ performance was comparable on the initial (visual cue) discrimination, with young and aged rats requiring similar numbers of trials to reach criterion performance. (B) In contrast to initial discrimination learning, aged rats required significantly more trials than young to reach criterion performance on the set shift (left/right) discrimination. (C) Aged rats also made significantly more errors than young before reaching criterion performance on the set shift. In both young and aged rats, errors corresponded primarily to contingencies that had been reinforced previously (during the initial discrimination). Data are expressed as mean+SEM. *p<0.05.

Fig. 3

Age-related changes in GABA(B) receptor protein expression and relationship to performance in the set shifting task. (A) Representative immunoreactive bands from young and aged mPFC homogenates following incubation with antibodies to GABA(B)R1, VGAT, and loading control β-tubulin. (B) Bar graph shows GABA(B) R1a expression in young and aged mPFC. While expression was numerically reduced in the aged mPFC, this reduction approached but did not reach significance (p=0.05). (C) Among aged rats, expression of GABA(B)R1a was significantly associated with performance in the set shifting task such that that lower expression was associated with worse behavioral flexibility (more TTC). (D) Bar graph shows that GABA(B)R1b expression was significantly reduced in the aged compared to the young mPFC. (E) Among aged rats, expression of GABA(B)R1b was significantly associated with set shifting performance, such that lower expression robustly predicted worse behavioral flexibility. (F) Bar graph shows that VGAT expression in mPFC did not differ between young and aged rats. (G) Among aged rats, expression of VGAT was not associated with performance in the set shifting task. Data are expressed as mean+SEM. tr. p=0.05, *p<0.05.

Fig. 4

Intra-mPFC baclofen administration enhanced set shifting performance in aged rats. (A) Schematic shows bilateral cannula placements in mPFC for each rat included in this experiment (illustrations adapted from Paxinos and Watson (2007)). (B) Bar graph shows that aged rats’ performance on the initial (visual cue) discrimination prior to baclofen administration did not differ between drug conditions. (C) Intra-mPFC baclofen significantly enhanced set shifting performance in aged rats, with rats receiving intra-mPFC infusion of 0.5-nmol baclofen requiring significantly fewer trials to reach criterion performance on the set shift in comparison to the rats receiving vehicle. (D) Rats receiving intra-mPFC infusion of 0.5-nmol baclofen also made significantly fewer errors before reaching criterion performance on the set shift compared to those receiving vehicle. Error analysis revealed that rats in the 0–5 nmol condition made fewer previously reinforced errors than rats in the vehicle condition, but did not differ from the vehicle condition in the number of never reinforced errors. Data are expressed as mean+SEM. *p<0.05 compared to vehicle.

Fig. 5

GABAergic signaling may mediate an inverse relationship between behavioral flexibility and working memory in aged rats. (A) Line graph shows performance of young and aged rats cross-characterized on both the set shifting task used in the current study and a delayed response task that assesses working memory (Beas et al., 2013). In this prior study, aged rats that were unimpaired relative to young on the set shifting task (Shift-U) showed impaired performance relative to young on the delayed response task. In contrast, aged rats that were impaired on the set shifting task (Shift-I) showed intact performance on the delayed response task. (B) Working model of potential relationships between PFC excitatory/inhibitory signaling and PFC-supported executive functions that could account for the inverse relationship between set shifting and delayed response performance in aged rats. See text for detailed explanation. (C) Scatter plot reproduced from Baņuelos et al. (2014) showing the negative correlation between performance on a mPFC-dependent delayed response working memory task and mPFC GABA(B)R1b expression in aged rats. (D) Data from Fig. 3E plotted with the X-axis reversed to illustrate the positive correlation between set shifting performance and mPFC GABA(B)R1b expression.