AAV-Mediated Overexpression of the CB1 Receptor in the mPFC of Adult Rats Alters Cognitive Flexibility, Social Behavior, and Emotional Reactivity

Abstract

The endocannabinoid (ECB) system is strongly involved in the regulation of cognitive processing and emotional behavior and evidence indicates that ECB signaling might affect these behavioral abilities by modulations of prefrontal cortical functions. The aim of the present study was to examine the role of the CB1 receptor in the medial prefrontal cortex (mPFC) on cognitive flexibility and emotional behavior. Therefore, the CB1 receptor was overexpressed by adeno-associated virus vector-mediated gene transfer specifically in the mPFC of adult Wistar rats. Animals were then tested in different anxiety-related paradigms for emotional reactivity [e.g., elevated plus maze (EPM), light/dark emergence test (EMT), social interaction] and the attentional set shift task (ASST) - an adaptation of the human Wisconsin card sorting test - for cognitive abilities and behavioral flexibility. A subtle increase in exploratory behavior was found in CB1 receptor overexpressing animals (CB1-R) compared to Empty vector injected controls (Empty) in the EMT and EPM, although general locomotor activity did not differ between the groups. During social interaction testing, social contact behavior toward the unknown conspecific was found to be decreased, whereas social withdrawal was increased in CB1-R animals and they showed an inadequate increase in exploratory behavior compared to control animals. In the ASST, impaired reversal learning abilities were detected in CB1-R animals compared to controls, indicating reduced behavioral flexibility. In conclusion, upregulation of the CB1 receptor specifically in the rat mPFC induces alterations in emotional reactivity, leads to inadequate social behavior, and impairs cognitive flexibility. These findings might be relevant for neuropsychiatric disorders, since higher cortical CB1 receptor expression levels as well as similar behavioral impairments as observed in the present study have been described in schizophrenic patients.

Keywords: CB1 receptor; attentional set shift task; cognitive flexibility; emotional behavior; mPFC; social interaction.

Figure 1

Timeline of the ASST paradigm. Pre: pretraining; SD: simple discrimination; CD: compound discrimination; CDrev: compound discrimination reversal; CDrep: compound discrimination repetition; IDS; intradimensional shift; EDS: extradimensional shift.

Figure 2

Adeno-associated virus-mediated CB1 expression in the mPFC. The cartoon (adapted from Paxinos and Watson, 1998) shows the representative transduced area (gray) in animals injected with AAV-CB1. Representative immunostaining for CB1 in the mPFC of AAV-CB1-injected rats at low (10× for A–C) and high (20× for A′–C′) magnification. (A) Extent of CB1 immunoreactivity showing robust transduction in the target area. (B) Same section stained for the nuclear stain DAPI. (C) Overlay of (A) and (B). The box in (C) indicates the area magnified in (A′–C′). Note that adjustment of the exposure time for visualization of ectopic CB1 precludes visualization of endogenous CB1. The counterstaining with DAPI delineates the tissue and shows correct targeting of ectopic CB1 expression (C). (C′) High power micrograph showing CB1 immunoreactivity in the neuronal soma and neuropil of the mPFC. (D) Representative brain section (left hemisphere) of an AAV-Empty-injected animal showing endogenous CB1 immunoreactivity at low power (10×). Note that the exposure time was 2.5× more than for the visualization of transgenic CB1 shown in (A). (E) DAPI stain. (F) Merger of (D) and (E). Prl, prelimbic cortex; Cg1, area 1 of the cingulate cortex; IL, infralimbic cortex; DP, dorsal peduncular cortex; M1/2, motor cortex; cc, corpus callosum; I, II, III, cortical layers.

Figure 3

Locomotor activity in an open field. No significant differences were found between CB1 receptor overexpressing animals (CB1-R) and Empty vector injected controls (Empty). Values are expressed as mean ± SEM.

Figure 4

Behavioral performance during social interaction with an unknown social partner. Significant differences between CB1-R and Empty animals were detected for anogenital exploration, approach/following (A), social contact behavior (B) and evade upon social contact (D). No differences were observed for non-anogenital exploration (A) and self-grooming behavior (C). Values are expressed as mean ± SEM (p < 0.05 is indicated by asterisks).

Figure 5

Behavioral performance in the ASST. CB1-R rats differed significantly from Empty animals in the CDrev stage. Values are expressed as mean ± SEM (p < 0.05 is indicated by asterisks).