Antagonism of muscarinic acetylcholine receptors in medial prefrontal cortex disrupts the context preexposure facilitation effect

Abstract

Cholinergic function plays a role in a variant of context fear conditioning known as the context preexposure facilitation effect (CPFE; Robinson-Drummer, Dokovna, Heroux, & Stanton, 2016). In the CPFE, acquisition of a context representation, the context-shock association, and expression of context fear occur across successive phases, usually 24h apart. Systemic administration of scopolamine, a muscarinic acetylcholine receptor antagonist, prior to each phase (context preexposure, immediate-shock training, and testing) disrupts the CPFE in juvenile rats (Robinson-Drummer et al., 2016). Dorsal hippocampal (dHPC) cholinergic function contributes significantly to this effect, as local infusion of scopolamine into the dHPC prior to any individual phase of the CPFE produces a disruption identical to systemic administration (Robinson-Drummer et al., 2016). The current experiment extended these findings to another forebrain region implicated in the CPFE, the medial prefrontal cortex (mPFC). Adolescent rats received bilateral infusions of scopolamine (35μg/side) or PBS 10min before all three phases of the CPFE or only prior to a single phase. Intra-mPFC administration of scopolamine prior to all three phases significantly impaired fear conditioning suggesting that mPFC cholinergic function is necessary for successful CPFE performance. Analyses of the individual infusion days revealed a significant impairment of the CPFE when infusions occurred prior to preexposure or training (i.e. immediate footshock) but not prior to testing. In total, these findings suggests a role of mPFC cholinergic function in the acquisition and/or consolidation of a contextual representation and the context-shock association but not in retrieval or expression of fear memory. Implications for mPFC involvement in contextual fear conditioning and neurological dysfunction following neonatal alcohol exposure are discussed.

Keywords: CPFE; Fear; Muscarinic; Prefrontal; Spatial learning.

Figure 1

Schematic representation of injection cannula tip placement in the mPFC for Experiment 1. Animals included in final analyses are represented by filled black circle while animals excluded as mPFC placement misses are filled black triangle. Extremely anterior (Bregma 4.68mm or more) or posterior (Bregma 2.28 or less) were automatically excluded and are not represented in the following figure. Coronal brain images are adapted from the rat brain atlas of Paxinos and Watson (2007).

Figure 2

Mean (±SEM) percent time freezing during a 5min test of conditioned fear. Animals were given either PBS or scopolamine prior to all three conditioning phases of the CPFE. Comparisons reflect a one-way ANOVA for group (Pooled Alt-Pre, Pre Scop and Pre PBS). Scopolamine significantly impaired CPFE performance on the testing day. Asterisks indicate significance relative to Pre PBS group. *** = p < .001

Figure 3

Schematic representation of injection cannula tip placement in the mPFC for Experiment 2. Animals included in final analyses are represented by filled black circle while animals excluded as mPFC placement misses are filled black triangle. Extremely anterior (Bregma 4.68mm or more) or posterior (Bregma 2.28 or less) were automatically excluded and are not represented in the following figure. Coronal brain images are adapted from the rat brain atlas of Paxinos and Watson (2007).

Figure 4

Mean (±SEM) percent time freezing during a 5min test of conditioned fear following pre-preexposure drug infusion (Experiment 2). Comparisons reflect an ANOVA for group (Alt- Pre PBS, Alt-Pre Scop, Pre PBS and Pre Scop) with planned comparisons. Scopolamine administered prior to context preexposure significantly impaired CPFE performance on the testing day. Asterisks indicate significance relative to Pre PBS group. Ampersand indicates significance within exposure condition. && = p < .01; * = p < .05; ** = p < .01; *** = p < .001.

Figure 5

Schematic representation of injection cannula tip placement in the mPFC for Experiment 3. Animals included in final analyses are represented by filled black circle while animals excluded as mPFC placement misses are filled black triangle. Extremely anterior (Bregma 4.68mm or more) or posterior (Bregma 2.28 or less) were automatically excluded and are not represented in the following figure. Coronal brain images are adapted from the rat brain atlas of Paxinos and Watson (2007).

Figure 6

Mean (±SEM) percent time freezing during a 5min test of conditioned fear following pre-training drug infusion (Experiment 3). Comparisons reflect a one-way ANOVA for group (Pooled Alt-Pre, Pre Scop and Pre PBS). Pre-training scopolamine significantly impaired CPFE performance on the testing day. Asterisks indicate significance relative to Pre PBS group. *** = p < .001

Figure 7

Schematic representation of injection cannula tip placement in the mPFC for Experiment 4. Animals included in final analyses are represented by filled black circle while animals excluded as mPFC placement misses are filled black triangle. Extremely anterior (Bregma 4.68mm or more) or posterior (Bregma 2.28 or less) were automatically excluded and are not represented in the following figure. Coronal brain images are adapted from the rat brain atlas of Paxinos and Watson (2007).

Figure 8

Mean (±SEM) percent time freezing during a 5min test of conditioned fear. Animals were given either PBS or scopolamine prior to the testing phase of the CPFE. 2 (Sex: Female, Male) × 3 (Condition: Alt-Pre, Pre Scop, Pre PBS) factorial ANOVA. Scopolamine significantly impaired CPFE performance in males. Asterisks indicate significance relative to Pre PBS group. Ampersand indicates significance relative to Alt-Pre. & = p < .05; *** = p < .001