Impaired extinction of cued fear memory and abnormal dendritic morphology in the prelimbic and infralimbic cortices in VPAC2 receptor (VIPR2)-deficient mice

Abstract

The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in stress regulation and learning and memory. Several bodies of research have shown the impact of the PACAP specific receptor PAC1 on fear memory, but the roles of other PACAP receptors in regulating fear stress responses remain to be elucidated. Here we aimed to investigate the effects of genetic deletion of VIPR2 encoding the VPAC2 receptor, which binds both VIP and PACAP, on fear-related memory and on dendritic morphology in the brain regions of the fear circuitry. Male VPAC2 receptor knockout (VPAC2-KO) and littermate wild-type control mice were subjected to Pavlovian fear conditioning paradigm. VPAC2-KO mice displayed normal acquisition of fear conditioning, contextual and cued fear memory, but impaired extinction of cued fear memory. Morphological analyses revealed reductions in cell body size and total branch number and length of apical and basal dendrites of prelimbic cortex neurons in VPAC2-KO mice. In addition, Sholl analysis indicated that the amount of dendritic material distal to the soma was decreased, while proximal dendritic material was increased. In the infralimbic cortex, the amount of apical dendritic material proximal to the soma was increased in VPAC2-KO mice, while other indices of morphology did not differ. Finally, there were no differences in dendritic morphology in basolateral amygdala neurons between genotypes. These findings suggest that the VPAC2 receptor plays an important role in the fear extinction processes and the regulation of the dendritic morphology in the prelimbic and infralimbic cortices.

Keywords: Dendritic morphology; Extinction; Fear conditioning; Prelimbic and infralimbic cortices; VPAC2 receptor (VIPR2).

Figure 1

Freezing behaviors of VPAC2-KO mice in the fear conditioning test. On Day 1, mice were trained with 5 tone-shock pairings (A). Then, mice were subjected to the contextual fear memory test on Day 2 (B), cued fear memory test on Day 3 (C), and cued fear extinction paradigm on Days 4 and 5 (D). Results are expressed as the mean ± S.E.M. of 14 mice per group. BL: Baseline. ^††P < 0.01, compared with the baseline. *P < 0.05, **P < 0.01, compared with wild-type.

Figure 2

Defective fear extinction in low shock-conditioned VPAC2-KO mice. On Day 1, mice were trained with 3 tone-shock pairings (A). Then, mice were subjected to the contextual fear memory test on Day 2 (B), cued fear memory test on Day 3 (C), and cued fear extinction paradigm on Days 4 and 5 (D). Results are expressed as the mean ± S.E.M. of 12 mice per group. BL: Baseline. ^††P < 0.01, compared with the baseline. *P < 0.05, compared with wild-type.

Figure 3

Dendritic morphology of PrL pyramidal neurons in VPAC2-KO mice. (A) Golgi-stained pyramidal neuron in PrL cortex and representative tracings of the dendrites of wild-type and VPAC2-KO mice. (B, C) Cell body size and total branch number and length of apical and basal dendrites are shown. The number of intersections of dendrites with 20 μm concentric spheres centered on the soma was measured by Sholl analysis. Results are expressed as the mean ± S.E.M. of 60 neurons from 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, compared with wild-type.

Figure 4

Dendritic morphology of IL pyramidal neurons in VPAC2-KO mice. (A) Golgi-stained pyramidal neuron in IL cortex and representative tracings of the dendrites of wild-type and VPAC2-KO mice. (B, C) Cell body size and total branch number and length of apical and basal dendrites are shown. The number of intersections of dendrites with 20 μm concentric spheres centered on the soma was measured by Sholl analysis. Results are expressed as the mean ± S.E.M. of 30 neurons from 3 mice per group. *P < 0.05, compared with wild-type.

Figure 5

Dendritic morphology of BLA pyramidal and stellate neurons in VPAC2-KO mice. (A) Golgi-stained pyramidal and stellate neuron in BLA and representative tracings of the dendrites of wild-type and VPAC2-KO mice. (B) Cell body size and total branch number and length of dendrites are shown. The number of intersections of dendrites with 20 μm concentric spheres centered on the soma was measured by Sholl analysis. Results are expressed as the mean ± S.E.M. of 8–10 pyramidal neurons and 15–17 stellate neurons from 3 mice per group.