Decreased corticolimbic allopregnanolone expression during social isolation enhances contextual fear: A model relevant for posttraumatic stress disorder

Abstract

Mice subjected to social isolation (3-4 weeks) exhibit enhanced contextual fear responses and impaired fear extinction. These responses are time-related to a decrease of 5alpha-reductase type I (5alpha-RI) mRNA expression and allopregnanolone (Allo) levels in selected neurons of the medial prefrontal cortex, hippocampus, and basolateral amygdala. Of note, the cued fear response was not different between group housed and socially isolated mice. In socially isolated mice, S-norfluoxetine, a selective brain steroidogenic stimulant (SBSS), in doses (0.45-1.8 mumol/kg) that increase brain Allo levels but fail to inhibit serotonin reuptake, greatly attenuates enhanced contextual fear response. SKF 105,111 (a potent 5alpha-RI inhibitor) decreases corticolimbic Allo levels and enhances the contextual fear response in group housed mice, which suggests that social isolation alters emotional responses by reducing the positive allosteric modulation of Allo at GABA(A) receptors in corticolimbic circuits. Thus, these procedures model emotional hyperreactivity, including enhanced contextual fear and impaired contextual fear extinction, which also is observed in posttraumatic stress disorder (PTSD) patients. A recent clinical study reported that cerebrospinal fluid Allo levels also are down-regulated in PTSD patients and correlate negatively with PTSD symptoms and negative mood. Thus, protracted social isolation of mice combined with tests of fear conditioning may be a suitable model to study emotional behavioral components associated with neurochemical alterations relating to PTSD. Importantly, drugs like SBSSs, which rapidly increase corticolimbic Allo levels, normalize the exaggerated contextual fear responses resulting from social isolation, suggesting that selective activation of neurosteroidogenesis may be useful in PTSD therapy.

Fig. 1.

In male mice socially isolated for 3–4 weeks, contextual freezing time is increased and 5α-RI mRNA expression in the hippocampus (Hip) and amygdala (Amy) is decreased. Total duration of freezing time was measured 24 h after a training session. The training session consisted of a CS (acoustic tone, 30 s, 85 dB) paired with a US (electric foot shock, 2 s, 0.5 mA) three times every 2 min. Each value is the mean ± SEM of five animals. †, P < 0.01 when hippocampus 5α-RI mRNA content in mice socially isolated for 3–4 weeks is compared with mice group housed (0 week of social isolation); ‡, P < 0.01 when amygdala 5α-RI mRNA content in mice socially isolated for 3–4 weeks is compared with mice group housed (0 week of social isolation); *, P < 0.01 freezing time at a given social isolation time period compared with a social isolation period of 0 week (one-way ANOVA followed by Bonferroni comparison). ¶, 5αRI mRNA values are expressed as fmol 5α-RI mRNA/pmol NSE mRNA.

Fig. 2.

Cued fear-conditioning response is not altered in socially isolated mice. The duration of cued freezing time was measured 24 h after a training session. The training session consisted of a CS (acoustic tone, 30 s, 85 dB) paired with a US (electric foot shock, 2 s, 0.5 mA) three times every 2 min. Mice previously trained were exposed after 24 h to a modified context (using a smaller Plexiglas box and a few drops of lemon scent in the contextual chamber) and 11 tones (cue) without footshocks. Each value is the mean ± SEM of five animals.

Fig. 3.

Extinction of contextual freezing behavior is impaired in socially isolated mice. Contextual freezing time was measured for six consecutive days starting 24 h after the training session. During the extinction trials, mice were reexposed daily to the conditional context without receiving footshocks. Two-way repeated measures ANOVA with factors for housing, time, and their interactions with the duration of freezing time revealed that mice socially isolated for 3 weeks expressed an overall higher duration of freezing time compared with group housed mice. †, P < 0.01 (two-way repeated measures ANOVA, followed by Bonferroni comparison). The slope values for the extinction curve (expressed in percentage of total duration of freezing time) during the first 3 days of the extinction trials were calculated for each mouse. The results show a delayed extinction in socially isolated compared with group housed mice (slope values: group housed, −41.0 ± 2.2; socially isolated, −27.2 ± 5.2, mean ± SEM; n = 5; P = 0.04, Student's t test).

Fig. 4.

Extinction of cued freezing behavior is not altered in socially isolated mice. Cued freezing time was measured for 3 consecutive days starting 24 h after the training session. During the cued extinction trials, mice were exposed daily to a modified context (using a smaller Plexiglas box and a few drops of lemon scent in the contextual chamber), and 11 tones without footshocks were delivered. Each value is the mean ± SEM of five animals. *, P < 0.04; **, P < 0.02 when freezing time at a given extinction trial was compared with the freezing time of the extinction trial at day 1 (one-way ANOVA, followed by Bonferroni test).

Fig. 5.

Decrease of Allo content in selected corticolimbic structures of socially isolated mice. Each value is the mean ± SEM of five animals. *, P < 0.05 and †, P < 0.01 Allo content of socially isolated mice compared with group housed mice (Student's t test). OB, olfactory bulb; FC, frontal cortex; Amy, amygdala; Hip, hippocampus; Str, striatum; CB, cerebellum.

Fig. 6.

SKF 105,111 induces increased contextual freezing behaviors and reduces hippocampal Allo content in group-housed male mice. Total duration of contextual freezing time was measured 24 h after the contextual training session. SKF 105,111 was injected 4 h before the training fear-conditioning session or before measurements of Allo content in the hippocampus (Hip). Each value is the mean ± SEM of five animals. *, P < 0.05 or †, P < 0.01 in SKF 105,111-treated groups compared with the respective vehicle-treated group (one-way ANOVA, followed by Bonferroni test).

Fig. 7.

Allo reverses increased contextual fear conditioning due to SKF 105,111 treatment. SKF 105,111 was injected 4 h before and Allo 1 h before the conditioning training session. Each value is the mean ± SEM of four animals. *, P < 0.05 in SKF 105,111-treated groups compared with control group or SKF plus Allo-treated group (one-way ANOVA, followed by Bonferroni test).