Social isolation and chronic handling alter endocannabinoid signaling and behavioral reactivity to context in adult rats

Abstract

Social deprivation in early life disrupts emotionality and attentional processes in humans. Rearing rats in isolation reproduces some of these abnormalities, which are attenuated by daily handling. However, the neurochemical mechanisms underlying these responses remain poorly understood. We hypothesized that post-weaning social isolation alters the endocannabinoid system, a neuromodulatory system that controls emotional responding. We characterized behavioral consequences of social isolation and evaluated whether handling would reverse social isolation-induced alterations in behavioral reactivity to context and the endocannabinoid system. At weaning, pups were single or group housed and concomitantly handled or not handled daily until adulthood. Rats were tested in emotionality- and attentional-sensitive behavioral assays (open field, elevated plus maze, startle and prepulse inhibition). Cannabinoid receptor densities and endocannabinoid levels were quantified in a separate group of rats. Social isolation negatively altered behavioral responding. Socially-isolated rats that were handled showed less deficits in the open field, elevated plus maze, and prepulse inhibition tests. Social isolation produced site-specific alterations (supraoptic nucleus, ventrolateral thalamus, rostral striatum) in cannabinoid receptor densities compared to group rearing. Handling altered the endocannabinoid system in neural circuitry controlling emotional expression. Handling altered endocannabinoid content (prefrontal and piriform cortices, nucleus accumbens) and cannabinoid receptor densities (lateral globus pallidus, cingulate and piriform cortices, hippocampus) in a region-specific manner. Some effects of social isolation on the endocannabinoid system were moderated by handling. Isolates were unresponsive to handling-induced increases in cannabinoid receptor densities (caudal striatum, anterior thalamus), but were sensitive to handling-induced changes in endocannabinoid content (piriform, prefrontal cortices), compared to group-reared rats. Our findings suggest alterations in the endocannabinoid system may contribute to the abnormal isolate phenotype. Handling modifies the endocannabinoid system and behavioral reactivity to context, but surmounts only some effects of social isolation. These data implicate a pivotal role for the endocannabinoid system in stress adaptation and emotionality-related disturbances.

Figure 1

Social isolation alters behavioral responsiveness to context in the (a - c) open field, (d) elevated plus maze and (e) prepulse inhibition test without altering (f) acoustic startle amplitude. Handling altered behavioral responsiveness to context in the (b) open field and (d) elevated plus maze in a manner opposite to that of isolates. Isolates deprived of handling exhibit hyperlocomotor activity measured by (c) line crossings in the open field and a decreased time spent in the (d) open arms of the elevated plus maze compared to group-reared rats; these differences were not present in isolates subjected to handling. Socially-isolated rats that were handled showed a lessened deficit in (e) % PPI compared to their non-handled counterparts, but were less able to inhibit responding to a prepulse compared to group-reared rats. Data are mean ± S.E.M. ***P < 0.001, **P < 0.01 vs. Group Reared (ANOVA); ^###P < 0.001, ^##P < 0.01 vs. Non-handled (ANOVA); ^xP < 0.05 vs. Group Reared/Non-Handled (ANOVA, Tukey post hoc); ^†††P < 0.001 vs. Group Reared/Non-Handled (t-test, two-tailed); ^{^}P < 0.05 vs. all other groups (ANOVA, Tukey post hoc).

Figure 2

Socially-isolated rats show increased cannabinoid receptor density in the (a) dorsomedial and (b) ventrolateral caudate putamen compared to group-reared rats. Representative photomicrographs show [³H]CP55,940 binding in brains of adult rats that were either (top: c, e) group or (bottom: d, f) isolation reared and concomitantly (right: e, f) handled or (left: c, d) not handled post-weaning. Sections were collected +1.70 mm from bregma. The rostral caudate putamen was divided into quadrants as previously reported (Hohmann and Herkenham, 2000). dm, dorsomedial; dl, dorsolateral; vm, ventromedial; vl, ventrolateral. Scale bar equals 1 mm. Data are mean ± S.E.M. *P < 0.05 vs. Group Reared (ANOVA).

Figure 3

Socially-isolated rats show decreased cannabinoid receptor density in the (a) supraoptic nucleus of the hypothalamus and (b) ventrolateral thalamus compared to group-reared rats. Data are mean ± S.E.M. *P < 0.05 vs. Group Reared (ANOVA). Representative photomicrographs are shown in Fig. 4–5. SO, supraoptic nucleus; vlTN, ventrolateral thalamic nuclei.

Figure 4

Representative photomicrographs show [³H]CP55,940 binding in brains from adult rats that were either (top: a, c) group or (bottom: b, d) isolation reared and concomitantly (right: c, d) handled or (left: a, b) not handled post-weaning. Sections were collected −1.30 mm from bregma. aTN, anterior thalamic nuclei; Cg, cingulate cortex; lGP, lateral globus pallidus; Pir, piriform cortex; cCPu, caudal caudate putamen; SO, supraoptic nucleus. Scale bar equals 1mm.

Figure 5

Representative photomicrographs show [³H]CP55,940 binding in brains derived from adult rats that were either (top: a, b) group or (bottom: c, d) isolation reared and concomitantly (right: b, d) handled or (left: a, c) not handled post-weaning. Sections were collected −2.30 mm from bregma. CA 1–3, molecular layers of hippocampus CA 1–3; DG, dentate gyrus; vlTN, ventrolateral thalamic nuclei. Scale bar equals 1mm.

Figure 6

Endocannabinoid content in brain punches derived from adult rats with manipulated rearing and handling histories post-weaning. Single hemisphere punches were obtained at the level of the (a – c) prefrontal cortex, (d – f) piriform cortex, (g – i) nucleus accumbens, and (j – l) hippocampus as outlined (a, d, g, j) and assayed for 2-arachidonoylglycerol (2-AG) and anandamide (AEA). Social isolation modified endocannabinoid content in rats that were non-handled; social isolation increased (b) 2-AG in the prefrontal cortex, without altering (c) AEA levels, and increased both (e) 2-AG and (f) AEA content in the piriform cortex, relative to group rearing. Handling altered endocannabinoid content in both a ligand and brain region specific manner. In the prefrontal cortex, handling selectively increased (b) 2-AG but not (c) AEA levels compared to non-handling. In the piriform cortex, handling decreased (f) AEA, but unaltered (e) 2-AG, relative to non-handling. In the nucleus accumbens, handling increased (i) AEA but not (h) 2-AG compared to non-handling. (k, l) Endocannabinoid content was unchanged in the hippocampus. Data are mean ± S.E.M. ^###P < 0.01, ^#P < 0.05 vs. Non-handled (ANOVA); ^††P < 0.01, ^†P < 0.05 vs. Group Reared/Non-Handled (t-test, two-tailed). Scale bar equals 1 mm.

Figure 7

Handled rats show altered cannabinoid receptor densities in the limbic loop of the basal ganglia. Handled rats exhibit increased cannabinoid receptor densities in the (a) lateral globus pallidus, (b) cingulate cortex, and (c) piriform cortex, but decreased cannabinoid receptor density in the (d) hippocampus, relative to non-handled rats. Data are mean ± S.E.M. ^##P < 0.01, ^#P < 0.05 vs. Non-Handled (ANOVA). Representative photomicrographs are shown in Fig. 4–5. Cg, cingulate cortex; Hippo, hippocampus CA 1–3 and dentate gyrus; lGP, lateral globus pallidus; Pir, piriform cortex.

Figure 8

Socially-isolated rats fail to exhibit handling-induced increases in cannabinoid receptor density exhibited by their group-reared counterparts in the (a) caudal caudate putamen and (b) anterior thalamic nuclei. Data are mean ± S.E.M. ⁺⁺P < 0.01, ⁺P < 0.05 vs. Group Reared/Handled (ANOVA). Representative photomicrographs are shown in Fig. 4–5. aTN, anterior thalamic nuclei; cCPu, caudal caudate putamen.

Figure 9

Neuroanatomical circuitry altered by post-weaning rearing and handling manipulations alone and in interaction. Thick arrows connect structures of Papez circuitry whereas thin arrows connect input-output loops through the basal ganglia. Key indicates treatment groups that are both color and underline coded to summarize changes in the endocannabinoid system. The key corresponds to the following group comparisons: Group Reared (Handled) vs. All other groups, Handled vs. Non-Handled, Isolation vs. Group Reared, and Isolation (Non-Handled) vs. Group Reared (Non-Handled). AEA, anandamide; 2-AG, 2-arachidonoylglycerol; CB_R, cannabinoid receptor.