Amygdala transcriptome and cellular mechanisms underlying stress-enhanced fear learning in a rat model of posttraumatic stress disorder

Abstract

Severe stress or trauma can cause permanent changes in brain circuitry, leading to dysregulation of fear responses and the development of posttraumatic stress disorder (PTSD). To date, little is known about the molecular mechanisms underlying stress-induced long-term plasticity in fear circuits. We addressed this question by using global gene expression profiling in an animal model of PTSD, stress-enhanced fear learning (SEFL). A total of 15 footshocks were used to induce SEFL and the volatile anesthetic isoflurane was used to suppress the behavioral effects of stress. Gene expression in lateral/basolateral amygdala was measured using microarrays at 3 weeks after the exposure to different combinations of shock and isoflurane. Shock produced robust effects on amygdalar transcriptome and isoflurane blocked or reversed many of the stress-induced changes. We used a modular approach to molecular profiles of shock and isoflurane and built a network of regulated genes, functional categories, and cell types that represent a mechanistic foundation of perturbation-induced plasticity in the amygdala. This analysis partitioned perturbation-induced changes in gene expression into neuron- and astrocyte-specific changes, highlighting a previously underappreciated role of astroglia in amygdalar plasticity. Many neuron-enriched genes were highly correlated with astrocyte-enriched genes, suggesting coordinated transcriptional responses to environmental challenges in these cell types. Several individual genes were validated using RT-PCR and behavioral pharmacology. This study is the first to propose specific cellular and molecular mechanisms underlying SEFL, an animal model of PTSD, and to nominate novel molecular and cellular targets with potential for therapeutic intervention in PTSD, including glycine and neuropeptide systems, chromatin remodeling, and gliotransmission.

Figure 1

Fear conditioning responses measured on day 25 in contexts A (left panel) and on day 24 in context B (right panel). The two-way ANOVAs showed significant effects of shock, isoflurane, and an interaction between the two factors (All P<0.05). The asterisks indicate significant difference between the 15 shock/0% isoflurane group and each of the other three groups (^*P<0.01; ^**P<0.001).

Figure 2

A meta-network of amygdalar regulatome shows top results of K-mean clustering and functional group over-representation analysis. Gene clusters are numbered as 1–24 (SEFL: 1–8; isoflurane: 9–16; and shock: 17–24). Only clusters linked to a highly over-represented functional group or cell type (P<0.0001; FDR<1%) are shown and only significantly overlapping clusters (χ² P<0.005; at least 10% overlap) are connected. Encircled are modules of overlapping clusters linked to a highly over-represented functional group, which is over-represented to some degree in each cluster of a given module. Color of the circle and the line connecting modules and over-represented groups corresponds to the color of the most significant cluster in the module. Thickness of lines between clusters and font sizes are proportional to the percentage of overlap and significance level of over-represented groups, respectively.

Figure 3

Expression patterns of genes enriched in neurons (a ,b) or astrocytes (c, d) and associated with SEFL behavior. Cell type- specific genes were identified based on literature (Cahoy et al, 2008). A clustergram of centered and normalized group means is shown. SEFL = SEFL group; C = control group (0% isoflurane, 0 shocks); IsoS = 0.6% isoflurane, 15 shocks; Iso = 0.6% isoflurane, 0 shocks. Gene symbols of several genes implicated in brain functions are shown to the right of the clustergram. FC% = percent fold change in the SEFL group compared with the average of the other three groups. Cell-type fold enrichment is shown on the right. Clustergram was created using Cluster 3.0 and Java TreeView 1.1.3.

Figure 4

Associative fear responses measured in context A (left panel) and SEFL measured in context B (right panel). Freezing in rats given 15 shocks and 0 mg/kg etomidate was reliably greater than in the other two groups (^*P<0.01; ^**P<0.001).