FKBP51 in the Oval Bed Nucleus of the Stria Terminalis Regulates Anxiety-Like Behavior

Expression and regulation of Fkbp5 in the ovBNST. A, Fkbp5 is among other regions expressed in the ovBNST at basal level and highly upregulated after exposure to acute stress. Stress regulation of FKBP51 is shown through β-gal upregulation (B) and on mRNA level (C). Furthermore, Fkbp5 is expressed and regulated in the majority of GABAergic neurons (D). Black arrow, GABAergic cell expressing Fkbp5; white arrow, non-GABAergic cell expressing Fkbp5. This was further confirmed by RNAscope (E), demonstrating a clear coexpression of Fkbp5 and GAD65. F, Fkbp5 was not significantly upregulated after exposure to CSDS. However, exposure to a different type of acute stress, acute fear conditioning, reliably resulted in a significant upregulation of Fkbp5 in the ovBNST (G). A time course of Fkbp5 revealed a significant upregulation, with Fkbp5 regulation peaking at its highest after 4 h (H). Data are mean ± SEM; *p < 0.05, **p < 0.01, #p < 0.001.

Fkbp5+ cells in the ovBNST coexpress with the neuropeptides Crh and Tac2 and their number is significantly increased after exposure to ASR. A, Fkbp5 and Tac2 are coexpressed in the ovBNST, as can be seen in detail (violet outline arrow). Fkbp5 and Crh are also coexpressed in the ovBNST as shown in detail (violet arrow). B, Expression patterns of Fkbp5 with Tac2 and Crh in the ovBNST also strongly overlapped (gray outline arrow). In addition, there were some cells that expressed Fkbp5 only (gray arrow). C, Quantification of the number of cells expressing Fkbp5 only, coexpressing Fkbp5 and Tac2, coexpressing Fkbp5 and Crh, and coexpressing Fkbp5, Tac2, and Crh after exposure to ASR resulted in significant upregulation across all cell types. Scale bar (A, B): 25 μm. Data are mean ± SEM; *p < 0.05, **p < 0.01.

OE of Fkbp5 in the BNST. A, Schematic representation of viral manipulation and experimental timeline including testing battery. B, Fkbp5 ISH demonstrating correct viral expression (scale bar: 250 μm). C, Quantification of Fkbp5 OE. The right panel illustrates the virus injection sites. D, There were no significant differences in open-arm time spent and number of open-arm entries in the EPM. E, FKBP51^BNST-OE and control animals also did not differ in the total distance covered during the OF test. F, FKBP51^BNST-OE animals entered the lit compartment of the DALI more frequently than control animals, but there was no significant difference in the distance covered within the light zone. G, FKBP51^BNST-OE animals demonstrated significantly lower corticosterone levels after a 15-min ARS. H, Both the coexpressed neuropeptides Tac2 and Crh were significantly upregulated in FKBP51^BNST-OE animals. Data are mean ± SEM; *p < 0.05, **p < 0.01.

KO of Fkbp5 in the BNST. A, Schematic of viral manipulation as well as experimental timeline and testing battery. B, Example of virus expression and correct targeting through Cre and Fkp5 ISH. Scale bar: 250 μm. C, Quantification of Fkbp5 knock-down in the ovBNST. The right panel illustrates the virus injection sites. D, In the EPM, FKBP51^BNST-KO animals (n = 13) spent significantly less time in the open arms and entered open arms less frequently than control animals (n = 10). E, FKBP51^BNST-KO animals covered significantly less distance within the light area of the dark-light box (DALI) test. F, Total distance covered within the OF test was not significantly different. G, FKBP51^BNST-KO animals demonstrated a matching neuroendocrine phenotype, exposing significantly higher corticosterone levels after a 15-min ASR. H, In line with previous results, the coexpressed neuropeptides Tac2 and Crh were significantly downregulated in FKBP51^BNST-KO animals. Data are mean ± SEM; *p < 0.05, **p < 0.01, #p < 0.001.

Activity-dependent KO of Fkbp5 in the BNST. A, Schematic representation of ESARE promoter driven and 4OHT-dependent conditional KO of previously activated neurons in the ovBNST, as well as subsequent experimental timeline. B, Fkbp5 ISH to validate correct viral manipulation. Scale bar: 250 μm (C–F). Activity-dependent KO of Fkpb5 in the ovBNST indicated an anxiogenic phenotype. C, FKBP51^ovBNST-KO (KO) animals spent significantly less time in the open arms of the EPM. In addition, they showed a tendency to travel less distance within the light zone of the dark-light box (DALI). The OF test (E) and the ASR response (F) did not show any significant differences between the two groups. Data are mean ± SEM; *p < 0.05, **p < 0.01.