Functional Characterization of the Basal Amygdala-Dorsal BNST Pathway during Contextual Fear Conditioning

The TetTag mouse and retrograde tracing are combined to investigate BA-dBNST circuitry function in contextual fear memory. A, Schematic of the double transgenic TetTag mouse line. Tetracycline transcription activator (tTA) is expressed under control of the c-fos promoter. In the absence of doxycycline (DOX), tTA binds to the tet operator (TetO) in the second transgene and drives expression of histone2B-GFP (His-GFP). His-GFP is long lasting and localized to the nucleus of cells activated during the off-DOX period. B, Experimental paradigm for the CTB-injected FC group (n = 9) versus HC group (n = 8). Lower panel shows the corresponding expression timeline of the CTB, His-GFP, Zif. C, The FC group showed typical levels of freezing during fear conditioning trials (S1, S2, S3) and retrieval session (RET). Freezing percentages are an average of the second and third minutes of each session. D, CTB-647 expression near the injection site in the dBNST. Scale bar = 200 μm. E, CTB-positive cells (CTB+, grayscale) in the BA with Zif mask (red), GFP+ mask (green), and colocalizing GFP+Zif+ cells (yellow). Scale bar = 100 μm. F, Percentage of CTB+ cells among DAPI cells in the aBA and pBA were similar between FC and HC groups. Graphs show means ± SEM.

Contextual fear engram neurons in the aBA do not project to the dBNST. A–D, Representative images of (A) GFP, (B) Zif, (C) CTB, and (D) overlay from a section of aBA with DAPI (blue). White arrow indicates a GFP+Zif+CTB– cell. Overlay displays an example of masks from thresholded images of GFP and Zif that were used for quantification and to determine colocalization. Scale bar = 50 μm. E, Contextual fear conditioning increased the percentage of GFP+ neurons among DAPI cells in the aBA (p = 0.0069). F, There was no difference between FC and HC in total percentage of Zif+ neurons in the aBA (p = 0.24) due to fear retrieval. G, Fear conditioning and retrieval increased the percentage of GFP+Zif+ reactivated fear neurons compared with chance level in the aBA (p = 0.016). H, The number of GFP+Zif+ cells minus chance level was significantly higher in the FC group compared with HC in the aBA (p = 0.013). I, Contextual fear conditioning increased the percentage of GFP+CTB– neurons among CTB negative (CTB–) cells in the aBA (p = 0.0043). J, Fear retrieval increased the percentage of Zif+CTB– neurons among CTB– cells in the aBA (p = 0.048). K, Fear conditioning and retrieval increased the percentage of GFP+Zif+CTB– reactivated fear neurons among CTB– cells in the aBA compared with chance level (HC: p = 0.38; FC: p = 0.030). L, The number of GFP+Zif+CTB– cells minus chance level was significantly higher in the FC group, indicating reactivation of fear neurons during retrieval (p = 0.0082). M–P, Fear conditioning and retrieval did not significantly change any levels of activated cells in the CTB+ population in the aBA; M, GFP+CTB+ (p = 0.15); N, Zif+CTB+ (p = 0.65); O, GFP+Zif+CTB+ versus chance (HC: p = 0.64; FC: p = 0.14); P, GFP+Zif+CTB+ cells minus chance level (p = 0.25). Graphs show mean ± SEM. *p < 0.05, **p < 0.01.

The pBA-dBNST circuit is active during contextual fear conditioning, but is not incorporated into the fear engram. A–D, Representative images of GFP, Zif, CTB, and overlay from a section of pBA with DAPI (blue). White arrow indicates a GFP+Zif-CTB+ cell. Scale bar = 50 μm. E, Contextual fear conditioning increased the percentage of GFP+ neurons among DAPI cells in the pBA (p = 0.0005). F, There was no difference between FC and HC in total percentage of Zif+ neurons in the pBA (p = 0.89) due to fear retrieval. G, Fear conditioning and retrieval did not increase the percentage of GFP+Zif+ reactivated fear neurons compared with chance level in the pBA (p = 0.23). H, The number of GFP+Zif+ cells minus chance level was not significantly different in the FC group compared with HC in the pBA (p = 0.70). I–L, Fear conditioning increased levels of GFP+ in the CTB– population in the pBA, but Zif+ and GFP+Zif+ did not differ between the groups; E, GFP+CTB– (p = 0.0036); F, Zif+CTB– (p = 0.41); G, GFP+Zif+CTB– versus chance (HC: p = 0.58; FC: p = 0.52); H, GFP+Zif+CTB– cells minus chance (p = 0.42). M–P, Fear conditioning increased levels of GFP+ in the CTB+ population in the pBA, but Zif+ and GFP+Zif+ did not differ between the groups; M, GFP+CTB+ (p = 0.025); N, Zif+CTB+ (p = 0.47); O, GFP+Zif+CTB+ versus chance (HC: p = 0.84; FC: p = 0.82); P, GFP+Zif+CTB+ cells minus chance level (p = 0.48). Graphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

The ovBNST is active during contextual fear conditioning but is not incorporated into the fear engram. A, Experimental outline for FC (n = 7) versus HC (n = 7) groups. Lower panel shows the corresponding expression timeline of the His-GFP and Zif proteins. B, The FC group showed typical levels of freezing during fear conditioning trials (S1, S2, S3) and retrieval session (RET). Freezing percentages are an average of the second and third minutes of each session. C, His-GFP expression in the dBNST after fear conditioning. Scale bar = 200 μm. D, Fear conditioning increased the number of GFP+ fear neurons in the ovBNST (p = 0.011), but not in the adBNST (p = 0.54). E, There was no significant difference in the number of Zif+ cells after retrieval in the FC group in either the ovBNST (p = 0.90) or adBNST (p = 0.71). F, There was no significant difference in the number of GFP+Zif+ cells compared with chance in the ovBNST (p = 0.094) or adBNST (p = 0.56) in the FC group. G, In neither region was the number of GFP+Zif+ cells minus chance level significantly different between groups (ovBNST p = 0.62; adBNST p > 0.99). Graphs show mean ± SEM. *p < 0.05.