Dynamic changes in neural circuitry during adolescence are associated with persistent attenuation of fear memories

Abstract

Fear can be highly adaptive in promoting survival, yet it can also be detrimental when it persists long after a threat has passed. Flexibility of the fear response may be most advantageous during adolescence when animals are prone to explore novel, potentially threatening environments. Two opposing adolescent fear-related behaviours-diminished extinction of cued fear and suppressed expression of contextual fear-may serve this purpose, but the neural basis underlying these changes is unknown. Using microprisms to image prefrontal cortical spine maturation across development, we identify dynamic BLA-hippocampal-mPFC circuit reorganization associated with these behavioural shifts. Exploiting this sensitive period of neural development, we modified existing behavioural interventions in an age-specific manner to attenuate adolescent fear memories persistently into adulthood. These findings identify novel strategies that leverage dynamic neurodevelopmental changes during adolescence with the potential to extinguish pathological fears implicated in anxiety and stress-related disorders.

Figure 1. Imaging spine remodelling in mPFC.

(a) Schematic for in vivo spine imaging in mPFC and FrA through a prism. The majority of analysed mPFC neurons reside in PL, although some may reside in the cingulate area of mPFC, dorsal to PL. IL neurons were excluded because they lie ventral to the prism. (b) YFP-expressing PL pyramidal neurons in Thy1/YFP-H line transgenic mice. (c) Spine remodelling was quantified in repeated images acquired at 1-day intervals. Eliminated spines (red arrow) were present at P30 but not P31. Formed spines (blue arrows) were present at P31 but not P30. (d) There were significant main effects of age (F(2,23)=87.3, P<0.0001) and cortical region (F(1,23)=41.4, P<0.0001) on spine formation (age X region interaction: F(2,23)=58.7, P<0.0001). Specifically, 1-day spine formation rates were selectively increased at P30 in mPFC compared with FrA (P<0.001, post hoc linear contrast). There were no significant regional differences at P45 or P90. (e) There was a significant main effect of age (F(2,23)=7.72, P=0.003) on spine elimination, but not of cortical region (F(1,23)=0.01, P=0.937). (f,g) Confocal images of YFP-expressing pyramidal cells in fixed tissue samples obtained at P24, P31 and P45. High-magnification images in g are from typical dendritic segments in PL (# spines/10 μm indicated at lower right). CP, Caudate/Putamen. (h) Quantification of developmental changes in spine density in PL, FrA and IL at P24, P31 and P45 (N=4–5 mice/age). There were significant main effects of region (F(2,31)=7.34, P=0.002) and age (F(2,31)=8.16, P=0.001), and an age X region interaction (F(4,31)=4.4, P=0.006), driven by an increase in spine density in PL at P31 relative to P24 (P=0.004) and P45 (P=0.007). Scale bars, 50 μm (b), 5 μm (c and g) and 150 μm (f). Error bars=s.e.m.; ***P<0.001; **P<0.01 for contrasts of P31 versus P24 and P45.

Figure 2. Dynamic developmental surge in neural connectivity to mPFC.

Representative images of FG-labelled neurons in (a) hippocampus ventral CA1 (vCA1) and (b) basolateral amygdala (BLA), Scale bars, 50 μm. (c) Schematic of FG injection in prelimbic cortex (PL) and FG+ cells throughout vCA1 and BLA. Outlined area denotes injection site, with surrounding diffusion maintained within PL boundaries. Quantification of density of FG (FG+) retrograde labelled neurons in showed a significant main effect of age in (d) vCA1, P<0.0005, F(3,16)=11.58, vCA1 (P23 8970.7±401.3; P30 11441.1±368.8; P45 8254.4±565.1; P90 8024.6±479.5) and a significant main effect of age in (e) BLA, P <0.0005, F(3, 16)=12.2, BLA (P23 14809.1±847.3; P30 20124.1±165.6; P45 16995.1±973.0; P90 15114.8±520.5).

Figure 3. Combined contextual and cue extinction effects on long-term memory.

(a) Behavioural paradigm depicting four groups designed to assess if there is an interaction between cued fear extinction and the context in which it is presented for mice fear conditioned at P29 and tested during late adolescence (P44). (b) Three tone test at P44 for all four behavioural treatment groups demonstrates significantly less freezing in the group that underwent cued fear extinction in the fear-conditioning context (F(3,12)=14.7, P<0.0005).