Genetic strain differences in learned fear inhibition associated with variation in neuroendocrine, autonomic, and amygdala dendritic phenotypes

Abstract

Mood and anxiety disorders develop in some but not all individuals following exposure to stress and psychological trauma. However, the factors underlying individual differences in risk and resilience for these disorders, including genetic variation, remain to be determined. Isogenic inbred mouse strains provide a valuable approach to elucidating these factors. Here, we performed a comprehensive examination of the extinction-impaired 129S1/SvImJ (S1) inbred mouse strain for multiple behavioral, autonomic, neuroendocrine, and corticolimbic neuronal morphology phenotypes. We found that S1 exhibited fear overgeneralization to ambiguous contexts and cues, impaired context extinction and impaired safety learning, relative to the (good-extinguishing) C57BL/6J (B6) strain. Fear overgeneralization and impaired extinction was rescued by treatment with the front-line anxiety medication fluoxetine. Telemetric measurement of electrocardiogram signals demonstrated autonomic disturbances in S1 including poor recovery of fear-induced suppression of heart rate variability. S1 with a history of chronic restraint stress displayed an attenuated corticosterone (CORT) response to a novel, swim stressor. Conversely, previously stress-naive S1 showed exaggerated CORT responses to acute restraint stress or extinction training, insensitivity to dexamethasone challenge, and reduced hippocampal CA3 glucocorticoid receptor mRNA, suggesting downregulation of negative feedback control of the hypothalamic-pituitary-adrenal axis. Analysis of neuronal morphology in key neural nodes within the fear and extinction circuit revealed enlarged dendritic arbors in basolateral amygdala neurons in S1, but normal infralimbic cortex and prelimbic cortex dendritic arborization. Collectively, these data provide convergent support for the utility of the S1 strain as a tractable model for elucidating the neural, molecular and genetic basis of persistent, excessive fear.

Figure 1

Fear overgeneralization and impaired safety learning. (a) S1 and B6 froze more to the conditioned context (Cxt A) than either a novel context (Cxt B) or slightly ambiguous context (Cxt C1) (^**p<0.01 vs Cxt A, n=8 per strain). (b) S1 froze more to the conditioned Cxt (Cxt A) than a novel Cxt (Cxt B) but not a highly ambiguous context (Cxt C2). B6 froze more to Cxt A than either Cxt B or Cxt C2 (^**p<0.01 vs Cxt A/same strain, n=8 per strain). (c) During Cxt extinction, there was a decrease in freezing from the first to the last timebin in B6 but not S1, and S1 froze more than B6 during all timebins except the first (^*p<0.05 vs B6/same timepoint, n=16 per strain). During the extinction retrieval test S1 froze more than B6 (^**p<0.01 vs B6), although freezing was lower than it was at the beginning of extinction training in S1 (^#p<0.05) and B6 ^(##p<0.01). (d) S1 and B6 were equally able to discriminate a novel CS– from a conditioned CS+ (n=13–14 per strain). (e) S1 were poorer than B6 at discriminating a partially reinforced CS– from a fully reinforced CS+ (^**p<0.01 vs B6, n=9–12 per strain). (f) S1 and B6 were equally able to discriminate a novel CS– from a conditioned CS+ after fear extinction, although discrimination in B6 was accounted for by a sub-population of poorly extinguishing mice (‘B6 high') (see inset) (^*p<0.05, ^**p<0.01 vs B6, ^##p<0.01 vs CS+/same strain, n=10–14 per strain). (g) Safety-trained B6, but not S1, showed inhibition of contextual fear during presentation of a CS safety signal ^(##p<0.01 vs Cxt/B6, n=9–10 per strain). (h) Safety-trained B6 and S1 mice showed retardation of new fear learning, as compared with previously naive controls (^**p<0.01 vs previously naive, n=6–9 per strain per group). (i) Following safety training in which the CS block always preceded the US block, S1 and B6 both showed inhibition of contextual fear during presentation of the CS safety signal (^**p<0.01 vs Cxt, n=7 per strain). Data are means±SEM.

Figure 2

FLX recue of impaired fear extinction and context overgeneralization. (a) Schematic of experimental design in which mice were FLX treated for 3 weeks before conditioning (Con), and remained on treatment through extinction training (Ext) and retrieval testing (Test). (b) S1 showed more freezing than B6 over extinction trials, regardless of treatment, although there was a nonsignificant trend for lesser freezing during the first trial-block in FLX-treated S1 mice (n=8 per strain per treatment). (c) S1 had higher freezing than B6 during extinction retrieval after water but not FLX treatment (^**p<0.01 vs B6/water, ^##p<0.01 vs S1/water, n=8 per strain per treatment). (d) Schematic of experimental design in which mice were FLX treated for 3 weeks starting after conditioning, and remained on treatment through testing. (e) S1 showed more freezing than B6 over extinction trials, regardless of treatment, although decreases in freezing were evident in all groups during the first four trial-blocks. (f) S1 had higher freezing than B6 during extinction retrieval after water but not (post-conditioning) FLX treatment (^**p<0.01 vs B6/water, ^##p<0.01 vs S1/water, n=7 per strain per treatment). (g) Schematic of experimental design in which mice were FLX treated for 3 weeks before context conditioning, and remained on treatment through the context generalization test. (h) S1 froze more to an ambiguous context than B6 after water but not FLX treatment (^**p<0.01 vs B6/water, ^#p<0.05 vs S1/water, n=6–8 per strain per treatment). (i) Schematic of experimental design in which mice were FLX treated for 3 weeks before acute forced swim stress. (j) S1 had higher acute swim stress-induced CORT than B6 after water but not FLX treatment. CORT was higher in B6 after FLX treatment relative to water (^**p<0.01 vs B6/water, ^##p<0.05 vs B6/water, n=7–8 per strain per treatment). Data are means±SEM.

Figure 3

Autonomic and neuroendocrine abnormalities. (a) S1 and B6 showed an increase in HR from baseline to the first-half and second-half blocks of extinction training, although this increase was greater in B6 than S1 (^##p<0.01 vs basal/same strain, ^**p<0.01 vs S1, n=8 per strain). (b) Both strains showed a reduction in HRV from baseline to the first-half block of extinction training, with a greater reduction in B6 than S1 and a recovery to baseline in B6, but not S1, on the second-half block (^##p<0.05 vs basal/same strain, ^*p<0.05 vs S1, n=8 per strain). (c) AM or PM basal CORT did not differ between strains, and PM CORT was generally higher than AM CORT (^#p<0.05 vs AM/same strain). CORT was increased 30 min after acute 2-h restraint stress, relative to basal PM (^‡p<0.05 same strain) but was elevated more so in S1 than B6 (^*p<0.05 vs B6). CORT returned to PM basal levels by 75 min (n=7–9 per strain per condition). (d) CORT was increased by acute swim stress ^(#p<0.05 vs basal/same strain) in previously stress-naive S1 and B6. In mice previously subjected to chronic restraint stress, acute swim produced an augmented CORT response in B6, but an attenuated response in S1, relative to the no restraint+swim groups (^‡p<0.05 vs no restraint+swim, ^*p<0.05 vs B6, 5–14 per strain per group) (from Mozhui et al, 2010 with permission). (e) Injection with DEX (0.05 and 0.1 mg/kg) reduced CORT relative to vehicle in B6, but not in S1 (^#p<0.05 vs 0 dose/B6, n=10–12 per strain per dose). (f) S1 had lower GR mRNA expression than B6 in the CA3, not dentate gyrus (DG) or CA1, hippocampal subregions (^*p<0.05 vs B6/same region, n=5–6 per strain). (g) Representative in situ hybridization images of GR mRNA from S1 and B6 (scale bar=1 mm). Data are means±SEM.

Figure 4

Increased dendritic arborization of BLA neurons. (a) Examples of reconstructed BLA neurons in S1 and B6. (b) Apical dendrites in BLA were on average longer in S1 than B6. (c) BLA spine density was no different between strains. (d) Sholl analysis showing greater apical dendritic material relatively distal from the soma in S1 as compared with B6. n=6–8 neurons per mouse, n=6–8 mice per strain. Data are means±SEM. ^*p<0.05 vs B6.

Figure 5

Normal PL and IL dendritic morphology and spine density. (a) Examples of reconstructed PL neurons. Apical dendritic length of PL neurons did not differ between strains as measured by overall averages (b) or Sholl analysis (c). (d) PL spine density was similar in S1 and B6. (e) Examples of reconstructed IL neurons. Apical dendritic length of IL neurons did not differ between strains as measured by overall averages (f) or Sholl analysis (g). (h) IL spine density was similar in S1 and B6. n=6–8 mice per strain per region. Data are means±SEM.