We recently identified a common inbred mouse strain, 129S1, that exhibits impaired fear extinction and functional abnormalities in a cortico-amygdala circuit mediating extinction (
Hefner et al. 2008;
Sotres-Bayon et al. 2008). The major novel finding of the current study was that impaired fear extinction extends to four other 129 substrains. Similar to 129S1, these substrains displayed normal fear (as measured by freezing) as compared to B6, but failed to show significant within-session extinction learning. Consistent with poor extinction learning, 129S1, 129P1 and 129T2 showed no evidence of extinction retrieval. Although 129P3 and 129X1 did display a reduction in freezing during the retrieval test relative to pre-extinction, this between-session reduction is most parsimoniously explained by decay of the original fear response unrelated to extinction because these two substrains showed no extinction learning.
Our data suggest that impaired fear extinction is a phenotypic abnormality common to the four separate genetic lineages of the 129 strains. This conclusion must be qualified by the fact that we only examined one representative substrain from the Steel and Teratoma lines and two substrains from the Parental line. The other dozen or so substrains have yet to be tested for this behavior and we cannot exclude the possibility that some will exhibit intact extinction. Notwithstanding, the current data do not identify a 129 substrain that shows good fear extinction. Such a strain would be very useful to compare with the poor extinguishing 129S1 substrain as a means to expedite the discovery of the genes underlying the differences in extinction efficacy.
The finding that impaired fear extinction appears to be a common phenotypic feature of the 129 strain could have implications for the use of this strain as a source of embryonic stem (ES) cells in the generation of targeted gene mutant mice. The popularity of these strains for this purpose largely stems from the relative success with which 129 ES cells can be derived and then incorporated into the germline of host blastocysts (‘germline competency’) (
Evans & Kaufman 1981;
Thomas & Capecchi 1987). Due to various abnormal phenotypic features at the molecular (
Koike et al. 2006), neuroanatomical (
Wahlsten 1982) and behavioral level (
Wolfer et al. 1997), 129 strains have not been favored as a genetic background for mutant mice (
Crawley et al. 1997). Nonetheless, even with repeated backcrossing onto a more suitable strain, such as B6, a mutant line generated with 129 ES cells will still harbor 129 genes in the region flanking the target mutation (
Crusio et al. 2008;
Wolfer et al. 2002). Thus, this raises the possibility that impairments in fear extinction attributed to a targeted gene mutation could in fact be a false positive caused by extant 129 flanking genes. It would seem prudent to at least bear this caveat in mind in future studies of fear extinction in mutant mice.
The current study replicates our recent observation (
Hefner et al. 2008) of impaired fear extinction in the 129S1 substrain, relative to B6, and extends the finding in a number of ways. To ensure that elevated freezing in 129S1 during extinction training was not due to a fear response to the tone
per se, we tested mice under conditions in which they received tone presentations, but not shocks, during conditioning, and found that freezing was negligible in both strains under these conditions. We also tested the possibility that impaired extinction in 129S1 was due to abnormalities in non-associative processes known as fear sensitization and habituation. Fear sensitization produces an increased fear response to a stimulus (e.g., tone) after exposure to an aversive event (e.g., footshocks), even though the tone has not been previously experienced (
Kamprath et al. 2006;
Kamprath & Wotjak 2004;
McSweeney & Swindell 2002). A failure to habituate to this sensitized response with repeated tone presentations would manifest in the same way as impaired fear extinction. Indeed, impaired habitation has been found to contribute to apparent deficits in fear extinction in, for example, cannabinoid CB1 receptor knockout mice (
Kamprath et al. 2006). Current results indicate that the deficient extinction in these mice does not appear to reflect impaired habituation of a sensitized fear response, as exposure to the same number and intensity of footshocks (without pairing to tone) was not sufficient to produce sensitized fear in either 129S1 or B6. Taken together, these findings provide further support for the specificity of the fear extinction deficit in the 129S1 strain.
The extinction deficit in 129S1 was observed in the current study using a testing paradigm modified from that used in the
Hefner et al., 2008 study which produces more modest levels of fear (e.g., see
Norcross et al. 2008). This reduces the potential for ‘ceiling’ levels of fear to confound the assessment of extinction. It was interesting to note that against this modest level of fear, 129S1 showed not only a failure to reduce fear, but demonstrated a modest increase in fear during extinction training. This profile is reminiscent of fear incubation; defined by Eysenck as ‘an increment in conditioned responding over a period of time when the CS is applied once or a number of times, but without reinforcement’ (
Eysenck 1968). The mechanisms underlying fear incubation have not yet been as thoroughly described as for those mediating fear extinction (
Quirk & Mueller 2008). Previous studies have shown that various experimental manipulations, such as spaced extinction training, administration of the β-adrenergic receptor agonist propranolol or extensive tone-shock conditioning, can produce fear incubation in rats and C57BL/6Tac mice (
Cain et al. 2003;
Pickens et al. 2009). It will be of great interest to apply these manipulations in future studies to more directly test for a potential fear incubation phenotype in 129S1. Some initial insights into this issue, however, were made in the current study.
We have previously shown that 129S1 and B6 do not differ in fear expression using relatively mild conditioning protocols involving 1 or 3 pairings between tone and 0.6 mA footshock (
Hefner et al. 2008). This is consistent with most previous studies that have also found either minimal differences or lesser fear in 129 substrains (including 129P1, 129P3, 129S1, 129S2/SvHsd, 129/SvevTacfBr, 129S6/SvEvTac, 129T2, 129X1) as compared to B6 (
Balogh & Wehner 2003;
Bolivar et al. 2001;
Bothe et al. 2004;
Cook et al. 2002;
Holmes et al. 2002;
Nguyen et al. 2000;
Owen et al. 1997;
Schimanski & Nguyen 2005). These data do not, however, rule out the possibility that 129 substrains could exhibit increased fear under stronger fear conditioning protocols. Here, we found that 129S1 can exhibit increased freezing during fear conditioning and expression, relative to B6, under a stronger (i.e., more pairings, higher shock intensity) conditioning protocol. Importantly, however, close inspection of the increased freezing during the first extinction trial indicated that immediate fear during the first 30 seconds of the CS was normal (i.e., B6-like) in 129S1, but that fear in these mice rapidly increased above B6 levels over the next 90 seconds of the CS. Thus, elevated 129S1 freezing during the first CS presentation cannot simply be explained by an increase in fear, but instead reflects a more complex response involving a rapid rise in fear during exposure to a long continuous CS. This is again suggestive of an incubation of fear, similar to the effects seen across multiple short-CS exposures discussed above. Alternative interpretations (e.g., timing of shock onset at tone termination) cannot be ruled out at this point, and further studies will be needed to more fully explore fear incubation and possible mechanisms underlying it (e.g., abnormal prelimbicamygdala circuitry, see
Vidal-Gonzalez et al. 2006).
In summary, we extend our recent finding that the 129S1 inbred mouse strain exhibits impaired Pavlovian fear extinction by showing that deficiency in this behavior is common across different genetic lineages of the 129 strain. Current results also demonstrate that the extinction deficit in 129S1 was not likely due to non-associative fear sensitization or a fear response to the auditory tone
per se. Our data do, however, raise the intriguing possibility that impaired extinction in this strain may reflect (or unmask) a pro-fear incubation-like process. Further studies using this mouse model of impaired extinction could provide insight into the pathophysiology and improved treatment of extinction impairment in neuropsychiatric disorders such as PTSD (
Holmes & Wellman 2009).
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