Our behavioral results indicate that fear extinction is severely impaired in PN-1 KO mice. This deficit is accompanied by an abnormal pattern of activity-dependent signaling markers across different amygdala nuclei, including the BA, mITC and CEl.
Impaired fear extinction in PN-1 KO mice
The impaired extinction phenotype is unlikely to reflect a founder effect of the PN-1 KO line, as another line of mice with reduced PN-1 protein expression also show impaired extinction (data not shown). It may, however, have a developmental component that we cannot exclude. This impairment also cannot be considered as a general learning deficit of the PN-1 KO mice as their fear conditioning learning is comparable to their WT littermates. In addition, while we found no evidence that they are more susceptible to learning fear, we cannot exclude that the threshold for fear acquisition is lower for PN-1 KO mice.
Our study is the first demonstration as far as we know that a serpin can influence emotional learning such as fear extinction. Earlier reports have shown that serine proteases can influence fear conditioning. Acutely stressed mice lacking the protease tissue plasminogen activator exhibit reduced contextual fear learning compared with WT animals (Norris & Strickland, 2007). On the other hand, mice lacking another activity-dependent serine protease, neuropsin, display increased fear after cued fear conditioning compared with WT littermates, even in the absence of stress (Horii et al., 2008). Mice with a targeted deletion of the serine protease-activated receptor-1 (PAR-1), also known as the thrombin receptor, show reduced fear retrieval after cued fear conditioning (Almonte et al., 2007). PN-1 inhibits many of the above involved proteases and reduces PAR-1 activation (Scott et al., 1985; Stone et al., 1987; Kvajo et al., 2004; Feutz et al., 2008). In addition to a reduced proteolytic inhibition, a further impact of the absence of PN-1 could be an altered cellular signaling triggered by high molecular weight complexes between PN-1 and its target proteins (Vaillant et al., 2007; Fayard et al., 2009). Consequently, our results suggest a possible involvement of serine proteases in fear extinction as well.
Molecular correlates of impaired fear extinction in PN-1 KO mice
We evaluated short- and long-term patterns of neuronal activation in the amygdala by comparing Fos immunoreactivity and pαCamKII protein levels in the amygdala of WT and PN-1 KO mice to find cellular correlates of this behavioral deficit. We concentrated on the amygdala because of the striking pattern of PN-1 expression in GABAergic neurons as well as its central role in integrating fear inputs. It is possible that other affected brain areas contribute to the overall extinction deficit in the PN-1 KO mouse, e.g. the prefrontal cortex (Quirk & Mueller, 2008) or the hippocampus (Corcoran et al., 2005).
In WT mice, Fos immunoreactivity increased in the no extinction and extinction groups as expected in the LA and BA after fear retrieval and extinction acquisition, compared with the naive control group (Herry & Mons, 2004). The Fos-immunopositive cells possibly represent subsets of the two populations of cells recently shown to be activated differentially by fear and extinction protocols (Herry et al., 2008).
This response was shifted in PN-1 KO mice, namely the increase was higher than the WT response after fear retrieval in the no extinction group and lower than the WT in the extinction group. This shift could be the result of impaired sensory or higher brain input or as well as of local impairments resulting from the absence of PN-1 signaling in the BA. Although PN-1 is not prominently expressed by BA principal neurons, our immunohistochemical results indicate its presence in the extracellular matrix, presumably through glial secretion. Application of purified PN-1 has been shown to rescue primary cultured cerebellar granular neuron precursors derived from PN-1 KO mice, suggesting that extracellular sources of PN-1 can participate (at least in some measure) in normal neuronal signaling (Vaillant et al., 2007).
Surprisingly, PN-1 KO mice displayed a greater Fos protein expression under conditions where we would expect reduced NMDAR activity. One possible explanation for the apparently paradoxical finding is a lowered basic inhibitory activity in the BLA. Inhibitory GABAergic interneurons in the BLA exhibit NMDAR-mediated synaptic currents (Szinyei et al., 2000) and provide a strong inhibitory control over principal neurons (Lang & Paré, 1997). Reduced levels of NMDAR activity on inhibitory neurons could therefore have a proportionately greater impact on the net level of BLA activity. Concurrently, the net strength or balance of various inputs (e.g. cortical and hippocampal) to the amygdala could be affected, thereby changing the activation outcome. This altered Fos upregulation measured after fear retrieval may be an indication that the net levels of activity in the BA are abnormal in PN-1 KO mice. In fact, some of these neurons expressing cFos after fear conditioning may not be directly involved with fear expression but contribute to resistance to extinction similar to what has been described in the prelimbic cortex (Burgos-Robles et al., 2009).
No change in Fos immunoreactivity was detected in the CEA. This is unlike previous studies showing an increase in the CEA after extinction (Hefner et al., 2008; Kolber et al., 2008). One reason may be that these studies used a fear conditioning protocol with a stronger and longer foot shock US than ours.
To evaluate longer term neuronal activation, we measured the relative phosphorylation level of αCamKII by immunoblot analysis of laser-dissected amygdala subnuclei. Long-lasting increased levels of autophosphorylated αCamKII in specific brain areas have been associated with learning (Pollak et al., 2005; Singh et al., 2005). In addition, normal autophosphorylation of αCamKII has been reported to be essential for learning extinction of conditioned contextual fear (Kimura et al., 2008). We found no fear conditioning- or extinction-dependent changes in relative pαCamKII levels in the LA, BA, CEm or lITC. This may reflect an averaged sampling of heterogeneous neuronal populations. A trend of a lower pαCamKII/αCamKII ratio was, however, detected in the lITC of PN-1 KO mice.
In WT mice, behavior-dependent increases in pαCamKII levels were found in the mITC after fear retrieval and extinction, and in the CEl after extinction, suggesting that these behaviors induce distinct activity-dependent changes in cellular or synaptic function in these areas. The mITC receives excitatory input from the BA as well as other regions (Royer et al., 2000). The pattern of pαCamKII levels in the mITC correlates with the relative levels of Fos activation of the BA after fear retrieval and extinction. Moreover, as BA cells are functionally heterogeneous with distinct subpopulations active after fear conditioning and extinction (Herry et al., 2008), it is tempting to speculate that mITC neurons might exhibit a similar heterogeneity, and that the mITC might not only be involved in fear extinction (Jüngling et al., 2008; Likhtik et al., 2008) but also in the regulation of high fear states (Paréet al., 2004). In the rat brain, the CEl receives inputs from the cortex, BA and LA (Cassell et al., 1999). Therefore, the increased phosphorylation of αCamKII we detected in the WT CEl after extinction would be consistent with a sufficiently increased input from the BA as indicated by the increased density of Fos-immunopositive cells.
In contrast, PN1-KO mice exhibited a shift in the distribution of pαCamKII after extinction training relative to WT animals. The absence of a further increase over fear retrieval levels of phosphorylation in the mITC correlates with the unchanged Fos induction in the BA and is consistent with the behavioral readout of high freezing levels in PN-1 KO mice after the extinction training. The increased pαCamKII levels in the CEl of KO mice after extinction training could be explained by a reduced inhibitory input from the mITC, implied by the below WT phosphorylation level. This may serve to offset a decreased BA input, implied by the relatively low Fos immunoreactivity, leading to a net increased activation of the CEl. Indeed, connections between mITC and CEl have been described in the cat (Paré & Smith, 1993), and extracellular stimulation within the mITC was reported to activate synapses on the dendrites of CEl neurons in the rat (Delaney & Sah, 2001). Another consideration is that increased pαCamKII levels in the CEl of PN-1 KO mice might reflect activation of functionally distinct, fear-promoting subpopulations of neurons that are normally not active during extinction training.
Our study shows the usefulness of laser dissection to monitor changes in protein phosphorylation in small, specific regions of the brain and correlate them to learning. We show that WT mice, acquiring extinction with the associated reduced freezing response and increased Fos protein expression in BLA, also display corresponding increases in pαCamKII levels in mITC and CEl. PN-1 KO mice, which we show are capable of acquiring conditioned fear responses but are resistant to acquiring extinction, show impairments in these responses. Our results do not allow us to distinguish if altered NMDAR activity, denoted by the abnormal levels of activity indicators in the BA, CEl and mITC, is responsible for the behavioral impairment or is the consequence thereof. Nevertheless, these data are in line with recent studies demonstrating a link between impaired extinction learning and altered immediate-early gene expression patterns in the BA, mITC and CEA in select mouse and rat strains with inborn behavioral deficits (Hefner et al., 2008; Muigg et al., 2009) and with the recovery of conditioned fear responses in extinguished animals after attenuation of glutamatergic input to mITCs or targeted immunotoxic lesions of mITCs (Jüngling et al., 2008; Likhtik et al., 2008).
In summary, our results support a growing view that the emotional learning and memory system is not limited to the BLA but is distributed across BLA, ITC and CEA circuitry of the amygdala (Paréet al., 2004; Wilensky et al., 2006). Moreover, our study demonstrates that lack of PN-1 results in area-specific changes in signaling activity markers underlying fear extinction. Serine proteases and their inhibitors may thus represent new targets for intervention in various conditions associated with anxiety and stress.