Freezing to a tone following auditory fear conditioning is commonly considered as a measure of the strength of the tone-shock association. The decrease in freezing on repeated nonreinforced tone presentation following conditioning, in turn, is attributed to the formation of an inhibitory association between tone and shock that leads to a suppression of the expression of fear. This study challenges these concepts for auditory fear conditioning in mice. We show that acquisition of conditioned fear by a few tone-shock pairings is accompanied by a nonassociative sensitization process. As a consequence, the freezing response of conditioned mice seems to be determined by both associative and nonassociative memory components. Our data suggest that the intensity of freezing as a function of footshock intensity is primarily determined by the nonassociative component, whereas the associative component is more or less categorical. We next demonstrate that the decrease in freezing on repeated nonreinforced tone presentation following conditioning shows fundamental properties of habituation. Thus, it might be regarded as a habituation-like process, which abolishes the influence of sensitization on the freezing response to the tone without affecting the expression of the associative memory component. Taken together, this study merges the dual-process theory of habituation with the concept of classical fear conditioning and demonstrates that sensitization and habituation as two nonassociati

Traumatic stress can lead to long-term emotional alterations, which may result in Posttraumatic Stress Disorder (PTSD). Fear reactions triggered by conditioned cues and exacerbated emotional arousal in face of non-conditioned stimuli are among the most prominent features of PTSD. We hypothesized that long-term emotional alterations seen in PTSD may depend on the strength of context-trauma association. Here, we investigated the contribution of previous contextual exploration to the long-term emotional outcomes of an intense foot shock in rats. We exposed male Wistar rats to a highly stressful event (foot shock, 2 mA, 1 s) allowing them to explore or not the chamber prior to trauma. We, then, evaluated the long-term effects on emotionality. Fear was assessed by the time spent in freezing behavior either upon re-exposure to trauma context or upon exposure to an unknown environment made potentially more aversive by presentation of an acoustic stimulus. Behaviors on the elevated-plus-maze and acoustic startle response were also assessed. The possibility to explore the environment immediately before the aversive event led to differential long-term emotional effects, including a heightened freezing response to re-exposure to context, blunted exploratory behavior, fear sensitization and exacerbation of the acoustic startle response, in contrast to the minor outcomes of the foot shock with no prior context exploration. The data showed the strong contribution of contextual learning to Behaviors on the elevated-plus-maze and acoustic startle response were also assessed. The possibility to explore the environment immediately before the aversive event led to differential long-term emotional effects, including a heightened freezing response to re-exposure to context, blunted exploratory behavior, fear sensitization and exacerbation of the acoustic startle response, in contrast to the minor outcomes of the foot shock with no prior context exploration. The data showed the strong contribution of contextual learning to long-term behavioral effects of traumatic stress. Although it is not possible to assess spontaneous memory recall in non-human animals, reactions triggered by implicit memory retrieval are experimentally induced in animal studies using the fear conditioning paradigm (Sanford et al., 2003 ; Jha et al., 2005 ; Pawlyk et al., 2005 ; Siegmund and Wotjak, 2006 , 2007a , b ; Siegmund et In animal studies of PTSD, re-exposure to the paired environment is frequently employed to trigger detectable emotional responses that are believed to resemble, at least in basic aspects, the symptoms manifested in PTSD patients (Jha et al., 2005 ; Pawlyk et al., 2005 ; Siegmund and Wotjak, 2007a , b ; Golub et al., 2009 ; Pamplona et al., 2011 ). It has been well-established in the fear conditioning literature, that more than 20 sec of pre-shock context exploration are necessary to elicit a robust behavioral conditioned fear response during re-exposure (Fanselow, 1986 ; Landeira-Fernandez et al., 2006 ). When the shock is delivered immediately after placement of the animal in the context, with insufficient environmental exploration, conditioning is not effective, and this phenomenon is coined as immediate shock deficit. Therefore, environment familiarization prior to foot shock contributes to the strength of the fear response expressed upon re-exposure to the conditioned context (Fanselow, 1986 , 1990 ; Frankland et al., 2004 ). One question that arises from the above assumptions is: given that the duration of prior environmental exploration determines the amount of conditioned fear expressed afterwards, would the strength of the contextual memory also influence the non-associative deleterious outcomes of the aversive experience? To address this question, we applied the immediate shock paradigm, in order to test the contribution of context familiarization before foot shock to long-term fear sensitization, exploratory behavior and exaggerated acoustic startle response in a fear conditioning-based animal model of traumatic stress. NS rats were given the opportunity to explore the conditioning chamber for 2 min with no shock delivery. After the procedure, each rat was kept in an individual cage until all rats from the same cage underwent the procedure. After that they were all brought back to home cage. Re-exposure Re-exposure to the conditioning chamber occurred 14 days after the conditioning, for 5 min, with no shock delivery. To evaluate the expression of fear upon re-exposure to the context, we scored freezing behavior, a defensive behavior defined as complete absence of movement, except for breathing-related motion (Blanchard et al., 1986 ). There was no significant difference in the number of traveled segments in the open field ( F (2,26) = 1.067; p = 0.358) (Figure 4B ), nor when central ( F (2,26) = 0.329; p = 0.722) (Figure 4C ) and peripheral ( F (2,26) = 2.445; p = 0.106) (Figure 4D ) areas were analyzed separately (Figure 4B ). Figure 4 Open field analyzes . (A) Expression of fear in a mildly aversive situation. Percentage of time in freezing before and during tone presentation. Number of (B) total (C) central and (D) peripheral segments traveled. NS = no-shock group ( n = 9); IS = immediate shock group ( n = 10); DS = delayed shock group ( n = 10). Analyzing minute by minute freezing upon re-exposure, we found a comparable initial fear reaction to the conditioned context for the groups, but the reaction declined throughout the session in the IS group only. Despite this clear, albeit transient reaction to the context, the IS group did not exhibit as much inhibition of exploratory activity, fear sensitization or hyperarousal as the DS group in the subsequent emotional assessments. The minute by minute results suggest a frail conditioned contextual fear reaction in the IS group, which is subjected to intra-session suppression, while fear is maintained in the DS group throughout the whole session.

Maladaptive fear responses, including sensitized threat reactions and overgeneralization, contribute to anxiety disorders such as generalized anxiety disorder and post-traumatic stress disorder. Although stress intensity influences the generation and extent of these maladaptive fears, the underlying mechanisms remain unclear. The present study examined whether varying footshock stress intensity and inhibition of protein synthesis have differential effect on fear sensitization and generalization in mice. Mice were subjected to a classic fear conditioning protocol involving five different levels of footshock intensities. Prior to fear acquisition, the protein synthesis inhibitor cycloheximide (CHX) was administered intraperitoneally. Fear sensitization to white noise and fear generalization to tones with frequencies differing from the conditioned tone were assessed at either 2 or 4 days after fear acquisition. The results showed that, although varying shock intensities (except the lowest) led to a similar pattern of increased freezing during auditory cues in fear acquisition, the extent of both fear sensitization and generalization increased with the intensity of the footshock in the following days. As shock intensities increased, there was a proportional rise in sensitized fear to white noise and generalized freezing to tones with frequencies progressively closer to the conditioned stimulus. Mildest shocks did not induce discriminative conditioned fear memory, whereas the most intense shocks led to pronounced fear generalization. Administration of CHX before fear acquisition did not affect sensitized fear but reduced generalization of freezing to tones dissimilar from the conditioned stimulus in the group exposed to the most intense shock. Our results suggest that maladaptive fear responses elicited by varying stress intensities exhibit distinct characteristics. The effect of CHX to prevent overgeneralization without affecting discriminative fear memory points to potential therapeutic approaches for fear-related disorders, suggesting the possibility of mitigating overgeneralization while preserving necessary fear discrimination.