**1. Introduction**

### **1.1 Increased defensive reactions as a sign of PTSD**

Post-traumatic stress disorder (PTSD) is a multi-symptom condition that includes three primary psychological features: reexperiencing, avoidance and emotional numbing, and hyperarousal (American Psychiatric Association, 2000). Historically, reexperiencing and hyperarousal have been the most studied features, from a neurobiological perspective, using various animal models. In these animal models, changes in defensive reflexive behaviors serve as the assessment measures for these symptoms; thus, both startle reactivity and freezing are now commonly used measures. Freezing behavior is advantageous because of its easy implementation; either the naked eye or an automated motor-tracking system can determine the duration and/or frequency of freezing behavior. In addition, freezing can occur in response to a specific fear-eliciting stimulus or to a fear-experienced context (Doyle & Yule, 1959; Bouton & Bolles, 1980; Fanselow, 1980). Because of these stimulus-response properties, freezing is the response commonly used to assess the experiencing of memories of conditioned stimuli that previously caused a heightened state of fear. At times, the acoustic startle response is used as an assessment of stimulus-elicited fear reactions (Davis, 1986; Hitchcock & Davis, 1987). Under this guise, similar stimuli used in conditioned freezing are experienced by the animal prior to a quick onset, relatively loud, acoustic stimulus. The result is a startle response that is enhanced over baseline levels, which is termed fear-potentiated startle.

However, in the case of PTSD, arousal is not necessarily tied to a memory or triggered by an explicit learned association. There are several examples of patients with PTSD exhibiting exaggerated startle responses in the absence of a known trigger (Butler et al., 1990; Orr et al., 1995; Yehuda et al., 1998; Orr et al., 2002). In fact, human longitudinal studies have found changes in startle reactivity occur over a period of time following the associated trauma (Shalev et al., 1998). Although there are possible confounding variables with any repeated test, such as developing an aversion to the startle testing context, there is a difference with PTSD patients as they fail to habituate to the startle test over months (Shalev et al., 1998). Increases in startle magnitudes can be elicited in rats in order to model this feature of PTSD by exposing them to inescapable shock. Interestingly, like some of the symptoms of PTSD,

Acquisition of Active Avoidance Behavior as a Precursor

characteristics that are exacerbated by experience.

2011b; Jiao et al., 2011; Beck et al., 2011).

with greater amplitudes.

to Changes in General Arousal in an Animal Model of PTSD 75

elevation in arousal, an increase in arousal due to experience, or a combination of innate

In developing our model of anxiety vulnerability, we sought a rodent that reliably exhibited a set of behaviors that are analogous to an identified human vulnerability condition. Behavioral inhibition (or inhibited temperament) is extreme withdrawal in face of novel social and nonsocial situations (Kagan et al., 1987; Kagan et al., 1989; Hirshfeld et al., 1992) and is identified as a risk factor for future symptoms of anxiety disorders (Hirshfeld-Becker et al., 2008; Jovanovic et al., 2010). The Wistar-Kyoto (WKY) rat demonstrates aspects of behavioral inhibition in terms of withdrawal in tests of social interaction (Pardon et al., 2002) and lack of exploration in the open field test (Pare, 1994; Servatius et al., 2008). Hence, we tested WKY rats in a multi-intensity startle procedure that allows for assessment of startle sensitivity (percentage of startles elicited at various acoustic intensities) and startle responsivity (magnitude of responses to the highest intensity). Given startle is a defensivereflex, increased responsivity can be viewed as an increase in general arousal (i.e. more energy used to respond to threat), whereas increased sensitivity can be viewed as an enhancement of vigilance (i.e. greater signal-detection of threats). With no prior manipulations, WKY rats exhibited similar startle sensitivity measures as Sprague Dawley (SD) rats, but exhibited higher startle responsivity (see Figure 2). These temperament and reactivity characteristics are reasonable analogs of the predisposing factors for developing symptoms of anxiety disorders; therefore, we adopted the WKY rat as our anxiety vulnerability model (Servatius et al., 2008; Beck et al., 2010; Ricart et al., 2011a; Ricart et al.,

Fig. 2. Box plots of initial startle responsivity and latency of male SD and WKY rats (left) and female SD and WKY rats (right). Notched regions depict median ± 95% confidence interval. Boxed region depicts interquartile range (middle fifty). Whiskers depict range of data 1.5\*interquartile range. Outliers depicted by '\*'. WKY rats exhibit innate differences in ASRs,

this procedure increases startle magnitude in male rats several days following stressor exposure (Servatius et al., 1994; Servatius et al. 1995; Beck et al., 2002; Manion et al., 2007; Manion et al., 2010); in other words, there is a delayed sensitization of the startle response (Beck et al., 2002). As shown in Figure 1, enhanced startle reactivity may then be observed up to several days later (Servatius et al., 1995; Beck et al., 2002; Manion et al., 2007; Manion et al., 2010). The delayed expression of this enhanced defensive response could be due to competing processes that are similarly elicited by inescapable shock. For instance, some female rats exhibit a suppression of the startle response following stressor exposure that is clearly linked to the immune response elicited by shock exposure (Beck & Servatius, 2005; Beck & Servatius, 2006), but this suppression of startle reactivity is only evident under certain ovarian hormone conditions (Beck et al., 2008). Although males have not been shown to exhibit a reduction in startle reactivity, there may be similar acute physiological reactions that negate an immediate increase in startle reactivity. Hence, these inescapable stress models of enhanced startle reactivity are largely based on the concept that PTSD-like hyperarousal is a product of a single, uncontrollable, traumatic event (or period of repeated trauma over consecutive days).

Fig. 1. Following 3 days of repeated tailshock exposure (40, 3 s, 2 mA shocks over 2 h ), startle magnitudes are elevated in male Sprague Dawley rats 4 days thereafter [Stress x Day F (4, 776) = 7.6, p < .001]. The delayed expression of this increase has been used as a model of the emergent aspects of PTSD symptom expression. Startle magnitude is represented in arbitrary units (AU), as the amplitude is corrected for by body weight.

These models of hyperarousal in PTSD fail to account for potential innate differences in reactivity. The diathesis model of anxiety disorders indicates that anxiety symptomology are a combination of innate characteristics that are influenced by life events. Recent prospective research on PTSD symptomology has indicated elevated arousal may be predictive of anxiety symptoms (Guthrie & Bryant, 2005; Pole et al., 2009). Startle reactivity, as one measure, is elevated in children that upon follow-up were diagnosed with clinical anxiety (Merikangas et al., 1999). Thus, pathologic arousal exhibited in PTSD may reflect an innate

this procedure increases startle magnitude in male rats several days following stressor exposure (Servatius et al., 1994; Servatius et al. 1995; Beck et al., 2002; Manion et al., 2007; Manion et al., 2010); in other words, there is a delayed sensitization of the startle response (Beck et al., 2002). As shown in Figure 1, enhanced startle reactivity may then be observed up to several days later (Servatius et al., 1995; Beck et al., 2002; Manion et al., 2007; Manion et al., 2010). The delayed expression of this enhanced defensive response could be due to competing processes that are similarly elicited by inescapable shock. For instance, some female rats exhibit a suppression of the startle response following stressor exposure that is clearly linked to the immune response elicited by shock exposure (Beck & Servatius, 2005; Beck & Servatius, 2006), but this suppression of startle reactivity is only evident under certain ovarian hormone conditions (Beck et al., 2008). Although males have not been shown to exhibit a reduction in startle reactivity, there may be similar acute physiological reactions that negate an immediate increase in startle reactivity. Hence, these inescapable stress models of enhanced startle reactivity are largely based on the concept that PTSD-like hyperarousal is a product of a single, uncontrollable, traumatic event (or period of repeated

Fig. 1. Following 3 days of repeated tailshock exposure (40, 3 s, 2 mA shocks over 2 h ), startle magnitudes are elevated in male Sprague Dawley rats 4 days thereafter [Stress x Day F (4, 776) = 7.6, p < .001]. The delayed expression of this increase has been used as a model of the emergent aspects of PTSD symptom expression. Startle magnitude is represented in

These models of hyperarousal in PTSD fail to account for potential innate differences in reactivity. The diathesis model of anxiety disorders indicates that anxiety symptomology are a combination of innate characteristics that are influenced by life events. Recent prospective research on PTSD symptomology has indicated elevated arousal may be predictive of anxiety symptoms (Guthrie & Bryant, 2005; Pole et al., 2009). Startle reactivity, as one measure, is elevated in children that upon follow-up were diagnosed with clinical anxiety (Merikangas et al., 1999). Thus, pathologic arousal exhibited in PTSD may reflect an innate

arbitrary units (AU), as the amplitude is corrected for by body weight.

trauma over consecutive days).

elevation in arousal, an increase in arousal due to experience, or a combination of innate characteristics that are exacerbated by experience.

In developing our model of anxiety vulnerability, we sought a rodent that reliably exhibited a set of behaviors that are analogous to an identified human vulnerability condition. Behavioral inhibition (or inhibited temperament) is extreme withdrawal in face of novel social and nonsocial situations (Kagan et al., 1987; Kagan et al., 1989; Hirshfeld et al., 1992) and is identified as a risk factor for future symptoms of anxiety disorders (Hirshfeld-Becker et al., 2008; Jovanovic et al., 2010). The Wistar-Kyoto (WKY) rat demonstrates aspects of behavioral inhibition in terms of withdrawal in tests of social interaction (Pardon et al., 2002) and lack of exploration in the open field test (Pare, 1994; Servatius et al., 2008). Hence, we tested WKY rats in a multi-intensity startle procedure that allows for assessment of startle sensitivity (percentage of startles elicited at various acoustic intensities) and startle responsivity (magnitude of responses to the highest intensity). Given startle is a defensivereflex, increased responsivity can be viewed as an increase in general arousal (i.e. more energy used to respond to threat), whereas increased sensitivity can be viewed as an enhancement of vigilance (i.e. greater signal-detection of threats). With no prior manipulations, WKY rats exhibited similar startle sensitivity measures as Sprague Dawley (SD) rats, but exhibited higher startle responsivity (see Figure 2). These temperament and reactivity characteristics are reasonable analogs of the predisposing factors for developing symptoms of anxiety disorders; therefore, we adopted the WKY rat as our anxiety vulnerability model (Servatius et al., 2008; Beck et al., 2010; Ricart et al., 2011a; Ricart et al., 2011b; Jiao et al., 2011; Beck et al., 2011).

Fig. 2. Box plots of initial startle responsivity and latency of male SD and WKY rats (left) and female SD and WKY rats (right). Notched regions depict median ± 95% confidence interval. Boxed region depicts interquartile range (middle fifty). Whiskers depict range of data 1.5\*interquartile range. Outliers depicted by '\*'. WKY rats exhibit innate differences in ASRs, with greater amplitudes.

Acquisition of Active Avoidance Behavior as a Precursor

that bias their behavioral strategies to such an extreme.

**2.2 Avoidance susceptibility as a model of anxiety vulnerability** 

As mentioned above, it is well documented that approximately 10% of those people who experience a significant trauma develop PTSD; therefore, there has been recent interest in

to Changes in General Arousal in an Animal Model of PTSD 77

strategy for the animal, it does not allow for the individual to be sensitive to contingency changes (i.e. when the warning signal no longer reliably predicts the noxious stimulus). At that point, the avoidance responses are being emitted to remove a possible (not probable) threat. Therefore, the animal may be expending energy, by moving to the lever and subsequently depressing it, trial after trial to avoid a threat that the warning signal no longer reliably predicts. For individuals with severe anxiety disorders, this strategy of avoiding possible threats can become very disruptive if 1) the individuals expend more energy to avoid situations than what would be required to actually deal with them and 2) the perceived warning signals become generalized, which narrows their ability to interact with the world. Therefore, identifying an animal model that will acquire an exceptionally high asymptotic level of avoidant behavior, and subsequently exhibits the predictable slow extinction of the response, can provide us with a valuable system for identifying the vulnerability factors that predict such avoidant behaviors as well as the neural mechanisms

There are various forms of active avoidance that can be modeled in rats, but the desire to track the development of increased avoidant behavior over time led us to adopt distinct lever-press avoidance as our active avoidance procedure. Lever-press avoidance has been utilized for decades to study learning, but it also has a history as a prominent model of anxiety (Pearl, 1963; D'Amato & Fazzaro, 1966; Hurwitz & Dillow, 1968; Gilbert, 1971; Dillow et al., 1972; Berger & Brush, 1975). Derived initially from the 2-factor theory of threat/fear motivation and learned avoidance (Mowrer, 1939a; Mowrer, 1939b; Mowrer & Lamoreaux, 1942; Mowrer & Lamoreaux, 1946), the general premise of this approach is that a learned fear of signals is sufficient to support avoidant behavior without requiring a continued re-exposure to the actual noxious stimulus or event. Others have provided alternative interpretations of the development of active avoidance learning. Herrnstein, Hineline, and Sidman all focused on the reduction in shock density over time and a second internal factor (e.g fear or anxiety) need not be required in order to explain the acquisition of avoidance behavior (Sidman, 1962; Herrnstein & Hineline, 1966; Hineline & Herrnstein, 1970). This is an important consideration, for without the theoretical need for an internal state, there is no reason to assume a general state of arousal should be evident in the absence of shock exposure. In short, once asymptotic performance is attained, because of the adaptation of the instrumental response to minimize shock frequency, general arousal should be reduced compared to early acquisition (when shocks are more frequent). Still, others have suggested that there may be another component to this acquired behavior – the attainment of perceived safety (Dinsmoor, 1977; Dinsmoor, 2001). This is an interesting proposition because it also does not require any rumination upon the animal's part to "know" the shock is coming. In this approach, the animal learns to exhibit the behavior because it leads to the attainment of perceived safety, which could be in the form of an explicit stimulus only present during periods of non-threat or simply as the absence of the warning signal. At the foundation of this theoretical discussion is a fundamental difference in the view of how animals perceive learning: a molecular (trial by trial, stimulus by stimulus) or molar (general state) analysis (Hineline, 2001; Bersh, 2001). One could argue that lingering changes in general arousal outside of the avoidance learning context may reflect overall changes in the animals that would be proposed by molar analysis theory.
