Pavlovian fear conditioning does not readily occur in rats in naturalistic environments

Pavlovian fear conditioning, which offers the advantage of simplicity in both the control of conditioned and unconditioned stimuli (CS, US) presentation and the analysis of specific conditioned and unconditioned responses (CR, UR) in a controlled laboratory setting, has been the standard model in basic and translational fear research. Despite 100 years of experiments, the utility of fear conditioning has not been trans-situationally validated in real-life contexts. We thus investigated whether fear conditioning readily occurs and guides the animal’s future behavior in an ecologically-relevant environment. To do so, Long-Evans rats foraging for food in an open arena were presented with a tone CS paired with electric shock US to their dorsal neck/body that instinctively elicited escape UR to the safe nest. On subsequent test days, the tone-shock paired animals failed to exhibit fear CR to the CS. In contrast, animals that encountered a realistic agent of danger (a looming artificial owl) paired with a shock, simulating a realistic predatory strike, instantly fled to the nest when presented with a tone for the first time. These results illustrate the survival function and precedence of a nonassociative process, rather than associative conditioning, in life-threatening situations that animals are likely to encounter in nature.


44
Since the time of Watson and Morgan's (1) conception that emotions, such as fear, should be studied 45 as conditioned (acquired) reactions and Watson and Rayner's (2) demonstration that fear can be rapidly 46 learned in 9-month-old "Little Albert," Pavlovian (or classical) fear conditioning has been the paradigm par excellence for studying both normal and abnormal fear behaviors (3-7). Briefly, fear conditioning focuses 48 on how an initially innocuous conditioned stimulus (CS; e.g., auditory, visual, contextual cues), upon 49 pairing with a noxious unconditioned stimulus (US; usually electric shock) that reflexively elicits 50 unconditioned response (UR; namely defensive reactions), becomes capable of eliciting conditioned 51 response (CR; e.g., freezing in rodents, increased skin conductance in humans). A century of fear 52 conditioning research has led to wide-ranging discoveries. In particular, fear conditioning experiments 53 have fundamentally transformed learning theories from the archaic contiguity (or temporal) relationship 54 (8-10) to the modern contingency (or informational) relationship between the CS and US (11-14), 55 revealed detailed neurobiological mechanisms of learning and memory (15-17) and influenced 56 contemporary cognitive behavioral therapy for various anxiety and traumatic-stressor related disorders, 57 such as panic, phobic and posttraumatic stress disorders (18)(19)(20)(21)(22).

58
Despite its utility and appeal, fear conditioning paradigms nonetheless simplify behavioral analyses of 59 fear, ignoring the multitude of actions and decisions that animals and humans utilize to survive the 60 breadth of risky situations in the real world (23-28). Moreover, the prevalent notion that fear conditioning 61 produces adaptive associative fear memory has yet to be ecologically validated. In fact, some 62 researchers have questioned the evolutionary logic underlying fear conditioning; "No owl hoots or whistles 63 5 seconds before pouncing on a mouse…Nor will the owl give the mouse enough trials for the necessary 64 learning to occur…What keeps animals alive in the wild is that they have very effective innate defensive 65 reactions which occur when they encounter any kind of new or sudden stimulus" (29). Indeed, laboratory 66 rodents exhibit unlearned, instinctive fear responses to advancing artificial terrestrial and aerial predators 67 (30,31), overhead looming stimuli (32), and predator odors (33).

68
Here, we investigated for the first time whether fear conditioning readily transpires and modifies 69 subsequent behavior of animals in a naturalistic environment. To achieve this, hunger-motivated rats 70 4 searching for a food pellet in a large arena-a purposive behavior (34)-were presented with a discrete 71 tone CS followed by a painful US to their dorsal neck/body region by means of chronically implanted 72 subcutaneous wires (Fig. 1A). A dorsal neck/body shock better simulates real predatory strike compared 73 to footshock used in standard fear conditioning studies, as it is unlikely that predators direct their attacks 74 on small prey animal's paws. Additionally, in nature, bodily injuries are normally inflicted by external 75 agents (namely, predators in animals and perpetrators in humans). Thus, other groups of rats were 76 presented with a looming aerial predator (i.e., a lifelike great horned owl) preceded with and without a 77 tone CS and followed by the same US . A single trial tone-shock, tone-owl, tone/owl-shock and

84
Female and male rats were pseudo-randomly assigned to tone-shock (8 females, 8 males), owl-85 shock (8 females, 8 males), tone/owl-shock (6 females, 8 males), and tone-owl (4 females, 4 males) 86 groups and implanted with subcutaneous wires in their dorsal neck/body . After recovery 87 from the surgery, the rats were trained to exit a nest compartment upon gate opening to procure a 88 sizable 0.5 g food pellet placed at variable distances in a large, expanding open arena (Fig. 1D,top

99
On the training day, all rats first underwent three foraging trials with pellets fixed at the longest 100 distance (125 cm) to confirm comparable pre-fear conditioning foraging behavior between groups (Fig.

101
2A, Baseline). Afterwards, animals were exposed to a tone-shock, an owl-shock, a tone/owl-shock or a 102 tone-owl pairing in the manner shown in Fig. 1 (Supplementary materials, Movie S1). Those rats 103 presented with the tone CS 5-sec prior to the gate opening (i.e., tone-shock, tone-owl, tone/owl-shock 104 groups) took more time to enter the foraging arena in comparisons to owl-shock animals unexposed to 105 the tone (Fig. 2B, Leave nest latency); this indicates that the tone was a salient cue that animals were 106 attentive to and thus conditionable. Once in the foraging arena, all animals readily advanced toward the 107 pellet and breached the trigger zone (25 cm from the pellet) to activate the shock, owl, or owl-shock 108 stimuli (Fig. 2B,Trigger zone latency). In response to the shock, owl, or owl-shock, all rats promptly fled 109 from the foraging arena to the nest (Fig. 2B, Escape latency; Fig. 2D,E, Escape speed). Figure 2C shows 110 representative track plot examples of tone-shock, owl-shock, tone/owl-shock and tone-owl animals 111 successfully procuring the pellet during pre-tone baseline but not during tone conditioning. The fact that 112 the escape latency and running speed were not significantly different between the tone-owl and other 113 groups indicates that the looming owl-induced innate fear sans pain was just as effective in eliciting the 114 flight UR as the painful shock or shock-owl combination. However, inspections of the escape trajectories 115 revealed that the tone-shock and tone-owl groups tended to flee linearly to the nest, whereas the owl-116 shock and tone/owl-shock groups that experienced a dorsal neck/body shock 100 ms after the looming 117 owl (mimicking realistic predatory attack) and begun their flight to the nest inclined to escape circuitously 118 (Fig. 2F,H). This was supported by significant group differences in the escape distances (Fig. 2G) and 119 trajectory angles (Fig. 2I), where owl-shock and tone/owl-shock groups traveled longer distances and had 120 higher angle variances, respectively, during their escape routes than tone-shock and tone-owl groups.

122
Context (pre-tone) testing 123 On the following day, animals were placed back in the nest and underwent three pre-tone baseline 124 trials (maximum 300 sec to retrieve the food pellet placed at 125 cm) to assess whether previous 125 encounters with tone-shock, owl-shock, tone/owl-shock and tone-owl stimuli combinations produced fear 6 of the arena. As can be seen in Figure 3A, the owl-shock and tone/owl-shock groups took significantly 127 longer latencies to procure the pellet (i.e., the time from gate opening-to-return to nest with the pellet) 128 than the tone-shock and tone-owl groups on the first day of testing. The lengthened times to enter the 129 foraging arena exhibited by owl-shock and tone/owl-shock rats likely reflect inhibitory avoidance resulting 130 from the previous predatory attack experience in the arena (35). In contrast, the fact that the pre-tone test

137
Tone testing 138 Immediately after the pre-tone baseline, all groups were subjected to three successive tone test trials 139 (one minute apart). The owl-shock and tone/owl-shock animals continued to take longer latencies to exit 140 the nest compared to tone-shock and tone-owl animals (Fig. 3B, Leave nest latency). Once in the 141 foraging arena, the tone/owl-shock group's latency to approach 25 cm from the pellet to trigger the tone 142 were marginally but reliably longer than those of tone-shock and tone-owl groups, but not owl-shock 143 group (Fig. 3B, Trigger zone latency). Upon the activation of tone (60 s continuous), the majority of owl-144 shock and tone/owl-shock animals promptly fled to the nest, thereby significantly increasing the latency to 145 procure the pellet (60 s = unsuccessful), whereas the tone-shock and tone-owl animals were largely 146 unaffected by the tone and readily procured the pellet (Fig. 3B, Procure pellet latency). The second day of 147 tone testing yielded similar patterns of group differences (Fig. 3D). Figure 3E shows individual track plots 148 from all animals with the initial number of trial(s) necessitated for successful foraging. Further analyses 149 across tone testing days (3 trials/day) showed that the overall success rates of procuring the pellet were 150 significantly lower in owl-shock and tone/owl-shock groups compared to tone-shock and tone-owl groups 151 (Fig. 3F), and that owl-shock and tone/owl-shock animals required extended trials to reliably obtain the 152 pellet (Fig. 3G). Because the temporal interval between the CS and US is well known to be crucial in 153 various types of Pavlovian conditioning, including fear conditioning (36), we examined whether tone fear 7 conditioning transpired in a specific (optimal) range of interstimulus intervals (ISI) but was masked by 155 non-optimal ISIs. We found no significant correlation between the ISIs and the magnitudes of tone-156 induced suppression of pellet procurement in tone-shock animals, indicating that tone fear conditioning 157 failed to materialize across varying ISIs of delay conditioning (Fig. 3H). Conversely, in the tone/owl-shock 158 animals, the tone-induced suppression of pellet procurement was uniformly observed across different 159 ISIs, suggesting that the observed fear in these animals may not necessarily reflect Pavlovian 160 conditioning ( Fig 3H). These results of delayed tone-shock paired animals failing to show conditioned 161 tone fear and contextual fear suggest that standard fear conditioning does not readily occur in naturalistic 162 environment. Instead, the finding of owl-shock animals displaying robust fear to a novel tone, which the 163 animals never heard before, suggests that non-associative sensitization-like processes play a crucial role 164 in protecting animals in the real world.

167
It is generally believed (though never validated) that there is behavioral continuity of Pavlovian fear

180
foraging rats that experienced a looming owl and shock pairing (owl-shock group) later exhibited robust 181 fear (escape) behavior to a novel tone presentation. In the tone/owl-shock animals, the escape behavior 8 was uniformly observed across different ISIs, suggesting that the observed fear to the tone stimulus 183 in this group may not be a Pavlovian response. These findings then point to a nonassociative 184 sensitization (or sensitization-like) process, rather than associative fear conditioning, as playing a vital 185 function in risky (i.e., predatory attack) situations that animals encounter in nature.

186
The tone CS (3 kHz, 80 dB, ranging 9-86.6 s) and shock US (2.5 mA, 1 s) employed in the present 187 study were effective in eliciting orienting and fleeing responses, respectively, and were presented to 188 animals in the manner (i.e., a delay conditioning) that satisfied the stimuli saliency, intensity, surprising,

200
Animals tested in naturalistic paradigms are given choices that do not force their behaviors into 201 dichotomies (i.e., freezing or no freezing; drug craving or no drug craving). Allowing for an expanded 202 behavioral repertoire, while more difficult to study, may thus yield a greater understanding of behaviors 203 and their underlying brain mechanisms.

204
It should also be noted that fear encounters in real life generally occur in the presence of external 205 agents or forms (i.e., predators/conspecifics in animals and assailants/combatants in humans), which is 206 virtually nonexistent in standard Pavlovian fear conditioning paradigms. Thus, the effects of a discernable 207 entity in associative fear learning have never been investigated. By simulating a realistic life-threatening 208 situation, i.e., a looming aerial predator that instinctively elicited flight behavior followed by somatic pain,

209
we found that rats engaged in purposive behavior utilize nonassociative sensitization as their primary 9 defensive mechanism. The fact that the owl-shock and tone/owl-shock animals exhibited relatively 211 nonlinear, erratic escape trajectories to the nest compared to linear escape trajectories in tone-shock 212 animals  suggests the intriguing possibility that the same dorsal neck/body shock US may be 213 interpreted as a life-or-death (panic) situation in the presence of an external threat agent versus a mere 214 startling (nociceptive) situation in the absence of an external threat agent. The erratic flight behavior in the 215 presence of a looming owl may represent the penultimate stage of circa-strike, or "life-or-death," behavior 216 within the "predatory imminence continuum" theory (47). Functionally, a sensitized fear system may 217 intensify avoidance behavior, which in turn effectively transposes novel, neutral cues into "false positives" 218 to prioritize survival in natural environment (29). In other words, nonspecific sensitization-based 219 overestimation of danger may be a more prudent course for survival than relatively more specific 220 association-based prediction of danger.

221
Clark Hull (48) has posited that Pavlovian fear conditioning offers biological utility by circumventing a 222 "bad biological economy" of defense reaction always necessitating injury. This prevailing view that 223 ascribes preeminent importance of fear conditioning as the primary defensive mechanism is likely to be a 224 theoretical simplification and provides an incomplete picture of fear, as its function in a natural 225 environment may be rather limited (i.e., lacks face validity). It may well be possible to produce fear 226 conditioning in naturalistic settings with further CS-US trials but then this too would be a bad biological 227 economy as such learning will dramatically reduce biological fitness. It is also important to recognize  inserted in the dorsal neck/back region of body. The wire tips were exposed (~1 cm), bent to a V-shape, 254 and hooked to subcutaneous tissue (36). The other ends of the wires were affixed to a headstage 255 (Plastics One, MS303-120), which was then cemented to the animal's skull embedded with 6 anchoring 256 screws. While still under anesthesia, animals were connected to a shock-apparatus and given a mild 257 shock to observe muscle twitching; 6 rats that showed no reaction to shock were removed from the 258 experiment. Animals were given 4 days of postoperative recovery and were adapted to handling for 5 259 days before nest habituation.

282
(Baseline days) After 1 minute in the nest sans food pellets, the gate opened, and the animal was 283 allowed to explore the large foraging arena and find a pellet placed 25 cm away from the nest (first trial).

284
As soon as the animal took the sizeable 0.5 g pellet back to the nest, the gate closed. Once the animal 285 finished eating, the second trial with the pellet placed 50 cm and then the third trial with the pellet placed 286 75 cm commenced in the same manner. Animals underwent 3-5 consecutive baseline days, with the 287 pellet distances gradually extending to 75, 100 and 125 cm, and they were also accustomed to tethering 288 beginning on baseline day 3 onward.

291
On the 4 th trial, the tone-shock, tone-owl and tone/owl-shock animals were exposed to a tone CS that 292 came on 5 seconds before the gate opened and remained on until they reached the trigger zone (25 cm 293 to the pellet). For tone-shock and tone-owl animals, the tone co-terminated with the shock US and the owl 294 looming, respectively. For tone/owl-shock animals, the shock occurred 0.1s sec after the owl looming and 12 co-terminated with the tone. Two animals in the tone/owl-shock group were excluded because they 296 failed to leave the nest within 2 min. The owl-shock animals were subjected to the same owl looming-297 shock pairing (as the tone/owl-shock animals) but in the absence of tone. All rats fled to the nest in 298 reaction to the shock and/or looming owl, at which time the gate was closed. After 1 minute in the 299 nest, the animals were placed back into their homecage.

300
(Testing days) All rats underwent 3 baseline trials (a maximum of 300 sec to retrieve the pellet) to 301 assess whether shock and/or looming owl encounter the previous day resulted in the fear of the 302 arena (i.e., contextual fear). Afterwards, animals were presented with the tone cue when they 303 approached the trigger zone (25 cm to the pellet). The tone played continuously for 60 sec, after 304 which the tone test trial ended. Animals underwent 3 tone tests daily until they successfully attained 305 the pellet (i.e., fear extinction).