Faced with potential harm, individuals must estimate the probability of threat and initiate an appropriate fear response. In the prevailing view, threat probability estimates are relayed to the ventrolateral periaqueductal gray (vlPAG) to organize fear output. A straightforward prediction is that vlPAG single-unit activity reflects fear output, invariant of threat probability. We recorded vlPAG single-unit activity in male, Long Evans rats undergoing fear discrimination. Three 10 s auditory cues predicted unique foot shock probabilities: danger (p=1.00), uncertainty (p=0.375) and safety (p=0.00). Fear output was measured by suppression of reward seeking over the entire cue and in one-second cue intervals. Cued fear non-linearly scaled to threat probability and cue-responsive vlPAG single-units scaled their firing on one of two timescales: at onset or ramping toward shock delivery. VlPAG onset activity reflected threat probability, invariant of fear output, while ramping activity reflected both signals with threat probability prioritized.

Aversive, positive prediction error (+PE) provides a mechanism to update and increase future fear to uncertain threat predictors. The ventrolateral periaqueductal grey (vlPAG) has been offered as a neural locus for +PE computation. Yet, a causal demonstration of vlPAG +PE activity to update fear remains elusive. We devised a fear discrimination procedure in which a danger cue predicts shock deterministically and an uncertainty cue predicts shock probabilistically, requiring prediction errors to achieve an appropriate fear response. Recording vlPAG single‐unit activity during fear discrimination in Long‐Evans rats, we reveal activity related to shock is consistent with +PE and updates subsequent fear to uncertainty at the trial level. We further demonstrate that vlPAG inhibition during shock selectively decreases future fear to uncertainty, but not danger, and temporal emergence of this effect is consistent with single‐unit activity. These findings provide causal evidence that vlPAG +PE is necessary for fear updating.

Early life adversity increases anxiety in adult rodents and primates, and increases the risk for developing post-traumatic disorder (PTSD) in humans. We hypothesized that early adversity impairs the use of learning signals -negative, aversive prediction errors–to reduce fear in uncertainty. To test this hypothesis, we gave adolescent rats a battery of adverse experiences then assessed adult performance in probabilistic Pavlovian fear conditioning and fear extinction. Rats were confronted with three cues associated with different probabilities of foot shock: one cue never predicted shock, another cue predicted shock with uncertainty, and a final cue always predicted shock. Control rats initially acquired fear to all cues, but rapidly reduced fear to the non-predictive and uncertain cues. Early adversity rats were slower to reduce fear to the non-predictive cue and never fully reduced fear to the uncertain cue. In extinction, all cues were presented in the absence of shock. Fear to the uncertain cue in discrimination, but not early adversity itself, predicted the reduction of fear in extinction. These results demonstrate early adversity impairs the use of negative aversive prediction errors to reduce fear, especially in situations of uncertainty.

Discriminating safety from danger must be accurate and rapid. Yet, the rapidity with which fear discrimination emerges remains unknown. Rapid fear discrimination in adulthood may be susceptible to impairment by adolescent heavy alcohol drinking, which increases incidence of anxiety disorders. Rats were given voluntary, adolescent alcohol access, and heavy drinkers were identified. In adulthood, rapid fear discrimination of safety, uncertainty, and danger cues was assessed. Normal rats, but not heavy drinkers, showed discriminative fear <1 sec following cue onset. This provides the first demonstration of subsecond fear discrimination and its adult impairment in adolescent heavy alcohol drinkers.

The ventrolateral periaqueductal gray (vlPAG) is proposed to mediate fear responses to imminent danger. Previously we reported that vlPAG neurons showing short-latency increases in firing to a danger cue – the presumed neural substrate for fear output – signal threat probability in male rats (Wright et al., 2019). Here, we scrutinize the activity vlPAG neurons that decrease firing to danger. One cue-inhibited population flipped danger activity from early inhibition to late excitation: a poor neural substrate for fear output, but a better substrate for threat timing. A second population showed differential firing with greatest inhibition to danger, less to uncertainty and no inhibition to safety. The pattern of differential firing reflected the pattern of fear output, and was observed throughout cue presentation. The results reveal an expected vlPAG signal for fear output in an unexpected, cue-inhibited population.