A fear conditioned cue orchestrates a suite of behaviors in rats.

Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior theoretically linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that - in addition to jumping, and rearing - is theoretically linked to imminent or occurring predation. A criticism of studies observing fear conditioned cue-elicited locomotion is that responding is non-associative. We gave rats Pavlovian fear discrimination over a baseline of reward seeking. TTL-triggered cameras captured 5 behavior frames/s around cue presentation. Experiment 1 examined the emergence of danger-specific behaviors over fear acquisition. Experiment 2 examined the expression of danger-specific behaviors in fear extinction. In total, we scored 112,000 frames for nine discrete behavior categories. Temporal ethograms show that during acquisition, a fear conditioned cue suppresses reward seeking and elicits freezing, but also elicits locomotion, jumping, and rearing - all of which are maximal when foot shock is imminent. During extinction, a fear conditioned cue most prominently suppresses reward seeking, and elicits locomotion that is timed to shock delivery. The independent expression of these behaviors in both experiments reveals a fear conditioned cue to orchestrate a temporally organized suite of behaviors.

Knowing that an animal is fearful is crucial for many psychology and neuroscience studies. For instance, this knowledge allows researchers to examine the brain pathways involved in processing and responding to fear. Typically, researchers consider that a rodent is experiencing fear if it 'freezes' ­ a response which, in the wild, helps to evade detection by predators. In Pavlovian fear conditioning experiments, for example, rats and mice freeze when exposed to a stimulus (often a specific sound) previously associated with unpleasant sensations. However, rodents can also respond more actively to threats, for instance by running or jumping away. It remains unclear whether the 'fearful stimuli' used in Pavlovian approaches specifically elicits only freezing, or other fear-related behaviors as well. To investigate this, Chu et al. used high-speed cameras to record rats' responses to a sound cue they had 'learned' to associate with a mild foot shock. In addition to freezing, the animals ran, jumped, stood on their hind legs and stopped their usual reward-seeking behavior in response to the cue. Crucially, these reactions were absent when the rats were exposed to sound cues not associated with pain. Overall, these experiments demonstrate that Pavlovian conditioning can elicit a full range of fear-related behaviors beyond freezing. Understanding the neural activity behind these diverse responses could lead to more targeted therapies and interventions addressing the various ways stress and anxiety manifest in people.

Pavlovian Fear Conditioning Is More than You Think It Is.

A common neuroscience application of Pavlovian fear conditioning is to manipulate neuron-type activity, pair a cue with foot shock, then measure cue-elicited freezing in a novel context. If the manipulation reduces freezing, the neuron type is implicated in Pavlovian fear conditioning. This application reduces Pavlovian fear conditioning to a single concept. In this Viewpoint, I describe experiments supporting the view that Pavlovian fear conditioning refers to three distinct concepts: procedure, process, and behavior. An experimenter controls procedure, observes behavior, but infers process. Distinguishing these concepts is essential because: (1) a shock-paired cue can engage numerous processes and behaviors; (2) experimenter decisions about procedure influence the processes engaged and behaviors elicited; and (3) many processes are latent, imbuing the cue with properties that only manifest outside of the original conditioning setting. This means we could understand the complete neural basis of freezing, yet know little about the neural basis of fear. Neuroscientists can choose to use a variety of procedures to study a diversity of processes and behaviors. Manipulating neuron-type activity in multiple procedures can reveal specific, general, or complex neuron-type contributions to cue-elicited processes and behaviors. The results will be a broader and more detailed neural basis of fear with greater relevance to the spectrum of symptoms defining anxiety and stressor-related disorders.

Optogenetic inhibition of the caudal substantia nigra inflates behavioral responding to uncertain threat and safety.

Defensive responding is adaptive when it approximates the current threat but maladaptive when it exceeds the current threat. Here we asked if the substantia nigra, a region consistently implicated in reward, is necessary to show appropriate levels of defensive responding in Pavlovian fear discrimination. Rats received bilateral transduction of the caudal substantia nigra with halorhodopsin or a control fluorophore and bilateral ferrule implants. Rats then behaviorally discriminated cues predicting unique foot shock probabilities (danger, p = 1; uncertainty, p = .25; and safety, p = 0). Green-light illumination (532 nm) during cue presentation inflated defensive responding of halorhodopsin rats-measured by suppression of reward seeking-to uncertainty and safety beyond control levels. Green-light illumination outside of cue presentation had no impact on halorhodopsin or control rat responding. The results reveal caudal substantia nigra cue activity is necessary to inhibit defensive responding to nonthreatening and uncertain threat cues. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Timing of behavioral responding to long-duration Pavlovian fear conditioned cues.

Behavioral responding is most beneficial when it reflects event timing. Compared to reward, there are fewer studies on timing of defensive responding. We gave female and male rats Pavlovian fear conditioning over a baseline of reward seeking. Two 100-s cues predicted foot shock at different time points. Rats acquired timing of behavioral responding to both cues. Suppression of reward seeking was minimal at cue onset and maximal before shock delivery. Rats also came to minimize suppres-sion of reward seeking following cue offset. The results reveal timing as a mechanism to focus defen-sive responding to shock-imminent, cue periods.

Optogenetic inhibition of the caudal substantia nigra inflates behavioral responding to uncertain threat and safety.

Defensive responding is adaptive when it approximates current threat, but maladaptive when it exceeds current threat. Here we asked if the substantia nigra, a region consistently implicated in reward, is necessary to show appropriate levels of defensive responding in Pavlovian fear discrimination. Rats received bilateral transduction of the caudal substantia nigra with halorhodopsin or a control fluorophore, and bilateral ferrule implants. Rats then behaviorally discriminated cues predicting unique foot shock probabilities (danger, p =1; uncertainty, p =0.25; and safety, p =0). Green-light illumination (532 nm) during cue presentation inflated defensive responding of halorhodopsin rats - measured by suppression of reward seeking - to uncertainty and safety beyond control levels. Green-light illumination outside of cue presentation had no impact on halorhodopsin or control rat responding. The results reveal caudal substantia nigra cue activity is necessary to inhibit defensive responding to non-threatening and uncertain threat cues.

Brainstem networks construct threat probability and prediction error from neuronal building blocks.

When faced with potential threat we must estimate its probability, respond advantageously, and leverage experience to update future estimates. Threat estimation is the proposed domain of the forebrain, while behaviour is elicited by the brainstem. Yet, the brainstem is also a source of prediction error, a learning signal to acquire and update threat estimates. Neuropixels probes allowed us to record single-unit activity across a 21-region brainstem axis in rats receiving probabilistic fear discrimination with foot shock outcome. Against a backdrop of diffuse behaviour signaling, a brainstem network with a dorsal hub signaled threat probability. Neuronal function remapping during the outcome period gave rise to brainstem networks signaling prediction error and shock on multiple timescales. The results reveal brainstem networks construct threat probability, behaviour, and prediction error signals from neuronal building blocks.

Dorsal Raphe 5-HT Neurons Utilize, But Do Not Generate, Negative Aversive Prediction Errors.

The dorsal raphe nucleus (DRN) contains the largest population of serotonin (5-HT) neurons in the central nervous system. 5-HT, synthesized via tryptophan hydroxylase 2 (Tph2), is a widely functioning neuromodulator implicated in fear learning. Here, we sought to investigate whether DRN 5-HT is necessary to reduce fear via negative prediction error (-PE). Using male and female TPH2-cre rats, DRNtph2+ cells were selectively deleted via cre-caspase (rAAV5-Flex-taCasp3-TEVp) in experiment 1. Rats then underwent fear discrimination during which three cues were associated with unique foot shock probabilities: safety p = 0.00, uncertainty p = 0.375, and danger p = 1.00. Rats then received selective extinction to the uncertainty cue, a behavioral manipulation designed to probe -PE. Deleting DRNtph2+ cells had no impact on initial discrimination but slowed selective extinction. In experiment 2, we used a within-subjects optogenetic inhibition design to causally implicate DRNtph2+ cells in prediction error signaling. Male and female TPH2-cre rats received intra-DRN infusions of cre-dependent halorhodopsin (rAAV5-Ef1a-DIO-eNpHR3.0-eYFP) or cre-YFP. DRNtph2+ cells were inhibited specifically during the time of prediction error or a control period. Illumination during either positive prediction error (+PE) or control periods had no impact on fear to the uncertainty cue. Inhibition of DRNtph2+ cells at the time of -PE did not impact immediate fear, but facilitated selective extinction in postillumination sessions. Together, these results demonstrate a role for DRNtph2+ cells in using, but not generating, -PE to weaken cue-shock associations.

Threat and Bidirectional Valence Signaling in the Nucleus Accumbens Core.

Appropriate responding to threat and reward is essential to survival. The nucleus accumbens core (NAcc) is known to support and organize reward behavior. The NAcc is also necessary to fully discriminate threat and safety cues. To directly reveal NAcc threat firing, we recorded single-unit activity from seven female rats undergoing pavlovian fear discrimination. Rats fully discriminated danger, uncertainty, and safety cues, and most NAcc neurons showed the greatest firing change to danger and uncertainty. Heterogeneity in cue and reward firing led us to identify distinct functional populations. One NAcc population signaled threat, specifically decreasing firing to danger and uncertainty cues. A separate population signaled Bidirectional Valence, decreasing firing to the danger cue (negative valence), but increasing firing to reward (positive valence). The results reveal the NAcc to be a source of threat information and a more general valence hub.SIGNIFICANCE STATEMENT The nucleus accumbens core (NAcc) is synonymous with reward. Yet, anatomy, neurotoxic lesions, and optogenetic manipulation implicate the NAcc in threat. Here, we directly revealed NAcc threat firing by recording single-unit activity during multicue fear discrimination. Most cue-responsive NAcc neurons markedly altered firing to threat cues. Finer analyses revealed a NAcc population signaling threat, specifically decreasing firing to danger and uncertainty cues; and a NAcc population signaling Bidirectional Valence, increasing firing to reward but decreasing firing to the danger cue. The results reveal the NAcc to be a source of threat information and a valence hub.

Decision making: Serotonin goes for goal.

Decision making is adaptive when our actions align with our goals. A new study shows that activity of dorsal raphe serotonin neurons is essential to adaptive decision making, permitting actions to reflect the current goal value.

Retrorubral field is a hub for diverse threat and aversive outcome signals.

Adaptive fear scales to the degree of threat and requires diverse neural signals for threat and aversive outcome. We propose that the retrorubral field (RRF), a midbrain region containing A8 dopamine, is a neural origin of such signals. To reveal these signals, we recorded RRF single-unit activity while male rats discriminated danger, uncertainty, and safety. Many RRF neurons showed firing extremes to danger and safety that framed intermediate firing to uncertainty. The remaining neurons showed unique, threat-selective cue firing patterns. Diversity in firing direction, magnitude, and temporal characteristics led to the detection of at least eight functional neuron types. Neuron types defined with respect to threat showed unique firing patterns following aversive outcome. The result was RRF signals for foot shock receipt, positive prediction error, anti-positive prediction error, persistent safety, and persistent threat. The diversity of threat and aversive outcome signals points to a key role for the RRF in adaptive fear.

Neural substrates of appetitive and aversive prediction error.

Prediction error, defined by the discrepancy between real and expected outcomes, lies at the core of associative learning. Behavioural investigations have provided evidence that prediction error up- and down-regulates associative relationships, and allocates attention to stimuli to enable learning. These behavioural advances have recently been followed by investigations into the neurobiological substrates of prediction error. In the present paper, we review neuroscience data obtained using causal and recording neural methods from a variety of key behavioural designs. We explore the neurobiology of both appetitive (reward) and aversive (fear) prediction error with a focus on the mesolimbic dopamine system, the amygdala, ventrolateral periaqueductal gray, hippocampus, cortex and locus coeruleus noradrenaline. New questions and avenues for research are considered.

Ventral pallidum neurons dynamically signal relative threat.

The ventral pallidum (VP) is anatomically poised to contribute to threat behavior. Recent studies report a VP population that scales firing increases to reward but decreases firing to aversive cues. Here, we tested whether firing decreases in VP neurons serve as a neural signal for relative threat. Single-unit activity was recorded while male rats discriminated cues predicting unique foot shock probabilities. Rats' behavior and VP single-unit firing discriminated danger, uncertainty, and safety cues. Two populations of VP neurons dynamically signaled relative threat, decreasing firing according to foot shock probability during early cue presentation, but disproportionately decreasing firing to uncertain threat as foot shock drew near. One relative threat population increased firing to reward, consistent with a bi-directional signal for general value. The second population was unresponsive to reward, revealing a specific signal for relative threat. The results reinforce anatomy to reveal the VP as a neural source of a dynamic, relative threat signal.

Early adolescent adversity alters periaqueductal gray/dorsal raphe threat responding in adult female rats.

Early adolescent adversity increases adult risk for anxiety disorders. The ventrolateral periaqueductal gray (vlPAG) and neighboring dorsal raphe (DR) are integral to threat prediction, and are responsive to acute stressors. Here, we tested the hypothesis that early adolescent adversity reshapes vlPAG/DR threat-related cue activity and threat probability signaling. Female, Long Evans rats experienced a battery of adverse adolescent experiences (n = 12), while controls did not (n = 8). Single-unit activity was recorded 50 + days following the final adverse experience, when the adult rats received fear discrimination consisting of danger, uncertainty and safety cues. Despite achieving fear discrimination that was equivalent to controls, vlPAG/DR threat responding was altered in adverse-experienced rats. Early adolescent adversity resulted in a greater proportion of cue-responsive neurons. Cue-excited neurons showed greater increases in firing and cue-inhibited neurons showed greater decreases. Even more, early adversity reduced flexible, threat probability signaling by cue-excited neurons and promoted more rigid, fear output signaling by cue-inhibited neurons. The results reveal long-lasting changes in vlPAG/DR threat responding resulting from early adolescent adversity.

The Nucleus Accumbens Core is Necessary to Scale Fear to Degree of Threat.

Fear is adaptive when the level of the response rapidly scales to degree of threat. Using a discrimination procedure consisting of danger, uncertainty, and safety cues, we have found rapid fear scaling (within 2 s of cue presentation) in male rats. Here, we examined a possible role for the nucleus accumbens core (NAcc) in the acquisition and expression of fear scaling. In experiment 1, male Long-Evans rats received bilateral sham or neurotoxic NAcc lesions, recovered, and underwent fear discrimination. NAcc-lesioned rats were generally impaired in scaling fear to degree of threat, and specifically impaired in rapid uncertainty-safety discrimination. In experiment 2, male Long-Evans rats received NAcc transduction with halorhodopsin (Halo) or a control fluorophore. After fear scaling was established, the NAcc was illuminated during cue or control periods. NAcc-Halo rats receiving cue illumination were specifically impaired in rapid uncertainty-safety discrimination. The results reveal a general role for the NAcc in scaling fear to degree of threat, and a specific role in rapid discrimination of uncertain threat and safety.SIGNIFICANCE STATEMENT Rapidly discriminating cues for threat and safety is essential for survival and impaired threat-safety discrimination is a hallmark of stress and anxiety disorders. In two experiments, we induced nucleus accumbens core (NAcc) dysfunction in rats receiving fear discrimination consisting of cues for danger, uncertainty, and safety. Permanent NAcc dysfunction, via neurotoxic lesion, generally disrupted the ability to scale fear to degree of threat, and specifically impaired one component of scaling: rapid discrimination of uncertain threat and safety. Reversible NAcc dysfunction, via optogenetic inhibition, specifically impaired rapid discrimination of uncertain threat and safety. The results reveal that the NAcc is essential to scale fear to degree of threat, and is a plausible source of dysfunction in stress and anxiety disorders.

The ventrolateral periaqueductal grey updates fear via positive prediction error.

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.

Cue-inhibited ventrolateral periaqueductal gray neurons signal fear output and threat probability in male rats.

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.

Ventrolateral periaqueductal gray neurons prioritize threat probability over fear output.

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.

Lateral orbitofrontal cortex partitions mechanisms for fear regulation and alcohol consumption.

Anxiety disorders and alcohol use disorder are highly comorbid, yet identifying neural dysfunction driving comorbidity has been challenging. Lateral orbitofrontal cortex (lOFC) dysfunction has been independently observed in each disorder. Here we tested the hypothesis that the lOFC is essential to partition mechanisms for fear regulation and alcohol consumption. Specifically, the capacity to regulate fear and the propensity to consume alcohol are unrelated when lOFC is intact, but become linked through lOFC dysfunction. Male Long Evans rats received bilateral, neurotoxic lOFC lesions or sham surgery. Fear regulation was determined by establishing discrimination to danger, uncertainty, and safety cues then shifting the shock probability of the uncertainty cue. Alcohol consumption was assessed through voluntary, intermittent access to 20% ethanol. The neurotoxic lesion approach ensured lOFC dysfunction spanned testing in fear regulation and alcohol consumption. LOFC-lesioned rats demonstrated maladaptive fear generalization during probability shifts, inverting normal prediction error assignment, and subsequently consumed more alcohol. Most novel, fear regulation and alcohol consumption were inextricably linked only in lOFC-lesioned rats: extreme fear regulation predicted excessive alcohol consumption. The results reveal the lOFC is essential to partition mechanisms for fear regulation and alcohol consumption and uncover a plausible source of neural dysfunction contributing to comorbid anxiety disorders and alcohol use disorder.

Early adolescent adversity inflates threat estimation in females and promotes alcohol use initiation in both sexes.

Childhood adversity is associated with exaggerated threat processing and earlier alcohol use initiation. Conclusive links remain elusive, as childhood adversity typically co-occurs with detrimental socioeconomic factors, and its impact is likely moderated by biological sex. To unravel the complex relationships among childhood adversity, sex, threat estimation, and alcohol use initiation, we exposed female and male Long-Evans rats to early adolescent adversity (EAA). In adulthood, >50 days following the last adverse experience, threat estimation was assessed using a novel fear discrimination procedure in which cues predict a unique probability of footshock: danger (p = 1.00), uncertainty (p = .25), and safety (p = .00). Alcohol use initiation was assessed using voluntary access to 20% ethanol, >90 days following the last adverse experience. During development, EAA slowed body weight gain in both females and males. In adulthood, EAA selectively inflated female threat estimation, exaggerating fear to uncertainty and safety, but promoted alcohol use initiation across sexes. Meaningful relationships between threat estimation and alcohol use initiation were not observed, underscoring the independent effects of EAA. Results isolate the contribution of EAA to adult threat estimation, alcohol use initiation, and reveal moderation by biological sex. (PsycINFO Database Record

Adolescent Alcohol Drinking Renders Adult Drinking BLA-Dependent: BLA Hyper-Activity as Contributor to Comorbid Alcohol Use Disorder and Anxiety Disorders.

Adolescent alcohol drinking increases the risk for alcohol-use disorder in adulthood. Yet, the changes in adult neural function resulting from adolescent alcohol drinking remain poorly understood. We hypothesized that adolescent alcohol drinking alters basolateral amygdala (BLA) function, making alcohol drinking BLA-dependent in adulthood. Male, Long Evans rats were given voluntary, intermittent access to alcohol (20% ethanol) or a bitter, isocaloric control solution, across adolescence. Half of the rats in each group received neurotoxic BLA lesions. In adulthood, all rats were given voluntary, intermittent access to alcohol. BLA lesions reduced adult alcohol drinking in rats receiving adolescent access to alcohol, but not in rats receiving adolescent access to the control solution. The effect of the BLA lesion was most apparent in high alcohol drinking adolescent rats. The BLA is essential for fear learning and is hyper-active in anxiety disorders. The results are consistent with adolescent heavy alcohol drinking inducing BLA hyper-activity, providing a neural mechanism for comorbid alcohol use disorder and anxiety disorders.