Orchestration of innate and conditioned defensive actions by the periaqueductal gray.

The midbrain periaqueductal gray (PAG) has been recognized for decades as having a central role in the control of a wide variety of defensive responses. Initial discoveries relied primarily on lesions, electrical stimulation and pharmacology. Recent developments in neural activity imaging and in methods to control activity with anatomical and genetic specificity have revealed additional streams of data informing our understanding of PAG function. Here, we discuss both classic and modern studies reporting on how PAG-centered circuits influence innate as well as learned defensive actions in rodents and humans. Though early discoveries emphasized the PAG's role in rapid induction of innate defensive actions, emerging new data indicate a prominent role for the PAG in more complex processes, including representing behavioral states and influencing fear learning and memory. This article is part of the Special Issue on "Fear, Anxiety and PTSD".

Anatomical and functional study of the cuneiform nucleus: A critical site to organize innate defensive behaviors.

The cuneiform nucleus (CUN) is a midbrain structure located lateral to the caudal part of the periaqueductal gray. In the present investigation, we first performed a systematic analysis of the afferent and efferent projections of the CUN using FluoroGold and Phaseolus vulgaris leucoagglutinin as retrograde and anterograde neuronal tracers, respectively. Next, we examined the behavioral responses to optogenetic activation of the CUN and evaluated the impact of pharmacological inactivation of the CUN in both innate and contextual fear responses to a predatory threat (i.e., a live cat). The present hodologic evidence indicates that the CUN might be viewed as a caudal component of the periaqueductal gray. The CUN has strong bidirectional links with the dorsolateral periaqueductal gray (PAGdl). Our hodological findings revealed that the CUN and PAGdl share a similar source of inputs involved in integrating information related to life-threatening events and that the CUN provides particularly strong projections to brain sites influencing antipredatory defensive behaviors. Our functional studies revealed that the CUN mediates innate freezing and flight antipredatory responses but does not seem to influence the acquisition and expression of learned fear responses.

Dissecting the brain's fear systems responding to snake threats.

We examined the behavioural responses and Fos expression pattern of rats that were exposed to snake threats from shed snakeskin and a live snake. We differentiated the behavioural responses and the pattern of Fos expression in response to the odour cues and mild threat from a live snake. Animals exposed to the snake odour alone or to the confined snake showed a great deal of risk assessment. Conversely, the intensification of odour during exposure to the live snake decreased the threat ambiguity, and the animals froze for a significantly longer period. Our Fos analysis showed that a pathway formed by the posteroventral part of the medial amygdalar nucleus to the central part of the ventromedial hypothalamic nucleus appeared to be solely responsive to odour cues. In addition, we showed increased Fos expression in a parallel circuit comprising the lateral amygdalar nucleus, ventral subiculum, lateral septum, and juxtadorsomedial region of the lateral hypothalamic area that is responsive to both the odour and mild threat from a live snake. This path is likely to process the environmental boundaries of the threat to be avoided. Both paths merge into the dorsal premammillary nucleus and periaqueductal grey sites, which all increase Fos expression in response to the snake threats and are likely to organize the defensive responses. Moreover, we found that the snake threat mobilized the Edinger-Westphal and supraoculomotor nuclei, which are involved in stress adaptation and attentional mechanisms.

Why do mice squeak? Toward a better understanding of defensive vocalization.

Although mice mostly communicate in the ultrasonic range, they also emit audible calls. We demonstrate that mice selectively bred for high anxiety-related behavior (HAB) have a high disposition for emitting sonic calls when caught by the tail. The vocalization was unrelated to pain but sensitive to anxiolytics. As revealed by manganese-enhanced MRI, HAB mice displayed an increased tonic activity of the periaqueductal gray (PAG). Selective inhibition of the dorsolateral PAG not only reduced anxiety-like behavior but also completely abolished sonic vocalization. Calls were emitted at a fundamental frequency of 3.8 kHz, which falls into the hearing range of numerous predators. Indeed, playback of sonic vocalization attracted rats if associated with a stimulus mouse. If played back to HAB mice, sonic calls were repellent in the absence of a conspecific but attractive in their presence. Our data demonstrate that sonic vocalization attracts both predators and conspecifics depending on the context.

Neural correlates of distinct levels of predatory threat in dorsal periaqueductal grey neurons.

The dorsal periaqueductal grey (PAG) is an important site for integrating predatory threats. However, it remains unclear whether predator-related activation in PAG primarily reflects threat itself and thus can distinguish between various degrees of threat, or rather reflects threat-oriented behaviours, with the PAG potentially orchestrating different types of defensive repertoire. To address this issue, we performed extracellular recording of dorsal PAG neurons in freely behaving rats and examined neuronal and behavioural responses to stimulus conditions with distinct levels of predatory threat. Animals were sequentially exposed to a nonthreatening stimulus familiar environment (exposure to habituated environment) and to a novel nonthreatening stimulus (i.e., a toy animal-plush) and to conditions with high (exposure to a live cat), intermediate (exposure to the environment just visited by the cat, with remnant predator scent), and low (exposure on the following day to the predatory context) levels of predatory threat. To test for contributions of both threat stimuli and behaviour to changes in firing rate, we applied a Poisson generalized linear model regression, using the different predator stimulus conditions and defensive repertoires as predictor variables. Analysis revealed that the different predator stimulus conditions were more predictive of changes in firing rate (primarily threat-induced increases) than the different defensive repertoires. Thus, the dorsal PAG may code for different levels of predatory threat, more than it directly orchestrates distinct threat-oriented behaviours. The present results open interesting perspectives to investigate the role of the dorsal PAG in mediating primal emotional and cognitive responses to fear-inducing stimuli.

Dissecting the brain’s fear systems responding to snake threats

We examined the behavioral responses and Fos expression pattern of rats that were exposed to snake threats from shed snakeskin and a live snake. We differentiated the behavioral responses and the pattern of Fos expression in response to the odor cues and mild threat from a live snake. Animals exposed to the snake odor alone or to the confined snake showed a great deal of risk assessment. Conversely, the intensification of odor during exposure to the live snake decreased the threat ambiguity, and the animals froze for a significantly longer period. Our Fos analysis showed that a pathway formed by the posteroventral part of the medial amygdalar nucleus to the central part of the ventromedial hypothalamic nucleus appeared to be solely responsive to odor cues. In addition, we showed increased Fos expression in a parallel circuit comprising the lateral amygdalar nucleus, ventral subiculum, lateral septum and juxtadorsomedial region of the lateral hypothalamic area that is responsive to both the odor and mild threat from a live snake. This path is likely to process the environmental boundaries of the threat to be avoided. Both paths merge into the dorsal premammillary nucleus and periaqueductal gray sites, which all increase Fos expression in response to the snake threats and are likely to organize the defensive responses. Moreover, we found that the snake threat mobilized the Edinger-Westphal and supraoculomotor nuclei, which are involved in stress adaptation and attentional mechanisms.

Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats.

Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats.

Orexin 1 and 2 Receptors in the Prelimbic Cortex Modulate Threat Valuation.

The ability to distinguish between threatening (repulsors), neutral and appetitive stimuli (attractors) stimuli is essential for survival. The orexinergic neurons of hypothalamus send projections to the limbic structures, such as different subregions of the medial prefrontal cortex (mPFC), suggesting that the orexinergic mechanism in the prelimbic cortex (PL) is involved in the processing of fear and anxiety. We investigated the role of orexin receptors type 1 (OX1R) and type 2 (OX2R) in the PL in such processes upon confrontation with an erratically moving robo-beetle in mice. The selective blockade of OX1R and OX2R in the PL with SB 334867 (3, 30, 300 nM) and TCS OX2 29 (3, 30, 300 nM), respectively, did not affect general exploratory behavior or reactive fear such as avoidance, jumping or freezing, but significantly enhances tolerance and approach behavior at the highest dose of each antagonist tested (300 nM). We interpret these findings as evidence for an altered cognitive appraisal of the potential threatening stimulus. Consequently, the orexin system seems to bias the perception of stimuli towards danger or threat via OX1R and OX2R in the PL.

Dorsal periaqueductal gray ensembles represent approach and avoidance states.

Animals must balance needs to approach threats for risk assessment and to avoid danger. The dorsal periaqueductal gray (dPAG) controls defensive behaviors, but it is unknown how it represents states associated with threat approach and avoidance. We identified a dPAG threatavoidance ensemble in mice that showed higher activity farther from threats such as the open arms of the elevated plus maze and a predator. These cells were also more active during threat avoidance behaviors such as escape and freezing, even though these behaviors have antagonistic motor output. Conversely, the threat approach ensemble was more active during risk assessment behaviors and near threats. Furthermore, unsupervised methods showed that avoidance/approach states were encoded with shared activity patterns across threats. Lastly, the relative number of cells in each ensemble predicted threat avoidance across mice. Thus, dPAG ensembles dynamically encode threat approach and avoidance states, providing a flexible mechanism to balance risk assessment and danger avoidance.

Shared Dorsal Periaqueductal Gray Activation Patterns during Exposure to Innate and Conditioned Threats.

The brainstem dorsal periaqueductal gray (dPAG) has been widely recognized as being a vital node orchestrating the responses to innate threats. Intriguingly, recent evidence also shows that the dPAG mediates defensive responses to fear conditioned contexts. However, it is unknown whether the dPAG displays independent or shared patterns of activation during exposure to innate and conditioned threats. It is also unclear how dPAG ensembles encode and predict diverse defensive behaviors. To address this question, we used miniaturized microscopes to obtain recordings of the same dPAG ensembles during exposure to a live predator and a fear conditioned context in male mice. dPAG ensembles encoded not only distance to threat, but also relevant features, such as predator speed and angular offset between mouse and threat. Furthermore, dPAG cells accurately encoded numerous defensive behaviors, including freezing, stretch-attend postures, and escape. Encoding of behaviors and of distance to threat occurred independently in dPAG cells. dPAG cells also displayed a shared representation to encode these behaviors and distance to threat across innate and conditioned threats. Last, we also show that escape could be predicted by dPAG activity several seconds in advance. Thus, dPAG activity dynamically tracks key kinematic and behavioral variables during exposure to threats, and exhibits similar patterns of activation during defensive behaviors elicited by innate or conditioned threats. These data indicate that a common pathway may be recruited by the dPAG during exposure to a wide variety of threat modalities.SIGNIFICANCE STATEMENT The dorsal periaqueductal gray (dPAG) is critical to generate defensive behaviors during encounters with threats of multiple modalities. Here we use longitudinal calcium transient recordings of dPAG ensembles in freely moving mice to show that this region uses shared patterns of activity to represent distance to an innate threat (a live predator) and a conditioned threat (a shock grid). We also show that dPAG neural activity can predict diverse defensive behaviors. These data indicate the dPAG uses conserved population-level activity patterns to encode and coordinate defensive behaviors during exposure to both innate and conditioned threats.

Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats.

Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is necessary for context-specific escape induced by moderate threats but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits.

Inactivation of the dorsolateral periaqueductal gray matter impairs the promoting influence of stress on fear memory during retrieval.

Exposure to stressful conditions induces long-lasting neurobiological changes in selected brain areas, which could be associated with the emergence of negative emotional responses. Moreover, the interaction of a stressful experience and the retrieval of an established fear memory trace enhance both fear expression and fear retention. Related to this, the stimulation of the dorsolateral part of the mesencephalic periaqueductal gray matter (dlPAG) prior to retrieval potentiates a fear memory trace previously acquired. Therefore, the question that arises is whether the dlPAG mediates the increased fear expression and fear retention after retrieval. Rats were subjected to a contextual fear conditioning paradigm using a single footshock, and 1 day later, rats were subjected to a stressful situation. As previously reported, there was an increase of freezing response only in those rodents that were re-exposed to the associated context at 1 and 5 days after stress exposure. Muscimol intra-dlPAG prior to the restraint event prevented such increase. Conversely, Muscimol intra-dlPAG infusion immediately after the stress experience had no effect on the resulting fear memory. When the neuroendocrine response to stress was explored, intra-dlPAG infusion of muscimol prior to stress decreased Fos expression in the paraventricular nucleus and serum corticosterone levels. Moreover, this treatment prevented the enhancement of the density of hippocampal "mature" spines associated with fear memory. In conclusion, the present results suggest that the dlPAG is a key neural site for the negative valence instruction necessary to modulate the promoting influence of stress on fear memory.

The periaqueductal gray and primal emotional processing critical to influence complex defensive responses, fear learning and reward seeking.

The periaqueductal gray (PAG) has been commonly recognized as a downstream site in neural networks for the expression of a variety of behaviors and is thought to provide stereotyped responses. However, a growing body of evidence suggests that the PAG may exert more complex modulation of a number of behavioral responses and work as a unique hub supplying primal emotional tone to influence prosencephalic sites mediating complex aversive and appetitive responses. Of particular relevance, we review how the PAG is involved in influencing complex forms of defensive responses, such as circa-strike and risk assessment responses in animals. In addition, we discuss putative dorsal PAG ascending paths that are likely to convey information related to threatening events to cortico-hippocampal-amygdalar circuits involved in the processing of fear learning. Finally, we discuss the evidence supporting the role of the PAG in reward seeking and note that the lateral PAG is part of the circuitry related to goal-oriented responses mediating the motivation to hunt and perhaps drug seeking behavior.

Integrated Control of Predatory Hunting by the Central Nucleus of the Amygdala.

Superior predatory skills led to the evolutionary triumph of jawed vertebrates. However, the mechanisms by which the vertebrate brain controls predation remain largely unknown. Here, we reveal a critical role for the central nucleus of the amygdala in predatory hunting. Both optogenetic and chemogenetic stimulation of central amygdala of mice elicited predatory-like attacks upon both insect and artificial prey. Coordinated control of cervical and mandibular musculatures, which is necessary for accurately positioning lethal bites on prey, was mediated by a central amygdala projection to the reticular formation in the brainstem. In contrast, prey pursuit was mediated by projections to the midbrain periaqueductal gray matter. Targeted lesions to these two pathways separately disrupted biting attacks upon prey versus the initiation of prey pursuit. Our findings delineate a neural network that integrates distinct behavioral modules and suggest that central amygdala neurons instruct predatory hunting across jawed vertebrates.

Evidence of a Role for the Lateral Hypothalamic Area Juxtadorsomedial Region (LHAjd) in Defensive Behaviors Associated with Social Defeat.

Our understanding of the extrinsic connections of the lateral hypothalamic area (LHA) has deepened in recent years. In particular, a series of studies using neural pathway-tracing methods to investigate the macroconnections of histologically differentiated LHA regions, have revealed that the neural connections of these regions are substantially distinct, and have robust connections with neural circuits controlling survival behaviors. To begin testing functional associations suggested by the distinct LHA region neural connections, the present study has investigated the role of the LHA juxtadorsomedial region (LHAjd) in the control of social defeat (a socially-relevant defensive behavior). Male rats received bilateral cytotoxic lesions targeted to the LHAjd. A resident-intruder paradigm was then employed to investigate the effect of these lesions on defensive behavioral responses. Behavioral data were collected during three phases of testing: (1) pre-encounter habituation to testing context; (2) encounter with a dominant conspecific in the testing context; and (3) post-encounter context. Statistical analysis of behavioral measures revealed a significant decrease in risk assessment behaviors during post-encounter context testing in lesioned intruders compared to sham-lesioned and intact rats. However, changes in defensive behavioral measures during the habituation, or during resident-intruder encounters, did not reach significance. We discuss these data in relation to LHAjd (and neighboring LHA region) neural connections, and in relation to current advances in understanding of the neural control of defensive behaviors. A refined model for the neural circuits that are central to the control of socially-relevant defensive behaviors is outlined. We also consider possible broader implications of these data for disorders of behavioral control.

Olfactory instruction for fear: neural system analysis.

Different types of predator odors engage elements of the hypothalamic predator-responsive circuit, which has been largely investigated in studies using cat odor exposure. Studies using cat odor have led to detailed mapping of the neural sites involved in innate and contextual fear responses. Here, we reviewed three lines of work examining the dynamics of the neural systems that organize innate and learned fear responses to cat odor. In the first section, we explored the neural systems involved in innate fear responses and in the acquisition and expression of fear conditioning to cat odor, with a particular emphasis on the role of the dorsal premammillary nucleus (PMd) and the dorsolateral periaqueductal gray (PAGdl), which are key sites that influence innate fear and contextual conditioning. In the second section, we reviewed how chemical stimulation of the PMd and PAGdl may serve as a useful unconditioned stimulus in an olfactory fear conditioning paradigm; these experiments provide an interesting perspective for the understanding of learned fear to predator odor. Finally, in the third section, we explored the fact that neutral odors that acquire an aversive valence in a shock-paired conditioning paradigm may mimic predator odor and mobilize elements of the hypothalamic predator-responsive circuit.

Executive and modulatory neural circuits of defensive reactions: implications for panic disorder.

The present review covers two independent approaches, a neuroanatomical and a pharmacological (focused on serotonergic transmission), which converge in highlighting the critical role of the hypothalamus and midbrain periaqueductal gray matter in the generation of panic attacks and in the mechanism of action of current antipanic medication. Accordingly, innate and learned fear responses to different threats (i.e., predator, aggressive members of the same species, interoceptive threats and painful stimuli) are processed by independent circuits involving corticolimbic regions (the amygdala, the hippocampus and the prefrontal and insular cortices) and downstream hypothalamic and brainstem circuits. As for the drug treatment, animal models of panic indicate that the drugs currently used for treating panic disorder should work by enhancing 5-HT inhibition of neural systems that command proximal defense in both the dorsal periaqueductal gray and in the medial hypothalamus. For the anticipatory anxiety, the reviewed evidence points to corticolimbic structures, such as the amygdala, the septo-hippocampus and the prefrontal cortex, as its main neural substrate, modulated by stimulation of 5-HT2C and 5-HT1A receptors.

Independent hypothalamic circuits for social and predator fear.

The neural circuits mediating fear to naturalistic threats are poorly understood. We found that functionally independent populations of neurons in the ventromedial hypothalamus (VMH), a region that has been implicated in feeding, sex and aggression, are essential for predator and social fear in mice. Our results establish a critical role for VMH in fear and have implications for selective intervention in pathological fear in humans.

Ventral premammillary nucleus as a critical sensory relay to the maternal aggression network.

Maternal aggression is under the control of a wide variety of factors that prime the females for aggression or trigger the aggressive event. Maternal attacks are triggered by the perception of sensory cues from the intruder, and here we have identified a site in the hypothalamus of lactating rats that is highly responsive to the male intruder--the ventral premammillary nucleus (PMv). The PMv is heavily targeted by the medial amygdalar nucleus, and we used lesion and immediate-early gene studies to test our working hypothesis that the PMv signals the presence of a male intruder and transfers this information to the network organizing maternal aggression. PMv-lesioned dams exhibit significantly reduced maternal aggression, without affecting maternal care. The Fos analysis revealed that PMv influences the activation of hypothalamic and septal sites shown to be mobilized during maternal aggression, including the medial preoptic nucleus (likely to represent an important locus to integrate priming stimuli critical for maternal aggression), the caudal two-thirds of the hypothalamic attack area (comprising the ventrolateral part of the ventromedial hypothalamic nucleus and the adjacent tuberal region of the lateral hypothalamic area, critical for the expression of maternal aggression), and the ventral part of the anterior bed nuclei of the stria terminalis (presently discussed as being involved in controlling neuroendocrine and autonomic responses accompanying maternal aggression). These findings reveal an important role for the PMv in detecting the male intruder and how this nucleus modulates the network controlling maternal aggression.