A subiculum-hypothalamic pathway functions in dynamic threat detection and memory updating.

Animals need to detect threats, initiate defensive responses, and, in parallel, remember where the threat occurred to avoid the possibility of re-encountering it. By probing animals capable of detecting and avoiding a shock-related threatening location, we were able to reveal a septo-hippocampal-hypothalamic circuit that is also engaged in ethological threats, including predatory and social threats. Photometry analysis focusing on the dorsal premammillary nucleus (PMd), a critical interface of this circuit, showed that in freely tested animals, the nucleus appears ideal to work as a threat detector to sense dynamic changes under threatening conditions as the animal approaches and avoids the threatening source. We also found that PMd chemogenetic silencing impaired defensive responses by causing a failure of threat detection rather than a direct influence on any behavioral responses and, at the same time, updated fear memory to a low-threat condition. Optogenetic silencing of the main PMd targets, namely the periaqueductal gray and anterior medial thalamus, showed that the projection to the periaqueductal gray influences both defensive responses and, to a lesser degree, contextual memory, whereas the projection to the anterior medial thalamus has a stronger influence on memory processes. Our results are important for understanding how animals deal with the threat imminence continuum, revealing a circuit that is engaged in threat detection and that, at the same time, serves to update the memory process to accommodate changes under threatening conditions.

An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept.

A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor (Oxtr) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species (n = 6) without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.

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.

Dynamics in brain activation and behaviour in acute and repeated social defensive behaviour.

In nature, confrontations between conspecifics are recurrent and related, in general, due to the lack of resources such as food and territory. Adequate defence against a conspecific aggressor is essential for the individual's survival and the group integrity. However, repeated social defeat is a significant stressor promoting several behavioural changes, including social defence per se. What would be the neural basis of these behavioural changes? To build new hypotheses about this, we here investigate the effects of repeated social stress on the neural circuitry underlying motivated social defence behaviour in male mice. We observed that animals re-exposed to the aggressor three times spent more time in passive defence during the last exposure than in the first one. These animals also show less activation of the amygdalar and hypothalamic nuclei related to the processing of conspecific cues. In turn, we found no changes in the activation of the hypothalamic dorsal pre-mammillary nucleus (PMD) that is essential for passive defence. Therefore, our data suggest that the balance between the activity of circuits related to conspecific processing and the PMD determines the pattern of social defence behaviour. Changes in this balance may be the basis of the adaptations in social defence after repeated social defeat.

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.

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.

Dissecting the brain's fear system reveals the hypothalamus is critical for responding in subordinate conspecific intruders.

Effective defense against natural threats in the environment is essential for the survival of individual animals. Thus, instinctive behavioral responses accompanied by fear have evolved to protect individuals from predators and from opponents of the same species (dominant conspecifics). While it has been suggested that all perceived environmental threats trigger the same set of innately determined defensive responses, we tested the alternate hypothesis that different stimuli may evoke differentiable behaviors supported by distinct neural circuitry. The results of behavioral, neuronal immediate early gene activation, lesion, and neuroanatomical experiments indicate that the hypothalamus is necessary for full expression of defensive behavioral responses in a subordinate conspecific, that lesions of the dorsal premammillary nucleus drastically reduce behavioral measures of fear in these animals, and that essentially separate hypothalamic circuitry supports defensive responses to a predator or a dominant conspecific. It is now clear that differentiable neural circuitry underlies defensive responses to fear conditioning associated with painful stimuli, predators, and dominant conspecifics and that the hypothalamus is an essential component of the circuitry for the latter two stimuli.