Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration.

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.

Sex-dependent and ontogenetic effects of low dose ethanol on social behavioral deficits induced by mouse maternal separation.

Early life stress can induce lifelong emotional and social behavioral deficits that may in some cases be alleviated by drugs or alcohol. A model for early life stress, rodent maternal separation, recapitulates these behavioral sequelae, which are not limited to potentiated anxiety-like behavior, attenuated social motivation, and altered reward-seeking. Here we employed mouse maternal separation with early weaning (MSEW), consisting of pup-dam separation lasting 4-8 hours on postnatal days (PD) 2-16, with early weaning on PD 17. Prior MSEW studies have limited subjects by age or sex, so we more comprehensively investigated MSEW effects in both sexes, during adolescence and adulthood. We found universal effects of MSEW to include lifelong enhancement of anxiety-like and despair behavior, as well as deficits in social motivation. We also observed some sex-dependent effects of MSEW, namely that female MSEW mice exhibited social habituation to a greater degree than their male counterparts. Low dose ethanol administration had no major effects on the social behavior of non-stressed mice. But interestingly, MSEW-induced social habituation was counteracted by low dose ethanol in adolescent female mice, and potentiated in adolescent male mice. These effects were absent in adult animals, suggesting that ethanol may exert differential effects on the developing brain in such a manner to produce age-, sex-, and stress-dependent effects upon social behavior. Together, results indicate that MSEW reliably produces long-lasting impairments in emotional and social behaviors in both sexes and across the lifespan, but may exert more salient social behavioral effects on female animals.

A prefrontal-bed nucleus of the stria terminalis circuit limits fear to uncertain threat.

In many cases of trauma, the same environmental stimuli that become associated with aversive events are experienced on other occasions without adverse consequence. We examined neural circuits underlying partially reinforced fear (PRF), whereby mice received tone-shock pairings on half of conditioning trials. Tone-elicited freezing was lower after PRF conditioning than fully reinforced fear (FRF) conditioning, despite an equivalent number of tone-shock pairings. PRF preferentially activated medial prefrontal cortex (mPFC) and bed nucleus of the stria terminalis (BNST). Chemogenetic inhibition of BNST-projecting mPFC neurons increased PRF, not FRF, freezing. Multiplexing chemogenetics with in vivo neuronal recordings showed elevated infralimbic cortex (IL) neuronal activity during CS onset and freezing cessation; these neural correlates were abolished by chemogenetic mPFC→BNST inhibition. These data suggest that mPFC→BNST neurons limit fear to threats with a history of partial association with an aversive stimulus, with potential implications for understanding the neural basis of trauma-related disorders.

While walking home alone late one night, you hear footsteps behind you. Your heart starts to beat faster as you wonder whether someone might be following you. Being able to identify and evade threats is essential for survival. A key part of this process is learning to recognize signals that predict potential danger: the sound of footsteps behind you, for example. But many such cues are unreliable. The person behind you might simply be heading in the same general direction as you. And if you spend too much time and energy responding to such false alarms, you may struggle to complete other essential tasks. To be useful, responses to cues that signal potential threats must thus be proportionate to the likelihood that danger is actually present. By studying threat detection in mice, Glover et al. have identified a brain circuit that helps ensure that this is the case. Two groups of mice learned to fear a tone that predicted the delivery of a mild footshock. In one group of animals, the tone was followed by a shock on every trial (it was said to be 'fully reinforced'). But in the other group, the tone was followed by a shock on only 50% of trials ('partially reinforced'). After training, both groups of mice froze whenever they heard the tone ­ freezing being a typical fear response in rodents. But the animals trained with the partially reinforced tone showed less freezing than their counterparts in the fully reinforced group. Moreover, freezing in response to the partially reinforced tone was accompanied by activity in a specific neural pathway connecting the frontal part of the brain to an area called the bed nucleus of the stria terminalis. Inhibiting this pathway made mice respond to the partially reinforced tone as though it had been reinforced on every trial. This suggests that activity in this pathway helps dampen responses to unpredictable threat cues. In people with anxiety disorders, cues that become associated with unpleasant events can trigger anxiety symptoms, even if the association is unreliable. The findings of Glover et al. suggest that reduced activity of circuits that constrain excessive responses to threats might contribute to anxiety disorders.

Phasic signaling in the bed nucleus of the stria terminalis during fear learning predicts within- and across-session cued fear expression.

While results from many past studies have implicated the bed nucleus of the stria terminalis (BNST) in mediating the expression of sustained negative affect, recent studies have highlighted a more complex role for BNST that includes aspects of fear learning in addition to defensive responding. As BNST is thought to encode ambiguous or unpredictable threat, it seems plausible that it may be involved in encoding early cued fear learning, especially immediately following a first tone-shock pairing when the conditioned stimulus-unconditioned stimulus (CS-US) contingency is not fully apparent. To investigate this, we conducted in vivo electrophysiological recording studies to examine neural dynamics of BNST units during cued fear acquisition and recall. We identified two functionally distinct subpopulations of BNST neurons that encode the intertrial interval (ITI) and may contribute to within- and across-session fear learning. "Ramping" cell activity during cued fear acquisition parallels the increase in freezing expression as mice learn the CS-US contingency, while "Phasic" cells encode postshock (USpost) periods (30 sec following encounter with footshock) only during early trials. Importantly, the magnitude of Phasic unit responsivity to the first USpost period predicted not only freezing expression in response to the subsequent CS during acquisition, but also CS freezing evoked 24 h later during CS retrieval. These findings suggest for the first time that BNST activity may serve as an instructive signal during cued fear learning.