Developmentally distinct architectures in top-down circuits.

The medial prefrontal cortex (mPFC) plays a key role in learning, mood and decision making, including in how individuals respond to threats 1-6 . mPFC undergoes a uniquely protracted development, with changes in synapse density, cortical thickness, long-range connectivity, and neuronal encoding properties continuing into early adulthood 7-21 . Models suggest that before adulthood, the slow-developing mPFC cannot adequately regulate activity in faster-developing subcortical centers 22,23 . They propose that during development, the enhanced influence of subcortical systems underlies distinctive behavioural strategies of juveniles and adolescents and that increasing mPFC control over subcortical structures eventually allows adult behaviours to emerge. Yet it has remained unclear how a progressive strengthening of top-down control can lead to nonlinear changes in behaviour as individuals mature 24,25 . To address this discrepancy, here we monitored and manipulated activity in the developing brain as animals responded to threats, establishing direct causal links between frontolimbic circuit activity and the behavioural strategies of juvenile, adolescent and adult mice. Rather than a linear strengthening of mPFC synaptic connectivity progressively regulating behaviour, we uncovered multiple developmental switches in the behavioural roles of mPFC circuits targeting the basolateral amygdala (BLA) and nucleus accumbens (NAc). We show these changes are accompanied by axonal pruning coinciding with functional strengthening of synaptic connectivity in the mPFC-BLA and mPFC-NAc pathways, which mature at different rates. Our results reveal how developing mPFC circuits pass through distinct architectures that may make them optimally adapted to the demands of age-specific challenges.

Brain-Wide Projections and Differential Encoding of Prefrontal Neuronal Classes Underlying Learned and Innate Threat Avoidance.

To understand how the brain produces behavior, we must elucidate the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) is critical for complex functions including decision-making and mood. mPFC projection neurons collateralize extensively, but the relationships between mPFC neuronal activity and brain-wide connectivity are poorly understood. We performed whole-brain connectivity mapping and fiber photometry to better understand the mPFC circuits that control threat avoidance in male and female mice. Using tissue clearing and light sheet fluorescence microscopy (LSFM), we mapped the brain-wide axon collaterals of populations of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC). We present DeepTraCE (deep learning-based tracing with combined enhancement), for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging), for quantifying cell bodies. Anatomical maps produced with DeepTraCE aligned with known axonal projection patterns and revealed class-specific topographic projections within regions. Using TRAP2 mice and DeepCOUNT, we analyzed whole-brain functional connectivity underlying threat avoidance. PL was the most highly connected node with functional connections to subsets of PL-cPL, PL-NAc, and PL-VTA target sites. Using fiber photometry, we found that during threat avoidance, cmPFC and NAc-projectors encoded conditioned stimuli, but only when action was required to avoid threats. mPFC-VTA neurons encoded learned but not innate avoidance behaviors. Together our results present new and optimized approaches for quantitative whole-brain analysis and indicate that anatomically defined classes of mPFC neurons have specialized roles in threat avoidance.SIGNIFICANCE STATEMENT Understanding how the brain produces complex behaviors requires detailed knowledge of the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) plays a key role in learning, mood, and decision-making, including evaluating and responding to threats. mPFC dysfunction is strongly linked to fear, anxiety and mood disorders. Although mPFC circuits are clear therapeutic targets, gaps in our understanding of how they produce cognitive and emotional behaviors prevent us from designing effective interventions. To address this, we developed a high-throughput analysis pipeline for quantifying bulk-labeled fluorescent axons [DeepTraCE (deep learning-based tracing with combined enhancement)] or cell bodies [DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging)] in intact cleared brains. Using DeepTraCE, DeepCOUNT, and fiber photometry, we performed detailed anatomic and functional mapping of mPFC neuronal classes, identifying specialized roles in threat avoidance.

Linking mPFC circuit maturation to the developmental regulation of emotional memory and cognitive flexibility.

The medial prefrontal cortex (mPFC) and its abundant connections with other brain regions play key roles in memory, cognition, decision making, social behaviors, and mood. Dysfunction in mPFC is implicated in psychiatric disorders in which these behaviors go awry. The prolonged maturation of mPFC likely enables complex behaviors to emerge, but also increases their vulnerability to disruption. Many foundational studies have characterized either mPFC synaptic or behavioral development without establishing connections between them. Here, we review this rich body of literature, aligning major events in mPFC development with the maturation of complex behaviors. We focus on emotional memory and cognitive flexibility, and highlight new work linking mPFC circuit disruption to alterations of these behaviors in disease models. We advance new hypotheses about the causal connections between mPFC synaptic development and behavioral maturation and propose research strategies to establish an integrated understanding of neural architecture and behavioral repertoires.

Hypothermia During General Anesthesia Interferes with Pain Assessment in Laboratory Rats (Rattus norvegicus).

Accurate pain assessment methods are necessary to ensure animal welfare and reliable data collection in animal research. The Rat Grimace Scale (RGS), a facial expression pain scale, allows effective identification of pain. However, the potential confounds of this method remain mostly unexplored. General anesthesia, which is used in many laboratory procedures, suppresses thermoregulation and results in hypothermia. We investigated the effects of isoflurane-induced hypothermia on RGS scores. Twenty (10 male and 10 female) Sprague-Dawley rats each received 30 min of anesthesia, followed by 30 min of observation after the return of sternal recumbency. Rats were randomized to receive warming with an electric heating pad or no warming during both periods. Unwarmed rats became hypothermic within 15 min after isoflurane exposure began and returned to normothermia within 15 min after returning to sternal recumbency. Warmed rats did not deviate from the normothermic range. The RGS scores of unwarmed rats were significantly higher than baseline levels for 3 h after anesthesia and were higher than those of warmed rats at 5 and 180 min after anesthesia. Hypothermia resulted in a larger proportion of rats crossing a predetermined analgesic intervention threshold. Our findings show that hypothermia induced by isoflurane anesthesia presents a confound to accurate RGS scoring. These results emphasize the importance of maintaining normothermia to avoid inflated pain scores and to obtain accurate pain assessment.

Comparing the Rat Grimace Scale and a composite behaviour score in rats.

There is a growing interest in the use of voluntarily displayed ongoing behaviours in laboratory animals to assess the pain experience. In rats, two behavioural pain scales, the Rat Grimace Scale (RGS, a facial expression scale) and a composite behaviour score (CBS, a behavioural ethogram reliant on postural changes), are both promising pain assessment methods. Both scales have been used to assess pain in a laparotomy model, however, they have never been compared directly and the knowledge of how different analgesics may affect these two scales is limited. This study aimed to provide a comparison to discriminate the temporal and analgesic response in a laparotomy model. Female Wistar (n = 26) and Sprague Dawley rats (n = 26) were block randomized to receive saline, meloxicam (2 mg/kg) or buprenorphine (0.05 mg/kg) 30 minutes before laparotomy. Rats were video-recorded before surgery (BL) and at 30, 150, 270, and 390 minutes post-operatively. Videos were assessed according to both scales by a trained, blinded observer. Both CBS and RGS scores increased significantly at all post surgical timepoints in the saline group. Both buprenorphine and meloxicam reduced CBS scores to baseline levels following laparotomy; however, RGS scores were only reduced following buprenorphine. RGS scores in the meloxicam group remained similar to scores of the saline group. These findings suggest that the CBS and RGS differ in their sensitivity to discriminating analgesic effects.