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.

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.

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, 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, for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT, 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) or cell bodies (DeepCOUNT) in intact cleared brains. Using DeepTraCE, DeepCOUNT, and fiber photometry, we performed detailed anatomical and functional mapping of mPFC neuronal classes, identifying specialized roles in threat avoidance.

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 potentialconfounds 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 onRGS scores. Twenty (10 male and 10 female) Sprague–Dawley rats each received 30 min of anesthesia, followed by 30 minof observation after the return of sternal recumbency. Rats were randomized to receive warming with an electric heatingpad or no warming during both periods. Unwarmed rats became hypothermic within 15 min after isoflurane exposure beganand returned to normothermia within 15 min after returning to sternal recumbency. Warmed rats did not deviate from thenormothermic range. The RGS scores of unwarmed rats were significantly higher than baseline levels for 3 h after anesthesiaand were higher than those of warmed rats at 5 and 180 min after anesthesia. Hypothermia resulted in a larger proportion ofrats crossing a predetermined analgesic intervention threshold. Our findings show that hypothermia induced by isofluraneanesthesia presents a confound to accurate RGS scoring. These results emphasize the importance of maintaining normothermiato avoid inflated pain scores and to obtain accurate pain assessment.

Long-range axonal projections provide the foundation for functional connectivity between brain regions and are critical in the modulation of behavior. Descending projections from medial prefrontal cortex (mPFC) to various target regions regulate critical behavioral functions including decision making, social behavior and mood. While specific mPFC projections have distinct behavioral roles, individual mPFC projection neurons can also innervate multiple target regions. Yet how mPFC projection neurons divide their axons across the brain is poorly understood. In this study, we mapped the axon collaterals of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC) in mice. We used tissue clearing and light sheet fluorescence microscopy to visualize the 3-D structure of axonal arbors across the intact brain. While machine learning can automate analysis of axons in images of cleared tissue, it is challenging to train a model that generalizes to all axonal structures because the appearance of axons varies by target region. In this study, we present DeepTraCE (Deep learning-based image Tracing with Combined-model Enhancement), a new strategy for axon segmentation and quantification in images of cleared tissue. DeepTraCE is based on the deep-learning framework TRAILMAP; it achieves highly accurate axon detection by combining multiple machine learning models that are each applied to different brain regions. Using DeepTraCE, we find that cmPFC, NAc, and VTA-projecting mPFC neurons represent largely separable classes with unique axon collaterals in cortical, olfactory, and thalamic regions, respectively.