Stress circuitry: mechanisms behind nervous and immune system communication that influence behavior.

Inflammatory processes are increased by stress and contribute to the pathology of mood disorders. Stress is thought to primarily induce inflammation through peripheral and central noradrenergic neurotransmission. In healthy individuals, these pro-inflammatory effects are countered by glucocorticoid signaling, which is also activated by stress. In chronically stressed individuals, the anti-inflammatory effects of glucocorticoids are impaired, allowing pro-inflammatory effects to go unchecked. Mechanisms underlying this glucocorticoid resistance are well understood, but the precise circuits and molecular mechanisms by which stress increases inflammation are not as well known. In this narrative review, we summarize the mechanisms by which chronic stress increases inflammation and contributes to the onset and development of stress-related mood disorders. We focus on the neural substrates and molecular mechanisms, especially those regulated by noradrenergic signaling, that increase inflammatory processes in stressed individuals. We also discuss key knowledge gaps in our understanding of the communication between nervous and immune systems during stress and considerations for future therapeutic strategies. Here we highlight the mechanisms by which noradrenergic signaling contributes to inflammatory processes during stress and how this inflammation can contribute to the pathology of stress-related mood disorders. Understanding the mechanisms underlying crosstalk between the nervous and immune systems may lead to novel therapeutic strategies for mood disorders and/or provide important considerations for treating immune-related diseases in individuals suffering from stress-related disorders.

Chronic paternal morphine exposure increases sensitivity to morphine-derived pain relief in male progeny.

Parental history of opioid exposure is seldom considered when prescribing opioids for pain relief. To explore whether parental opioid exposure may affect sensitivity to morphine in offspring, we developed a "rat pain scale" with high-speed imaging, machine learning, and mathematical modeling in a multigenerational model of paternal morphine self-administration. We find that the most commonly used tool to measure mechanical sensitivity in rodents, the von Frey hair, is not painful in rats during baseline conditions. We also find that male progeny of morphine-treated sires had no baseline changes in mechanical pain sensitivity but were more sensitive to the pain-relieving effects of morphine. Using RNA sequencing across pain-relevant brain regions, we identify gene expression changes within the regulator of G protein signaling family of proteins that may underlie this multigenerational phenotype. Together, this rat pain scale revealed that paternal opioid exposure increases sensitivity to morphine's pain-relieving effects in male offspring.

Flying in the Face of Adversity: a Drosophila-Based Virtual CURE (Course-Based Undergraduate Research Experience) Provides a Semester-Long Authentic Research Opportunity to the Flipped Classroom.

A call for the integration of research experiences into all biology curricula has been a major goal for educational reform efforts nationally. Course-based undergraduate research experiences (CUREs) have been the predominant method of accomplishing this, but their associated costs and complex design can limit their wide adoption. In 2020, the COVID-19 pandemic forced programs to identify unique ways to still provide authentic research experiences while students were virtual. We report here a complete guide for the successful implementation of a semester-long virtual CURE that uses Drosophila behavioral assays to explore the connection between pain and addiction with the use of an at-home "lab-in-a-box." Individual components were piloted across three semesters and launched as a 100-level introductory course with 19 students. We found that this course increased science identity and successfully improved key research competencies as per the Undergraduate Research Student Self-Assessment (URSSA) survey. This course is ideal for flipped classrooms ranging from introductory to upper-level biology/neuroscience courses and can be integrated directly into the lecture period without the need for building a new course. Given the low cost, recent comfort with virtual learning environments, and current proliferation of flipped classrooms following the 2020 pandemic, this curriculum could serve as an ideal project-based active-learning tool for equitably increasing access to authentic research experiences.

Using high-speed videography for objective and reproducible pain measurement on a mouse pain scale.

Mouse models are essential for studying pain neurobiology and testing pain therapeutics. The reliance on assays that only measure the presence, absence, or frequency of a reflex have limited the reliability of preclinical pain studies. Our high-speed videography protocol overcomes this by projecting the discrete sub-second kinematic behavioral features induced by hind paw stimulation onto a "mouse pain scale." This provides a more objective and robust pain measurement in mice by quantifying the quality of the stimulus-induced hind paw reflex. For complete details on the use and execution of this protocol, please refer to Abdus-Saboor et al. (2019).

Development of a Mouse Pain Scale Using Sub-second Behavioral Mapping and Statistical Modeling.

Rodents are the main model systems for pain research, but determining their pain state is challenging. To develop an objective method to assess pain sensation in mice, we adopt high-speed videography to capture sub-second behavioral features following hind paw stimulation with both noxious and innocuous stimuli and identify several differentiating parameters indicating the affective and reflexive aspects of nociception. Using statistical modeling and machine learning, we integrate these parameters into a single index and create a "mouse pain scale," which allows us to assess pain sensation in a graded manner for each withdrawal. We demonstrate the utility of this method by determining sensations triggered by three different von Frey hairs and optogenetic activation of two different nociceptor populations. Our behavior-based "pain scale" approach will help improve the rigor and reproducibility of using withdrawal reflex assays to assess pain sensation in mice.

Region-specific disruption of the blood-brain barrier following repeated inflammatory dural stimulation in a rat model of chronic trigeminal allodynia.

Background The blood-brain barrier (BBB) has been hypothesized to play a role in migraine since the late 1970s. Despite this, limited investigation of the BBB in migraine has been conducted. We used the inflammatory soup rat model of trigeminal allodynia, which closely mimics chronic migraine, to determine the impact of repeated dural inflammatory stimulation on BBB permeability. Methods The sodium fluorescein BBB permeability assay was used in multiple brain regions (trigeminal nucleus caudalis (TNC), periaqueductal grey, frontal cortex, sub-cortex, and cortex directly below the area of dural activation) during the episodic and chronic stages of repeated inflammatory dural stimulation. Glial activation was assessed in the TNC via GFAP and OX42 immunoreactivity. Minocycline was tested for its ability to prevent BBB disruption and trigeminal sensitivity. Results No astrocyte or microglial activation was found during the episodic stage, but BBB permeability and trigeminal sensitivity were increased. Astrocyte and microglial activation, BBB permeability, and trigeminal sensitivity were increased during the chronic stage. These changes were only found in the TNC. Minocycline treatment prevented BBB permeability modulation and trigeminal sensitivity during the episodic and chronic stages. Discussion Modulation of BBB permeability occurs centrally within the TNC following repeated dural inflammatory stimulation and may play a role in migraine.

The Role of Adenosine Signaling in Headache: A Review.

Migraine is the third most prevalent disease on the planet, yet our understanding of its mechanisms and pathophysiology is surprisingly incomplete. Recent studies have built upon decades of evidence that adenosine, a purine nucleoside that can act as a neuromodulator, is involved in pain transmission and sensitization. Clinical evidence and rodent studies have suggested that adenosine signaling also plays a critical role in migraine headache. This is further supported by the widespread use of caffeine, an adenosine receptor antagonist, in several headache treatments. In this review, we highlight evidence that supports the involvement of adenosine signaling in different forms of headache, headache triggers, and basic headache physiology. This evidence supports adenosine A2A receptors as a critical adenosine receptor subtype involved in headache pain. Adenosine A2A receptor signaling may contribute to headache via the modulation of intracellular Cyclic adenosine monophosphate (cAMP) production or 5' AMP-activated protein kinase (AMPK) activity in neurons and glia to affect glutamatergic synaptic transmission within the brainstem. This evidence supports the further study of adenosine signaling in headache and potentially illuminates it as a novel therapeutic target for migraine.

Functional mitochondrial analysis in acute brain sections from adult rats reveals mitochondrial dysfunction in a rat model of migraine.

Mitochondrial dysfunction has been implicated in many neurological disorders that only develop or are much more severe in adults, yet no methodology exists that allows for medium-throughput functional mitochondrial analysis of brain sections from adult animals. We developed a technique for quantifying mitochondrial respiration in acutely isolated adult rat brain sections with the Seahorse XF Analyzer. Evaluating a range of conditions made quantifying mitochondrial function from acutely derived adult brain sections from the cortex, cerebellum, and trigeminal nucleus caudalis possible. Optimization of this technique demonstrated that the ideal section size was 1 mm wide. We found that sectioning brains at physiological temperatures was necessary for consistent metabolic analysis of trigeminal nucleus caudalis sections. Oxygen consumption in these sections was highly coupled to ATP synthesis, had robust spare respiratory capacities, and had limited nonmitochondrial respiration, all indicative of healthy tissue. We demonstrate the effectiveness of this technique by identifying a decreased spare respiratory capacity in the trigeminal nucleus caudalis of a rat model of chronic migraine, a neurological disorder that has been associated with mitochondrial dysfunction. This technique allows for 24 acutely isolated sections from multiple brain regions of a single adult rat to be analyzed simultaneously with four sequential drug treatments, greatly advancing the ability to study mitochondrial physiology in adult neurological disorders.

Quantitative characterization reveals three types of dry-sensitive corneal afferents: pattern of discharge, receptive field, and thermal and chemical sensitivity.

This study reveals that the cold-sensitive (CS) + dry-sensitive (DS) corneal afferents reported in a previous study consist of two types: 1) low threshold (LT)-CS + DS neurons with <1°C cooling sensitivity, and 2) high threshold (HT)-CS + DS neurons with a wide range of cooling sensitivities (~1-10°C cooling). We also found DS neurons with no cooling sensitivity down to 19°C [cold-insensitive (CI) + DS neurons]. LT-CS + DS neurons showed highly irregular discharge patterns during the dry cornea characterized by numerous spiking bursts, reflecting small temperature changes in the cornea. Their receptive fields (RFs) were mainly located in the cornea's center, the first place for tears to ebb from the surface and be susceptible to external temperature fluctuations. HT-CS and CI + DS neurons showed a gradual rise in firing rate to a stable level over ~60 s after the dry stimulus onset. Their RFs were located mostly in the cornea's periphery, the last place for tears to evaporate. The exquisite sensitivity to cooling in LT-CS + DS neurons was highly correlated with heat sensitivity (~45°C). There was a perfect correlation between noxious heat sensitivity and capsaicin responsiveness in each neuron type. The high sensitivity to noxious osmotic stress was a defining property of the HT-CS and CI + DS neurons, while high sensitivity to menthol was a major characteristic of the LT-CS + DS neurons. These observations suggest that three types of DS neurons serve different innocuous and nociceptive functions related to corneal dryness.