Orofacial pain has been well-characterized clinically, but evaluation of orofacial pain in animals has not kept pace. The objective of this study was to describe behavioral responses to facial thermal stimulation and inflammation with/without an analgesic using a novel operant paradigm. Animals were trained to voluntarily place their face against a stimulus thermode (37.7-57.2 degrees C) providing access to positive reinforcement. These contingencies present a conflict between positive reward and tolerance for nociceptive stimulation. Inflammation was induced and morphine was provided as an analgesic in a subset of animals. Six outcome measures were determined: reward intake, reward licking contacts, stimulus facial contacts, facial contact duration, ratio of reward/stimulus contacts, and ratio of facial contact duration/event. Animals displayed aversive behaviors to the higher temperatures, denoted by a significant decrease in reward intake, total facial contact duration, and reward licking events. The number of facial contacts increased with increasing temperature, replacing long drinking bouts with more frequent short drinks, as reflected by a low ratio of facial contact duration/event. The number of reward licking/facial contact events was significantly decreased as the thermal stimulus intensity increased, providing another pain index derived from this operant method. These outcomes were significantly affected in the direction of increased nociception following inflammation, and these indices of hyperalgesia were reversed with morphine administration. These data reflect an orofacial pain behavior profile that was based on an animal's responses in an operant escape paradigm. This technique allows evaluation of nociceptive processing and modulation throughout the neuraxis.

Background: Rodent models of orofacial pain typically use methods adapted from manipulations to hind paw; however, limitations of these models include animal restraint and subjective assessments of behavior by the experimenter. In contrast to these methods, assessment of operant responses to painful stimuli has been shown to overcome these limitations and expand the breadth of interpretation of the behavioral responses. In the current study, we used an operant model based on a reward-conflict paradigm to assess nociceptive responses in three strains of mice (SKH1-Hr hr , C57BL/6J, TRPV1 knockout). We previously validated this operant model in rats and hypothesized in this study that wildtype mice would demonstrate a similar thermal stimulus-dependent response and similar operant pain behaviors. Additionally, we evaluated the effects on operant behaviors of mice manipulated genetically (e.g., TRPV1 k.o.) or pharmacologically with resiniferatoxin (RTX), a lesioning agent for TRPV1-expressing neurons. During the rewardconflict task, mice accessed a sweetened milk reward solution by voluntarily position their face against a neutral or heated thermode (37-55°C).

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The brain-gut axis plays a critical role in the regulation of different diseases, many of which are characterized by sympathetic dysregulation. However, a direct link between sympathetic dysregulation and gut dysbiosis remains to be illustrated. Bone marrow (BM)-derived immune cells continuously interact with the gut microbiota to maintain homeostasis in the host. Their function is largely dependent upon the sympathetic nervous system acting via adrenergic receptors present on the BM immune cells. In this study, we utilized a novel chimera mouse that lacks the expression of BM beta1/2 adrenergic receptors (b1/2-ARs) to investigate the role of the sympathetic drive to the BM in gut and microbiota homeostasis. Fecal analyses demonstrated a shift from a dominance of Firmicutes to Bacteroidetes phylum in the b1/2-ARs KO chimera, resulting in a reduction in Firmicutes/Bacteroidetes ratio. Meanwhile, a significant reduction in Proteobacteria phylum was determined. No changes in the abundance of acetate-, butyrate-, and lactate-producing bacteria, and colon pathology were observed in the b1/2-ARs KO chimera. Transcriptomic profiling in colon identified Killer Cell Lectin-Like Receptor Subfamily D, Member 1 (Klrd1), Membrane-Spanning 4-Domains Subfamily A Member 4A (Ms4a4b), and Casein Kinase 2 Alpha Prime Polypeptide (Csnk2a2) as main transcripts associated with the microbiota shifts in the b1/2-ARs KO chimera. Suppression of leukocyte-related transcriptome networks (i.e., function, differentiation, migration), classical compliment pathway, and networks associated with intestinal function, barrier integrity, and excretion was also observed in the colon of the KO chimera. Moreover, reduced expression of transcriptional networks related to intestinal diseases (i.e., ileitis, enteritis, inflammatory lesions, and stress) was noted. The observed suppressed transcriptome networks were associated with a reduction in NK cells, macrophages, and CD4+ T cells in the b1/2-ARs KO chimera colon. Thus, sympathetic regulation of BM-derived immune cells plays a significant role in modifying inflammatory networks in the colon and the gut microbiota composition. To our knowledge, this study is the first to suggest a key role of BM b1/2-ARs signaling in host-microbiota interactions, and reveals specific molecular mechanisms that may lead to generation of novel anti-inflammatory treatments for many immune and autonomic diseases as well as gut dysbiosis across the board.

Activation of autonomic neural pathways by chronic hypertensive stimuli plays a significant role in pathogenesis of hypertension. Here, we proposed that even a single acute hypertensive stimulus will activate neural and immune pathways that may be important in initiation of memory imprinting seen in chronic hypertension. We investigated the effects of acute angiotensin II (Ang II) administration on blood pressure, neural activation in cardioregulatory brain regions, and central and systemic immune responses, at 1 and 24 h post-injection. Administration of a single bolus intra-peritoneal (I.P.) injection of Ang II (36 μg/kg) resulted in a transient increase in the mean arterial pressure (MAP) (by 22 ± 4 mmHg vs saline), which returned to baseline within 1 h. However, in contrast to MAP, neuronal activity, as measured by manganese-enhanced magnetic resonance (MEMRI), remained elevated in several cardioregulatory brain regions over 24 h. The increase was predominant in autonomic regions, such as the subfornical organ (SFO; ~20%), paraventricular nucleus of the hypothalamus (PVN; ~20%) and rostral ventrolateral medulla (RVLM; ~900%), among others. Similarly, systemic and central immune responses, as evidenced by circulating levels of CD4+/IL17+ T cells, and increased IL17 levels and activation of microglia in the PVN, respectively, remained elevated at 24 h following Ang II challenge. Elevated Fos expression in the PVN was also present at 24 h (by 73 ± 11%) following Ang II compared to control saline injections, confirming persistent activation of PVN. Thus, even a single Ang II hypertensive stimulus will initiate changes in neuronal and immune cells that play a role in the developing hypertensive phenotype.