Rostral Ventral Medulla Research Articles

Rostral ventral medulla circuits regulate both the sensory and affective dimensions of neuropathic pain: a commentary on Dogrul et al.

Rostral ventral medulla circuits regulate both the sensory and affective dimensions of neuropathic pain: a commentary on Dogrul et al.

Open-Label Placebo Injection for Chronic Back Pain With Functional Neuroimaging

Chronic back pain (CBP) is a leading cause of disability. Placebo treatments often provide as much pain relief as bona fide treatments, such as steroid injections. Open-label (honestly prescribed) placebos (OLPs) may relieve CBP without deception, but OLP mechanisms remain poorly understood. To investigate the long-term efficacy and neurobiological mechanisms of OLP for CBP. A randomized clinical trial of CBP with longitudinal functional magnetic resonance imaging (MRI) comparing OLP with usual care, with 1-year follow-up, was conducted in a university research setting and a community orthopedic clinic. Participants were individuals aged 21 to 70 years with CBP. The trial was conducted from November 2017 to August 2018, with 1-year follow-up completed by November 2019. Data analysis was performed from April 2020 to May 2024. The primary analysis was conducted on an intention-to-treat sample. Participants randomized to OLP received a 1-time subcutaneous lumbar saline injection presented as placebo accompanied by information about the power of placebo to relieve pain, alongside their ongoing care. Usual care participants continued their ongoing care. The primary outcome was pain intensity (0-10, with 0 indicating no pain and 10 the most intense) at 1 month posttreatment. Secondary outcomes included pain interference, depression, anxiety, anger, and sleep quality. Functional MRI was performed before and after treatment during evoked and spontaneous back pain. A total of 101 adults (52 [51.4%] females; mean [SD] age, 40.4 [15.4] years) with moderate severity CBP (mean [SD], 4.10 [1.25] intensity; duration, 9.7 [8.5] years) were enrolled. Compared with usual care, OLP reduced CBP intensity posttreatment (relative reduction, 0.61; Hedges g = 0.45; 95% CI, -0.89 to 0.04; P = .02). Through 1-year follow-up, pain relief did not persist, although significant benefits were observed for depression, anger, anxiety, and sleep disruption (Hedges g = 0.3-0.5; all P < .03). Brain responses to evoked back pain for OLP vs usual care increased in rostral anterior cingulate and ventromedial prefrontal cortex and decreased in somatomotor cortices and thalamus. During spontaneous pain, functional connectivity analyses identified OLP vs usual care increases in ventromedial prefrontal cortex connectivity to the rostral ventral medulla, a pain-modulatory brainstem nucleus. No adverse effects of treatment were reported by participants. In this randomized clinical trial of OLP vs usual care, a single nondeceptive placebo injection reduced CBP intensity for 1 month posttreatment and provided benefits lasting for at least 1 year posttreatment. Brain mechanisms of OLP in a clinical population overlap with those of deceptive placebos in healthy volunteers, including engagement of prefrontal-brainstem pain modulatory pathways. ClinicalTrials.gov Identifier: NCT03294148.

RNA sequencing of the thalamus and rostral ventral medulla in rats with chronic orofacial pain.

Diagnosing and treating chronic orofacial pain is challenging due to its complex structure and limited understanding of its causes and mechanisms. In this study, we used RNA sequencing to identify differentially expressed genes (DEGs) in the rostral ventral medulla (RVM) and thalamus of rats with persistent orofacial pain, aiming to explore its development. DEGs were functionally analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results showed a significant association between immune response and pain in this model. Key DEG mRNA expression trends were further validated using real-time quantitative polymerase chain reaction (RT-PCR), confirming their crucial roles in chronic orofacial pain. After injecting complete Freund's adjuvant (CFA) into the bilateral temporomandibular joint cavity for 14 days, we observed 293 upregulated genes and 14 downregulated genes in the RVM, and 1086 upregulated genes and 37 downregulated genes in the thalamus. Furthermore, we identified 27 common DEGs with altered expression (upregulation) in both the thalamus and RVM, including Cd74, C3, Cxcl13, C1qb, Itgal, Fcgr2b, C5ar1, and Tlr2, which are pain-associated genes. Protein-protein interaction (PPI) analysis using Cytoscape revealed the involvement of Toll-like receptors, complement system, differentiation clusters, and antigen presentation-related proteins in the interaction between the thalamus and RVM. The results of this study show that the immune system seems to have a more significant influence on chronic orofacial pain. There may be direct or indirect influence between the thalamus and RVM, which may participate in the regulation of chronic orofacial pain.

Elevated Serotonin in Mouse Spinal Dorsal Horn Is Pronociceptive.

Serotonergic neurons in the rostral ventral medulla (RVM) contribute to bidirectional control of pain through modulation of spinal and trigeminal nociceptive networks. Deficits in this pathway are believed to contribute to pathologic pain states, but whether changes in serotonergic mechanisms are pro- or antinociceptive is debated. We used a combination of optogenetics and fiber photometry to examine these mechanisms more closely. We find that optogenetic activation of RVM serotonergic afferents in the spinal cord of naive mice produces mechanical hypersensitivity and conditioned place aversion (CPA). Neuropathic pain, produced by chronic constriction injury of the infraorbital nerve (CCI-ION), evoked a tonic increase in serotonin (5HT) concentrations within the spinal trigeminal nucleus caudalis (SpVc), measured with liquid chromatography-tandem mass spectroscopy (LC-MS/MS). By contract, CCI-ION had no effect on the phasic serotonin transients in SpVc, evoked by noxious pinch, and measured with fiber photometry of a serotonin sensor. These findings suggest that serotonin release in the spinal cord is pronociceptive and that an increase in sustained serotonin signaling, rather than phasic or event driven increases, potentiate nociception in models of chronic pain.

SAT596 Evidence That Corticotropin-releasing Hormone Receptor 2 Activation Mediates Stress-induced Hyperglycemia

Abstract Disclosure: R.B. McCosh: None. M.J. Kreisman: None. J. Jang: None. J. Jung: None. K.M. Breen Church: None. Stress-induced hyperglycemia refers to the transient increase in blood glucose (BG) levels during stress, which occurs in healthy and diabetic patients. In critically ill patients, stress-induced hyperglycemia is associated with a 2-fold increase in risk of incident diabetes. Thus, stress-induced hyperglycemia is a predictor of or may contribute to glucose dysregulation. In mice, activation of catecholamine neurons in the rostral ventral lateral medulla (rVLM) is necessary for stress-induced hyperglycemia; however, the mechanism by which these cells become activated during stress is not clear. Here, we tested the possibility that corticotropin-releasing hormone receptor 2 (CRHR2) signaling mediates stress-induced hyperglycemia in adult, ovariectomized mice. First, BG levels and rVLM cell activation were examined in two stress models. For psychosocial stress, BG levels were not altered by control treatment. In contrast, placement in a restraint device increased BG levels ∼2-fold, within 30 min, from pretreatment values and BG remained elevated for an additional 90 min. For immune stress, BG was significantly elevated (21% increase) at 30 min post IP injection of lipopolysaccharide (LPS, 10ng/g bw), then returned to baseline levels whereas BG levels were not altered from pretreatment levels in saline injected mice. In tissue collected 2 hr after initiation of restraint stress, the percentage of tyrosine hydroxylase (TH, marker of catecholamine cells) neurons in the rVLM that contained c-Fos (a marker of neural activation) was significantly greater than control mice. Next, we assessed the sufficiency of CRHR2 activation to induce hyperglycemia using the CRHR2 agonist urocortin 2 (UCN2), injected intracerebroventricular (ICV). BG was significantly increased 30 min after injection of both saline and UCN2 (60-80%); however, BG subsequently returned to baseline in saline-treated mice, but remained elevated in mice that received UCN2, similar to restraint stress. To test necessity of CRHR2 activation to induce hyperglycemia, we delivered ICV either the specific CRHR2 antagonist Astressin 2B (A2B) or saline 1 hr before psychosocial or immune stress. In no stress controls, A2B did not alter BG levels compared to saline, yet, BG levels in both groups decreased over time. BG levels were elevated in saline-treated controls 30 min after restraint stress; and, this increase was delayed, although not prevented, by 30 min by A2B pre-treatment. Following LPS injection, BG was increased at 30 min then decreased relative to baseline in saline-treated controls; yet, A2B pre-treatment blocked these changes. Together these data support the hypothesis that CRHR2 signaling is an important mediator of stress-induced hyperglycemia. Assessment of rVLM TH cells is ongoing. Presentation: Saturday, June 17, 2023

The chronification mechanism of orofacial inflammatory pain: Facilitation by GPER1 and microglia in the rostral ventral medulla.

Chronic orofacial pain is a common and incompletely defined clinical condition. The role of G protein-coupled estrogen receptor 1 (GPER1) as a new estrogen receptor in trunk and visceral pain regulation is well known. Here, we researched the role of GPER1 in the rostral ventral medulla (RVM) during chronic orofacial pain. A pain model was established where rats were injected in the temporomandibular joint with complete Freund's adjuvant (CFA) to simulate chronic orofacial pain. Following this a behavioral test was performed to establish pain threshold and results showed that the rats injected with CFA had abnormal pain in the orofacial regions. Additional Immunostaining and blot analysis indicated that microglia were activated in the RVM and GPER1 and c-Fos were significantly upregulated in the rats. Conversely, when the rats were injected with G15 (a GPER1 inhibitor) the abnormal pain the CFA rats were experiencing was alleviated and microglia activation was prevented. In addition, we found that G15 downregulated the expression of phospholipase C (PLC) and protein kinase C (PKC), inhibited the expression of GluA1, restores aberrant synaptic plasticity and reduces the overexpression of the synapse-associated proteins PSD-95 and syb-2 in the RVM of CFA rats. The findings indicate that GPER1 mediates chronic orofacial pain through modulation of the PLC-PKC signal pathway, sensitization of the RVM region and enhancement of neural plasticity. These results of this study therefore suggest that GPER1 may serve as a potential therapeutic target for chronic orofacial pain.

PS-BPB07-2: AFFERENT RENAL NERVES CONTRIBUTE TO LEFT VENTRICULAR DYSFUNCTION THROUGH HYPOTHALAMIC VASOPRESSIN-MEDIATED CENTRAL SYMPATHOEXCITATION IN HYPERTENSIVE HEART FAILURE MODEL

Objective: Afferent renal nerves may contribute to the development and progression of heart failure, which, however, has not been well investigated. This study aimed to examine whether the afferent renal nerve input contributes to hypertensive cardiac dysfunction. Design and method: Male Dahl salt-sensitive rats were fed 0.3% low-salt diet (LS) or 8% high-salt diet (HS) from 6 weeks of age. HS-fed rats were divided into selective afferent renal denervation (HS-ARDN) or sham operation (HS-Sham) at 9 weeks. The effects of ARDN on central sympathetic regulation were investigated at 12 weeks. In another cohort, its effects on the cardiac phenotypes were evaluated at 16 weeks, with adding total renal denervation (HS-TRDN) group. Results: At 12 weeks, systolic blood pressure (SBP) measured by tail-cuff method was significantly increased in HS-Sham compared to LS, while there was no significant difference between HS-Sham and HS-ARDN. Plasma norepinephrine concentration was increased in HS-Sham compared to LS and was attenuated in HS-ARDN. The c-Fos expression, a neuronal activation marker, in presympathetic neurons within hypothalamic paraventricular nucleus (PVN) and rostral ventral lateral medulla (RVLM) was also increased in HS-Sham and attenuated in HS-ARDN, which was in parallel with the c-Fos expression in vasopressin-expressing neurons within PVN. At 16 weeks, SBP was similarly increased in HS-Sham, HS-ARDN, and HS-TRDN compared to LS. Although the parameters of left ventricular (LV) hypertrophy, such as LV wall thickness by echocardiography and LV weight, were significantly increased in HS-Sham, HS-ARDN, and HS-TRDN compared to LS, there was no significant difference between HS-fed three groups. Echocardiographic LV fractional shortening was significantly decreased in HS-Sham compared to LS, and was attenuated to the same extent in HS-ARDN and HS-TRDN. Histological LV fibrosis determined by Masson trichrome staining and mRNA expression of ANP and CTGF in LV were significantly increased in HS-Sham compared to LS; these changes were similarly ameliorated in both HS-ARDN and HS-TRDN. Conclusion: The input from renal afferent nerves contributes to sympathoexcitation and cardiac dysfunction independently of SBP in Dahl salt-sensitive hypertensive heart failure model. Vasopressin production in PVN may be involved in the renal afferents-mediated sympathetic regulation.

Brainstem Damage Underlies Changes in Hypoxic Ventilatory Response Following Cardiac Arrest and Resuscitation in Rats.

People resuscitated after sudden cardiac arrest remain at high risk for mortality, with treatment for survivors varying from monitoring to life support. With respect to assessing survivability post cardiac arrest and resuscitation (CAR), we previously demonstrated the potential of the hypoxic ventilatory response (HVR) as a reliable indicator for discerning between survivors and non-survivors in the early stages of recovery following CAR in rats. Since HVR describes the increase in ventilation in response to hypoxia, we hypothesize that damage to cardiorespiratory regulatory centers in the brainstem underlie the loss of HVR observed post resuscitation in nonsurvivors. Wistar rats underwent cardiac arrest (12-min) and resuscitation. At 1day post-resuscitation, rats were perfused transcardially and the brains were harvested and processed for immunohistostaining of caspase-3, a marker of apoptosis. Positive caspase-3 staining was observed in brainstem regions such as the rostral ventral lateral medulla (RVLM); Co-localization of caspase-3 and NeuN was observed in the RVLM as well, suggesting that apoptosis most likely occurs in neurons. Our results showed positive markers for neuronal apoptosis present in pathways of the brainstem involved in respiratory and cerebrovascular regulation, suggesting brain stem damage underlies changes in HVR following CAR.

TRPV1: A Common Denominator Mediating Antinociceptive and Antiemetic Effects of Cannabinoids.

The most common medicinal claims for cannabis are relief from chronic pain, stimulation of appetite, and as an antiemetic. However, the mechanisms by which cannabis reduces pain and prevents nausea and vomiting are not fully understood. Among more than 450 constituents in cannabis, the most abundant cannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Cannabinoids either directly or indirectly modulate ion channel function. Transient receptor potential vanilloid 1 (TRPV1) is an ion channel responsible for mediating several modalities of pain, and it is expressed in both the peripheral and the central pain pathways. Activation of TRPV1 in sensory neurons mediates nociception in the ascending pain pathway, while activation of TRPV1 in the central descending pain pathway, which involves the rostral ventral medulla (RVM) and the periaqueductal gray (PAG), mediates antinociception. TRPV1 channels are thought to be implicated in neuropathic/spontaneous pain perception in the setting of impaired descending antinociceptive control. Activation of TRPV1 also can cause the release of calcitonin gene-related peptide (CGRP) and other neuropeptides/neurotransmitters from the peripheral and central nerve terminals, including the vagal nerve terminal innervating the gut that forms central synapses at the nucleus tractus solitarius (NTS). One of the adverse effects of chronic cannabis use is the paradoxical cannabis-induced hyperemesis syndrome (HES), which is becoming more common, perhaps due to the wider availability of cannabis-containing products and the chronic use of products containing higher levels of cannabinoids. Although, the mechanism of HES is unknown, the effective treatment options include hot-water hydrotherapy and the topical application of capsaicin, both activate TRPV1 channels and may involve the vagal-NTS and area postrema (AP) nausea and vomiting pathway. In this review, we will delineate the activation of TRPV1 by cannabinoids and their role in the antinociceptive/nociceptive and antiemetic/emetic effects involving the peripheral, spinal, and supraspinal structures.

Postsurgical Latent Pain Sensitization Is Driven by Descending Serotonergic Facilitation and Masked by µ-Opioid Receptor Constitutive Activity in the Rostral Ventromedial Medulla.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MORCA) in the dorsal horn of the spinal cord. To demonstrate LS, we conducted plantar incision in mice and then waited 3-4 weeks for hypersensitivity to resolve. At this time (remission), systemic administration of the opioid receptor antagonist/inverse agonist naltrexone reinstated mechanical and heat hypersensitivity. We first tested the hypothesis that LS extends to serotonergic neurons in the rostral ventral medulla (RVM) that convey pronociceptive input to the spinal cord. We report that in male and female mice, hypersensitivity was accompanied by increased Fos expression in serotonergic neurons of the RVM, abolished on chemogenetic inhibition of RVM 5-HT neurons, and blocked by intrathecal injection of the 5-HT3R antagonist ondansetron; the 5-HT2AR antagonist MDL-11 939 had no effect. Second, to test for MORCA, we microinjected the MOR inverse agonist d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and/or neutral opioid receptor antagonist 6β-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws; 6β-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MORCA, rather than an opioid ligand-dependent mechanism, maintains LS in remission. We conclude that incision establishes LS in descending RVM 5-HT neurons that drives pronociceptive 5-HT3R signaling in the dorsal horn, and this LS is tonically opposed by MORCA in the RVM. The 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.SIGNIFICANCE STATEMENT Surgery leads to latent pain sensitization and a compensatory state of endogenous pain control that is maintained long after tissue healing. Here, we show that either chemogenetic inhibition of serotonergic neuron activity in the RVM or pharmacological inhibition of 5-HT3 receptor signaling at the spinal cord blocks behavioral signs of postsurgical latent sensitization. We conclude that MORCA in the RVM opposes descending serotonergic facilitation of LS and that the 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.

The development of descending serotonergic modulation of the spinal nociceptive network: a life span perspective.

The nociceptive network, responsible for transmission of nociceptive signals that generate the pain experience, is not fully developed at birth. Descending serotonergic modulation of spinal nociception, an important part of the pain network, undergoes substantial postnatal maturation and is suggested to be involved in the altered pain response observed in human newborns. This review summarizes preclinical data of the development of descending serotonergic modulation of the spinal nociceptive network across the life span, providing a comprehensive background to understand human newborn pain experience and treatment. Sprouting of descending serotonergic axons, originating from the rostroventral medulla, as well as changes in receptor function and expression take place in the first postnatal weeks of rodents, corresponding to human neonates in early infancy. Descending serotonergic modulation switches from facilitation in early life to bimodal control in adulthood, masking an already functional 5-HT inhibitory system at early ages. Specifically the 5-HT3 and 5-HT7 receptors seem distinctly important for pain facilitation at neonatal and early infancy, while the 5-HT1a, 5-HT1b, and 5-HT2 receptors mediate inhibitory effects at all ages. Analgesic therapy that considers the neurodevelopmental phase is likely to result in a more targeted treatment of neonatal pain and may improve both short- and long-term effects. IMPACT: The descending serotonergic system undergoes anatomical changes from birth to early infancy, as its sprouts and descending projections increase and the dorsal horn innervation pattern changes. Descending serotonergic modulation from the rostral ventral medulla switches from facilitation in early life via the 5-HT3 and 5-HT7 receptors to bimodal control in adulthood. A functional inhibitory serotonergic system mainly via 5-HT1a, 5-HT1b, and 5-HT2a receptors at the spinal level exists already at the neonatal phase but is masked by descending facilitation.

Neuropathology of Perry Syndrome: Evidence of Medullary and Hypothalamic Involvement.

Perry syndrome is a rare genetic parkinsonian disorder with TAR DNA binding protein 43 (TDP-43) pathology clinically presenting with parkinsonism, neuropsychiatric features, weight loss, and central hypoventilation. As respiratory complications are often the cause of death, studies likely show the early stage of the neurodegenerative process. Because of the rarity of this condition, few studies exist, and each case provides insight into pathological findings in this neurodegenerative condition. To study the clinical and pathological correlations of an autopsy case of Perry syndrome. The patient was a woman in her 50s with Perry syndrome and a DCTN1 gene mutation. Between October 2016 and July 2019, she underwent postmortem and pathological examination at University Hospital in London, Ontario, Canada. Data were obtained through clinical pathological examination. Microscopy showed significant neuronal loss with pigmentary incontinence and gliosis in the substantia nigra. There was no atrophy elsewhere, including the frontal and cingulate cortex. Intraneuronal cytoplasmic TDP-43 inclusions and neurites were noticed in a moderate number in the substantia nigra and midbrain and were sparsely noticed in the basal ganglia, thalamus, thoracic motor neuron, posterior nucleus of the hypothalamus, and rostral ventral medulla. β-Amyloid, Lewy body, and tau pathologies were absent. Rare axonal swelling was identified at the rostral ventrolateral medulla. This study confirms that Perry syndrome is characterized by TDP-43 pathology with absent Lewy bodies or tau pathology. These findings support the hypothesis of dysfunctional neurons in the medulla and hypothalamus, which may respectively correlate to the clinical symptoms of hypoventilation and weight loss in Perry syndrome.

D2 Receptors in the Periaqueductal Gray/Dorsal Raphe Modulate Peripheral Inflammatory Hyperalgesia via the Rostral Ventral Medulla

Dopamine neurons in the periaqueductal gray (PAG)/dorsal raphe are key modulators of antinociception with known supraspinal targets. However, no study has directly tested whether these neurons contribute to descending pain inhibition. We hypothesized that PAG dopamine neurons contribute to the analgesic effect of D-amphetamine via a mechanism that involves descending modulation via the rostral ventral medulla (RVM). Male C57BL/6 mice showed increased c-FOS expression in PAG dopamine neurons and a significant increase in paw withdrawal latency to thermal stimulation after receiving a systemic injection of D-amphetamine. Targeted microinfusion of D-amphetamine, L-DOPA, or the selective D2 agonist quinpirole into the PAG produced analgesia, while a D1 agonist, chloro APB, had no effect. In addition, inhibition of D2 receptors in the PAG by eticlopride prevented the systemic D-amphetamine analgesic effect. D-amphetamine and PAG D2 receptor-mediated analgesia were inhibited by intra-RVM injection of lidocaine or the GABAA receptor agonist muscimol, indicating a PAG-RVM signaling pathway in this model of analgesia. Finally, both systemic D-amphetamine and local PAG microinjection of quinpirole, inhibited inflammatory hyperalgesia induced by carrageenan. This hyperalgesia was transiently restored by intra-PAG injection of eticlopride, as well as RVM microinjection of muscimol. We conclude that D-amphetamine analgesia is partially mediated by descending inhibition and that D2 receptors in the PAG are responsible for this effect via modulating neurons that project to the RVM. These results further our understanding of the antinociceptive effects of dopamine and elucidate a mechanism by which clinically available dopamine modulators produce analgesia.

Nociception alterations precede motor symptoms in a progressive model of parkinsonism induced by reserpine in middle-aged rats

Nociception alterations are frequent non-motor symptoms of the prodromal phase of Parkinson’s disease (PD). The period for the onset of symptoms and the pathophysiological mechanisms underlying these alterations remain unclear. We investigated the course of nociception alterations in a progressive model of parkinsonism induced by reserpine (RES) in rats. Male Wistar rats (6–7 months) received 5 or 10 subcutaneous injections of RES (0.1 mg/kg) or vehicle daily for 20 days. Motor evaluation and nociceptive assessment were performed throughout the treatment. At the end of the treatment rats were euthanized, the brains removed and processed for immunohistochemical analysis (TH and c-Fos). The RES-treated rats exhibited an increased nociceptive response to mechanical and chemical stimulation in the electronic von Frey and formalin tests, respectively. Moreover, these alterations preceded the motor impairment observed in the catalepsy test. In addition, the RES treatment reduced the TH-immunoreactivity in the ventral tegmental area (VTA) and increased the c-Fos expression in the ventral-lateral periaqueductal gray (vlPAG), rostral ventral medulla (RVM) and dorsal raphe nucleus (DRN) after noxious stimuli induced by formalin. Taken together, our results reinforce that nociceptive changes are one of the early signs of PD and monoamine depletion in basal ganglia can be involved in the abnormal processing of nociceptive information in PD.

Optogenetic Approach to Local Neuron Network Analysis of the Medullary Respiratory Center.

Using an optogenetic approach, we analyzed a local neuron network of the respiratory center in the medulla of a brainstem-spinal cord preparation isolated from neonatal rat. We developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch) or one of the step-function channelrhodopsin variants (ChRFR) under the control of Phox2b promoter-enhancer regions. Then, in en bloc preparations from 0- to 2-day-old Tg neonatal rats, we analyzed membrane potential changes of medullary respiratory-related neurons in response to photostimulation of the rostral ventral medulla. The photostimulation-induced inhibition or facilitation of the respiratory rhythm in Arch-expressing or ChRFR-expressing Tg rat preparations, respectively. Selective photoactivation of Phox2b-positive neurons expressing ChRFR in the rostral ventrolateral medulla of a neonatal rat en bloc preparation induced membrane potential changes of respiratory-related neurons that were dependent on heterogeneous properties of synaptic connections in the respiratory center. We concluded that the optogenetic approach is a powerful method of verifying a hypothetical model of local networks among respiratory-related neurons in the rostral ventrolateral medulla of neonatal rat.

Sex-steroid-dependent plasticity of brain-stem autonomic circuits.

In the central nervous system (CNS), nuclei of the brain stem play a critical role in the integration of peripheral sensory information and the regulation of autonomic output in mammalian physiology. The nucleus tractus solitarius of the brain stem acts as a relay center that receives peripheral sensory input from vagal afferents of the nodose ganglia, integrates information from within the brain stem and higher central centers, and then transmits autonomic efferent output through downstream premotor nuclei, such as the nucleus ambiguus, the dorsal motor nucleus of the vagus, and the rostral ventral lateral medulla. Although there is mounting evidence that sex and sex hormones modulate autonomic physiology at the level of the CNS, the mechanisms and neurocircuitry involved in producing these functional consequences are poorly understood. Of particular interest in this review is the role of estrogen, progesterone, and 5α-reductase-dependent neurosteroid metabolites of progesterone (e.g., allopregnanolone) in the modulation of neurotransmission within brain-stem autonomic neurocircuits. This review will discuss our understanding of the actions and mechanisms of estrogen, progesterone, and neurosteroids at the cellular level of brain-stem nuclei. Understanding the complex interaction between sex hormones and neural signaling plasticity of the autonomic nervous system is essential to elucidating the role of sex in overall physiology and disease.

Sodium pumps, ouabain and aldosterone in the brain: A neuromodulatory pathway underlying salt-sensitive hypertension and heart failure

Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The system involves distinct ion transport pathways including the alpha-2 isoform of the Na,K pump and epithelial sodium channels, as well as critical hormone elements such as angiotensin II, aldosterone, mineralocorticoid receptors and endogenous ouabain. Activation of this system either by circulating or central sodium ions and/or angiotensin II leads to a cascading sequence of events that begins in the hypothalamus and involves the participation of several brain nuclei including the subfornical organ, supraoptic and paraventricular nuclei and the rostral ventral medulla. Key events include heightened aldosterone synthesis and mineralocorticoid receptor activation, upregulation of epithelial sodium channels, augmented synthesis and secretion of endogenous ouabain from hypothalamic magnocellular neurons, and sustained increases in sympathetic outflow. The latter step depends upon increased production of angiotensin II and the primary amplification of angiotensin II type I receptor signaling from the paraventricular nucleus to the rostral ventral lateral medulla. The transmission of sympathetic traffic is secondarily amplified in the periphery by increased short- and long-term potentiation in sympathetic ganglia and by sustained actions of endogenous ouabain in the vascular wall that augment expression of sodium calcium exchange, increase cytosolic Ca2+ and heighten myogenic tone and contractility. Upregulation of this multi-amplifier system participates in forms of hypertension where salt, angiotensin and/or aldosterone are elevated and contributes to adverse outcomes in heart failure.

Cardiovascular Effects Of Acute Optogenetic Modulation Of Oxytocin‐PVN Neurons

The actions of oxytocin (OT), a neuropeptide that is largely synthesized in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus, has been implicated in a variety of homeostatic processes. Recent studies have demonstrated that chronic activation of OT neurons in the PVN prevents hypertension that is a consequence of intermittent hypoxia/hypercapnia. The present study combines genetically‐modified mice with in vivo optogenetics to determine the cardiovascular consequences of selective inhibition or excitation of OT neurons residing in the PVN. Male mice engineered to direct the expression of Cre recombinase to the OT gene had adenoassociated virus synthesizing an inhibitory opsin (Switch), an excitatory opsin (ChR2), or enhanced yellow fluorescent protein (eYFP) bilaterally injected into the PVN. Subsequently, mice were implanted with fiber optics targeting the PVN. One month later, mice were anesthetized with isoflurane and were implanted with a Millar catheter into their left common carotid artery for continuous recording of blood pressure and heart rate. Relative to the eYFP control group, OT‐Cre mice expressing Switch exhibited reduced blood pressure (−24±6 mmHg) and heart rate (−21±9 BPM) in response to optogenetic inhibition when compared with the eYFP group (4±2 mmHg and 5±4 bpm; n=3, p&lt;0.05). In contrast, in the ChR2‐injected group, blue light stimulation (1 min; 30 Hz; 20ms pulse width) significantly increased blood pressure by 15±2 mmHg while decreasing heart rate by −27±10 bpm when compared to controls (0±1mmHg and 9±5 bpm; n=5, p&lt;0.01. Neuroanatomical experiments revealed that neurons in the PVN that synthesize OT send dense axonal projections to the median eminence, the nucleus of the solitary tract, and the rostral ventral lateral medulla. Collectively, the results suggest that OT synthesizing neurons in the PVN mediate cardiovascular function possibly by affecting autonomic outflow and/or the activity of neuroendocrine axes.Support or Funding InformationNIH Funding supported the present studyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(237) Periaqueductal Gray Dopaminergic Neurons Modulate Peripheral Inflammatory Hyperalgesia

Neural dopamine (DA) modulators such as ᴅ-amphetamine and methylphenidate provide a promising new approach to treating pain. Though the mechanism by which they produce analgesia is poorly understood, these psychostimulants are antinociceptive and counteract the opioid-induced side effects of respiratory depression and sedation. Despite their potential, DA-modulating drugs have never been adopted clinically for pain relief, possibly due to a lack of understanding their mechanism of action. Considering previous evidence of a role of DA neurons from the periaqueductal gray (PAG) in pain modulation, we hypothesized that PAG DA neurons are antinociceptive and contribute significantly to the analgesic effect of ᴅ-amphetamine via descending spinal cord inhibition at the rostral ventral medulla (RVM). Male C57BL/6 mice that had received carrageenan in the hind paw were treated intraperitoneally with ᴅ-amphetamine, and its analgesic effect was compared with the analgesia produced by morphine. Separate groups of mice were stereotaxically prepared with infusion cannula targeted to either the PAG or RVM. We observed that the inhibition of thermal hyperalgesia in the mouse model of carrageenan-induced inflammatory pain by systemic ᴅ-amphetamine (6mg/kg) was similar to that produced by morphine (3mg/kg), and that ᴅ-amphetamine increased cFOS expression in PAG DA neurons. Targeted microinfusion of ᴅ-amphetamine, L-DOPA, or a selective D2 receptor agonist, but not a D1 agonist, into the PAG also attenuated thermal hyperalgesia, suggesting a role for D2 receptors in PAG-mediated analgesia. Finally, systemic ᴅ-amphetamine-mediated analgesia was prevented when neural transmission through the RVM was inhibited by local pretreatment with the GABAA receptor agonist muscimol. These findings indicate a modulatory role of D2 receptors and descending inhibition through GABAergic receptors at the RVM in ᴅ-amphetamine-mediated analgesia. Importantly, they indicate that the use of stimulants could enhance pain control, potentially as an alternative to opioids, avoiding the deleterious side-effects responsible for the current opioid crisis.

Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells

Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0–2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25–26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.