Molecularly defined circuits for cardiovascular and cardiopulmonary control.

The sympathetic and parasympathetic nervous systems regulate the activities of internal organs1, but the molecular and functional diversity of their constituent neurons and circuits remains largely unknown. Here we use retrograde neuronal tracing, single-cell RNA sequencing, optogenetics and physiological experiments to dissect the cardiac parasympathetic control circuit in mice. We show that cardiac-innervating neurons in the brainstem nucleus ambiguus (Amb) are comprised of two molecularly, anatomically and functionally distinct subtypes. The first, which we call ambiguus cardiovascular (ACV) neurons (approximately 35 neurons per Amb), define the classical cardiac parasympathetic circuit. They selectively innervate a subset of cardiac parasympathetic ganglion neurons and mediate the baroreceptor reflex, slowing heart rate and atrioventricular node conduction in response to increased blood pressure. The other, ambiguus cardiopulmonary (ACP) neurons (approximately 15 neurons per Amb) innervate cardiac ganglion neurons intermingled with and functionally indistinguishable from those innervated by ACV neurons. ACP neurons also innervate most or all lung parasympathetic ganglion neurons-clonal labelling shows that individual ACP neurons innervate both organs. ACP neurons mediate the dive reflex, the simultaneous bradycardia and bronchoconstriction that follows water immersion. Thus, parasympathetic control of the heart is organized into two parallel circuits, one that selectively controls cardiac function (ACV circuit) and another that coordinates cardiac and pulmonary function (ACP circuit). This new understanding of cardiac control has implications for treating cardiac and pulmonary diseases and for elucidating the control and coordination circuits of other organs.

Medullary kappa-opioid receptor neurons inhibit pain and itch through a descending circuit.

In perilous and stressful situations, the ability to suppress pain can be critical for survival. The rostral ventromedial medulla contains neurons that robustly inhibit nocioception at the level of the spinal cord through a top-down modulatory pathway. Although much is known about the role of the rostral ventromedial medulla in the inhibition of pain, the precise ability to directly manipulate pain-inhibitory neurons in the rostral ventromedial medulla has never been achieved. We now expose a cellular circuit that inhibits nocioception and itch in mice. Through a combination of molecular, tracing and behavioural approaches, we found that rostral ventromedial medulla neurons containing the kappa-opioid receptor inhibit itch and nocioception. With chemogenetic inhibition, we uncovered that these neurons are required for stress-induced analgesia. Using intersectional chemogenetic and pharmacological approaches, we determined that rostral ventromedial medulla kappa-opioid receptor neurons inhibit nocioception and itch through a descending circuit. Lastly, we identified a dynorphinergic pathway arising from the periaqueductal grey that modulates nociception within the rostral ventromedial medulla. These discoveries highlight a distinct population of rostral ventromedial medulla neurons capable of broadly and robustly inhibiting itch and nocioception.

Development of the human hypoglossal nucleus from mid-gestation to the perinatal period: A morphological study.

INTRODUCTION: The human hypoglossal nucleus (nXII) was morphologically examined from mid-gestation to the perinatal period. MATERIALS/METHODS: Serial brain sections from 6 preterm and 4 perinatal infants aged 21-43 postmenstrual weeks (PW) were stained with the Klüver-Barrera method. Following microscopic observation, morphometric parameters (volume, neuronal number, and neuronal profile area [PA]) were analysed. RESULTS: Two types of neurons, motor and non-motor neurons, were observed at 21 PW. The motor neurons were distributed into clusters, which were not completely separated. The non-motor neurons were dispersed among the motor neurons. Myelination of the hypoglossal nerve roots was noted at 21 PW, when degenerated neurons were sporadically encountered. To a lesser extent, they were seen until 35 PW. The nXII volume increased exponentially with age. Conversely, the neuronal numerical density decreased exponentially, while the total number remained relatively stable. The neuronal PA increased gradually, with a greater rate of increase measured in the caudal part. CONCLUSIONS: In the human nXII, motor and non-motor neurons are distinguishable from mid-gestation. Then, while the nXII expands exponentially in volume, the two types of neurons change in number and PA almost in parallel during the second half of gestation. Natural neuronal death may also occur.

Expression of nischarin, an imidazoline 1 receptor candidate protein, in the ventrolateral medulla of newborn rats.

The activation of imidazoline 1 (I1) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3-5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats.

Encoding of limb state by single neurons in the cuneate nucleus of awake monkeys.

The cuneate nucleus (CN) is among the first sites along the neuraxis where proprioceptive signals can be integrated, transformed, and modulated. The objective of the study was to characterize the proprioceptive representations in CN. To this end, we recorded from single CN neurons in three monkeys during active reaching and passive limb perturbation. We found that many neurons exhibited responses that were tuned approximately sinusoidally to limb movement direction, as has been found for other sensorimotor neurons. The distribution of their preferred directions (PDs) was highly nonuniform and resembled that of muscle spindles within individual muscles, suggesting that CN neurons typically receive inputs from only a single muscle. We also found that the responses of proprioceptive CN neurons tended to be modestly amplified during active reaching movements compared to passive limb perturbations, in contrast to cutaneous CN neurons whose responses were not systematically different in the active and passive conditions. Somatosensory signals thus seem to be subject to a "spotlighting" of relevant sensory information rather than uniform suppression as has been suggested previously.NEW & NOTEWORTHY The cuneate nucleus (CN) is the somatosensory gateway into the brain, and only recently has it been possible to record these signals from an awake animal. We recorded single CN neurons in monkeys. Proprioceptive CN neurons appear to receive input from very few muscles, and their sensitivity to movement changes reliably during reaching relative to passive arm perturbations. Sensitivity is generally increased, but not exclusively so, as though CN "spotlights" critical proprioceptive information during reaching.

Orexin-A directly depolarizes dorsomedial hypothalamic neurons, including those innervating the rostral ventrolateral medulla.

The dorsomedial hypothalamus (DMH) receives dense orexinergic innervation. Intra-DMH application of orexins increases arterial pressure and heart rate in rats. We studied the effects of orexin-A on DMH neurons, including those innervating the medullary cardiovascular center, the rostral ventrolateral medulla (RVLM), by using whole-cell recordings in brain slices. In the presence of tetrodotoxin, orexin-A (30-1000 nM) depolarized 56% of DMH neurons (EC50 82.4 ± 4.4 nM). Under voltage-clamp recording, orexin-A (300 nM) induced three types of responses characterized by different current-voltage relationships, namely unchanged, increased, and decreased slope conductance in 68%, 14%, and 18% of orexin-A-responsive neurons, respectively. The reversal potential of the decreased-conductance response was near the equilibrium potential of K+ and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is the underlying mechanism. In a low-Na+ solution, unchanged-, increased-, and decreased-conductance responses were observed in 56%, 11%, and 33% of orexin-A-responsive neurons, respectively, implying that a non-selective cation current (NSCC) underlies orexin-A-induced responses in a small population of DMH neurons. KBR-7943 (70 µM), an inhibitor of Na+-Ca2+ exchanger (NCX), suppressed orexin-A-induced depolarization in 7 of 10 neurons. In the presence of KBR-7943, the majority of orexin-A-responsive neurons exhibited decreased-conductance responses. These findings suggest that NCX activation may underlie orexin-A-induced depolarization in the majority of orexin-responsive DMH neurons. Of 19 RVLM-projecting DMH neurons identified by retrograde labeling, 17 (90%) were orexin-A responsive. In conclusion, orexin-A directly excited over half of DMH neurons, including those innervating the RVLM, through decreasing K+ conductance, activating NCX, and/or increasing NSCC.

The retrotrapezoid nucleus and the neuromodulation of breathing.

Breathing is regulated by a host of arousal and sleep-wake state-dependent neuromodulators to maintain respiratory homeostasis. Modulators such as acetylcholine, norepinephrine, histamine, serotonin (5-HT), adenosine triphosphate (ATP), substance P, somatostatin, bombesin, orexin, and leptin can serve complementary or off-setting functions depending on the target cell type and signaling mechanisms engaged. Abnormalities in any of these modulatory mechanisms can destabilize breathing, suggesting that modulatory mechanisms are not overly redundant but rather work in concert to maintain stable respiratory output. The present review focuses on the modulation of a specific cluster of neurons located in the ventral medullary surface, named retrotrapezoid nucleus, that are activated by changes in tissue CO2/H+ and regulate several aspects of breathing, including inspiration and active expiration.

A functional map for diverse forelimb actions within brainstem circuitry.

The brainstem is a key centre in the control of body movements. Although the precise nature of brainstem cell types and circuits that are central to full-body locomotion are becoming known1-5, efforts to understand the neuronal underpinnings of skilled forelimb movements have focused predominantly on supra-brainstem centres and the spinal cord6-12. Here we define the logic of a functional map for skilled forelimb movements within the lateral rostral medulla (latRM) of the brainstem. Using in vivo electrophysiology in freely moving mice, we reveal a neuronal code with tuning of latRM populations to distinct forelimb actions. These include reaching and food handling, both of which are impaired by perturbation of excitatory latRM neurons. Through the combinatorial use of genetics and viral tracing, we demonstrate that excitatory latRM neurons segregate into distinct populations by axonal target, and act through the differential recruitment of intra-brainstem and spinal circuits. Investigating the behavioural potential of projection-stratified latRM populations, we find that the optogenetic stimulation of these populations can elicit diverse forelimb movements, with each behaviour stably expressed by individual mice. In summary, projection-stratified brainstem populations encode action phases and together serve as putative building blocks for regulating key features of complex forelimb movements, identifying substrates of the brainstem for skilled forelimb behaviours.

A ventrolateral medulla-midline thalamic circuit for hypoglycemic feeding.

Marked deficits in glucose availability, or glucoprivation, elicit organism-wide counter-regulatory responses whose purpose is to restore glucose homeostasis. However, while catecholamine neurons of the ventrolateral medulla (VLMCA) are thought to orchestrate these responses, the circuit and cellular mechanisms underlying specific counter-regulatory responses are largely unknown. Here, we combined anatomical, imaging, optogenetic and behavioral approaches to interrogate the circuit mechanisms by which VLMCA neurons orchestrate glucoprivation-induced food seeking behavior. Using these approaches, we found that VLMCA neurons form functional connections with nucleus accumbens (NAc)-projecting neurons of the posterior portion of the paraventricular nucleus of the thalamus (pPVT). Importantly, optogenetic manipulations revealed that while activation of VLMCA projections to the pPVT was sufficient to elicit robust feeding behavior in well fed mice, inhibition of VLMCA-pPVT communication significantly impaired glucoprivation-induced feeding while leaving other major counterregulatory responses intact. Collectively our findings identify the VLMCA-pPVT-NAc pathway as a previously-neglected node selectively controlling glucoprivation-induced food seeking. Moreover, by identifying the ventrolateral medulla as a direct source of metabolic information to the midline thalamus, our results support a growing body of literature on the role of the PVT in homeostatic regulation.

Identification of a brainstem locus that inhibits tumor necrosis factor.

In the brain, compact clusters of neuron cell bodies, termed nuclei, are essential for maintaining parameters of host physiology within a narrow range optimal for health. Neurons residing in the brainstem dorsal motor nucleus (DMN) project in the vagus nerve to communicate with the lungs, liver, gastrointestinal tract, and other organs. Vagus nerve-mediated reflexes also control immune system responses to infection and injury by inhibiting the production of tumor necrosis factor (TNF) and other cytokines in the spleen, although the function of DMN neurons in regulating TNF release is not known. Here, optogenetics and functional mapping reveal cholinergic neurons in the DMN, which project to the celiac-superior mesenteric ganglia, significantly increase splenic nerve activity and inhibit TNF production. Efferent vagus nerve fibers terminating in the celiac-superior mesenteric ganglia form varicose-like structures surrounding individual nerve cell bodies innervating the spleen. Selective optogenetic activation of DMN cholinergic neurons or electrical activation of the cervical vagus nerve evokes action potentials in the splenic nerve. Pharmacological blockade and surgical transection of the vagus nerve inhibit vagus nerve-evoked splenic nerve responses. These results indicate that cholinergic neurons residing in the brainstem DMN control TNF production, revealing a role for brainstem coordination of immunity.

Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata.

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area - reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.

Neurons in the Nucleus papilio contribute to the control of eye movements during REM sleep.

Rapid eye movements (REM) are characteristic of the eponymous phase of sleep, yet the underlying motor commands remain an enigma. Here, we identified a cluster of Calbindin-D28K-expressing neurons in the Nucleus papilio (NPCalb), located in the dorsal paragigantocellular nucleus, which are active during REM sleep and project to the three contralateral eye-muscle nuclei. The firing of opto-tagged NPCalb neurons is augmented prior to the onset of eye movements during REM sleep. Optogenetic activation of NPCalb neurons triggers eye movements selectively during REM sleep, while their genetic ablation or optogenetic silencing suppresses them. None of these perturbations led to a change in the duration of REM sleep episodes. Our study provides the first evidence for a brainstem premotor command contributing to the control of eye movements selectively during REM sleep in the mammalian brain.

Firing properties of medullary expiratory neurons during fictive straining in cats.

Expiratory (E) neurons in the caudal nucleus retroambigualis extend descending spinal axons to the lumbar and sacral spinal cord. Discharge rates of single E neurons were recorded to examine differences in activity of E neurons projecting to the lumbar or sacral spinal cord during fictive straining induced by distention of the colon with a balloon. Firing frequencies of E neurons with descending axons in the thoracic and lumbar spinal cord increased during the repetitive rise of rectum pressure, whereas those of E neurons with descending axons in the sacral spinal cord decreased. E neurons with descending axons in the thoracic/lumbar and sacral spinal cord exhibit different firing characteristics during the repetitive rise of rectum pressure when straining during defecation. The activity of abdominal nerves during fictive straining is in phase with changes in rectum pressure, but out of phase with the activity of E neurons.

Rostral ventrolateral medullary catecholaminergic neurones mediate irregular breathing pattern in volume overload heart failure rats.

KEY POINTS: A strong association between disordered breathing patterns, elevated sympathetic activity, and enhanced central chemoreflex drive has been shown in experimental and human heart failure (HF). The aim of this study was to determine the contribution of catecholaminergic rostral ventrolateral medulla catecholaminergic neurones (RVLM-C1) to both haemodynamic and respiratory alterations in HF. Apnoea/hypopnoea incidence (AHI), breathing variability, respiratory-cardiovascular coupling, cardiac autonomic control and cardiac function were analysed in HF rats with or without selective ablation of RVLM-C1 neurones. Partial lesion (∼65%) of RVLM-C1 neurones reduces AHI, respiratory variability, and respiratory-cardiovascular coupling in HF rats. In addition, the deleterious effects of central chemoreflex activation on cardiac autonomic balance and cardiac function in HF rats was abolished by ablation of RVLM-C1 neurones. Our findings suggest that RVLM-C1 neurones play a pivotal role in breathing irregularities in volume overload HF, and mediate the sympathetic responses induced by acute central chemoreflex activation. ABSTRACT: Rostral ventrolateral medulla catecholaminergic neurones (RVLM-C1) modulate sympathetic outflow and breathing under normal conditions. Heart failure (HF) is characterized by chronic RVLM-C1 activation, increased sympathetic activity and irregular breathing patterns. Despite studies showing a relationship between RVLM-C1 and sympathetic activity in HF, no studies have addressed a potential contribution of RVLM-C1 neurones to irregular breathing in this context. Thus, the aim of this study was to determine the contribution of RVLM-C1 neurones to irregular breathing patterns in HF. Sprague-Dawley rats underwent surgery to induce volume overload HF. Anti-dopamine ß-hydroxylase-saporin toxin (DßH-SAP) was used to selectively lesion RVLM-C1 neurones. At 8 weeks post-HF induction, breathing pattern, blood pressures (BP), respiratory-cardiovascular coupling (RCC), central chemoreflex function, cardiac autonomic control and cardiac function were studied. Reduction (∼65%) of RVLM-C1 neurones resulted in attenuation of irregular breathing, decreased apnoea-hypopnoea incidence (11.1 ± 2.9 vs. 6.5 ± 2.5 events h-1 ; HF+Veh vs. HF+DßH-SAP; P < 0.05) and improved cardiac autonomic control in HF rats. Pathological RCC was observed in HF rats (peak coherence >0.5 between breathing and cardiovascular signals) and was attenuated by DßH-SAP treatment (coherence: 0.74 ± 0.12 vs. 0.54 ± 0.10, HF+Veh vs. HF+DßH-SAP rats; P < 0.05). Central chemoreflex activation had deleterious effects on cardiac function and cardiac autonomic control in HF rats that were abolished by lesion of RVLM-C1 neurones. Our findings reveal that RVLM-C1 neurones play a major role in irregular breathing patterns observed in volume overload HF and highlight their contribution to cardiac dysautonomia and deterioration of cardiac function during chemoreflex activation.

Persistent postoperative pain: mechanisms and modulators.

PURPOSE OF REVIEW: Persistent postoperative pain (PPP) is a significant source of morbidity in our population. An excellent opportunity to understand the transition from acute to chronic pain states. Understanding the mechanisms that drive this and modulators that influence this transition is essential to both prevent and manage this condition. RECENT FINDINGS: Although the exact mechanism for the development of PPP is still poorly understood, hypotheses abound. Basic science research with animal models implicates nociceptive and neuropathic pain signals leading to pain sensitization due to persistent noxious signaling. Effects on the inhibitory modulation of noxious signaling in medullary-spinal pathways and descending modulation have also been implicated. SUMMARY: Persistent maladaptive neuroplastic changes secondary to neurotrophic factors and interactions between neurons and microglia may well explain the phenomenon. This article reviews the current thought processes on mechanisms and modulators from a basic science and epidemiological perspective.

N-Cadherin Orchestrates Self-Organization of Neurons within a Columnar Unit in the Drosophila Medulla.

Columnar structure is a basic unit of the brain, but the mechanism underlying its development remains largely unknown. The medulla, the largest ganglion of the Drosophila melanogaster visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. In this study, using N-cadherin (Ncad) as a marker, we reveal the donut-like columnar structures along the 2D layer in the larval medulla that evolves to form three distinct layers in pupal development. Column formation is initiated by three core neurons, R8, R7, and Mi1, which establish distinct concentric domains within a column. We demonstrate that Ncad-dependent relative adhesiveness of the core columnar neurons regulates their relative location within a column along a 2D layer in the larval medulla according to the differential adhesion hypothesis. We also propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns.SIGNIFICANCE STATEMENT The columnar structure is a basic unit of the brain, but its developmental mechanism remains unknown. The medulla, the largest ganglion of the fly visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. We reveal that column formation is initiated by three core neurons that establish distinct concentric domains within a column. We demonstrate the in vivo evidence of N-cadherin-dependent differential adhesion among the core columnar neurons within a column along a 2D layer in the larval medulla. The 2D larval columns evolve to form three distinct layers in the pupal medulla. We propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns.

Afferent neuropeptide Y projections to the ventral tegmental area in normal-weight male Wistar rats.

The hypothalamic neuropeptide Y (NPY) circuitry is a key regulator of feeding behavior. NPY also acts in the mesolimbic dopaminergic circuitry, where it can increase motivational aspects of feeding behavior through effects on dopamine output in the nucleus accumbens (NAc) and on neurotransmission in the ventral tegmental area (VTA). Endogenous NPY in the NAc originates from local interneurons and afferent projections from the hypothalamic arcuate nucleus (Arc). However, the origin of endogenous NPY in the VTA is unknown. We determined, in normal-weight male Wistar rats, if the source of VTA NPY is local, and/or whether it is derived from VTA-projecting neurons. Immunocytochemistry, in situ hybridization and RT-qPCR were utilized, when appropriate in combination with colchicine treatment or 24 hr fasting, to assess NPY/Npy expression locally in the VTA. Retrograde tracing using cholera toxin beta (CTB) in the VTA, fluorescent immunocytochemistry and confocal microscopy were used to determine NPY-immunoreactive afferents to the VTA. NPY in the VTA was observed in fibers, but not following colchicine pretreatment. No NPY- or Npy-expressing cell bodies were observed in the VTA. Fasting for 24 hr, which increased Npy expression in the Arc, failed to induce Npy expression in the VTA. Double-labeling with CTB and NPY was observed in the Arc and in the ventrolateral medulla. Thus, VTA NPY originates from the hypothalamic Arc and the ventrolateral medulla of the brainstem in normal-weight male Wistar rats. These afferent connections link hypothalamic and brainstem processing of physiologic state to VTA-driven motivational behavior.

Ablation of brainstem C1 neurons improves cardiac function in volume overload heart failure.

Activation of the sympathetic nervous system is a hallmark of heart failure (HF) and is positively correlated with disease progression. Catecholaminergic (C1) neurons located in the rostral ventrolateral medulla (RVLM) are known to modulate sympathetic outflow and are hyperactivated in volume overload HF. However, there is no conclusive evidence showing a contribution of RVLM-C1 neurons to the development of cardiac dysfunction in the setting of HF. Therefore, the aim of this study was to determine the role of RVLM-C1 neurons in cardiac autonomic control and deterioration of cardiac function in HF rats. A surgical arteriovenous shunt was created in adult male Sprague-Dawley rats to induce HF. RVLM-C1 neurons were selectively ablated using cell-specific immunotoxin (dopamine-ß hydroxylase saporin [DßH-SAP]) and measures of cardiac autonomic tone, function, and arrhythmia incidence were evaluated. Cardiac autonomic imbalance, arrhythmogenesis and cardiac dysfunction were present in HF rats and improved after DßH-SAP toxin treatment. Most importantly, the progressive decline in fractional shortening observed in HF rats was reduced by DßH-SAP toxin. Our results unveil a pivotal role played by RVLM-C1 neurons in cardiac autonomic imbalance, arrhythmogenesis and cardiac dysfunction in volume overload-induced HF.

Microglia in the RVLM of SHR have reduced P2Y12R and CX3CR1 expression, shorter processes, and lower cell density.

The RVLM of spontaneously hypertensive rats (SHR) contains over-active C1 neurons, which model the pathology of essential hypertension. Hypertension involves chronic low-grade neuroinflammation. Inflammation in the brain is produced and maintained primarily by microglia. We assessed microglial gene expression (P2Y12R and CX3CR1) and morphology in the RVLM of SHR compared to normotensive Wistar-Kyoto rats (WKY). The gene expression of the metabotropic purinergic receptor P2Y12 and the fractalkine receptor CX3CR1 was downregulated in the RVLM of SHR compared to WKY (by 37.3% and 30.9% respectively). P2Y12R and CX3CR1 are required for normal microglial function, and reduced P2Y12R expression is associated with changes in microglial activity. Histological analysis showed a 22.9% reduction in microglial cell density, along with 18.7% shorter microglial processes, a phenotypic indicator of activation, in the RVLM of SHR compared to WKY. These results indicate a subtle loss of function, or a mild state of inflammation, in the RVLM microglia of SHR.

Serotonergic projections to the ventral respiratory column from raphe nuclei in rats.

The ventral respiratory column (VRC) generates rhythmical respiration and is divided into four compartments: the Bötzinger complex (BC), pre-Bötzinger complex (PBC), rostral ventral respiratory group (rVRG), and caudal ventral respiratory group (cVRG). Serotonergic nerve fibers are densely distributed in the rostral to caudal VRC and serotonin would be one of the important modulators for the respiratory control in the VRC. In the present study, to elucidate detailed distribution of serotonergic neurons in raphe nuclei projecting to the various rostrocaudal levels of VRC, we performed combination of retrograde tracing technique by cholera toxin B subunit (CTB) with immunohistochemistry for tryptophan hydroxylase 2 (TPH2). The double-immunoreactive neurons with CTB and TPH2 were distributed in the both rostral and caudal raphe nuclei, i.e. dorsal raphe nucleus, raphe magnus nucleus, gigantocellular reticular nucleus alpha and ventral parts, lateral paragigantocellular nucleus, parapyramidal area, raphe obscurus nucleus, and raphe pallidus nucleus. The distributions of double-immunoreactive neurons were similar among injection groups of BC, PBC, anterior rVRG, and posterior rVRG/cVRG. In conclusion, serotonergic neurons in both rostral and caudal raphe nuclei projected throughout the VRC and these serotonergic projections may contribute to respiratory responses to various environmental and vital changes.