Neurons In Medulla Research Articles

Rostral ventrolateral medulla neuron activity is suppressed by Klotho and stimulated by FGF23 in newborn Wistar rats.

Hypertension often occurs in patients with chronic kidney disease (CKD). Considering the decrease in serum Klotho and increase in serum FGF23 levels in such patients, decreased Klotho and increased FGF23 levels were thought to be associated with hypertension. Presympathetic neurons at the rostral ventrolateral medulla (RVLM) contribute to sympathetic activity and regulation of blood pressure. Therefore, we hypothesized that Klotho would reduce the activities of RVLM neurons and FGF23 would stimulate them. Accordingly, this study examined the effects of Klotho and FGF23 on bulbospinal neurons in the RVLM. We used a brainstem-spinal cord preparation to record from RVLM presympathetic neurons and to evaluate the effects of Klotho and FGF23 on firing rate and membrane potentials of these neurons. Our results showed that Klotho-induced RVLM neuron hyperpolarization, while ouabain, a Na+/K+-ATPase inhibitor, suppressed the effects of Klotho on such neurons. Moreover, FGF23 induced RVLM neuron depolarization, while SU5402, an FGF23 receptor (FGFR1) antagonist, induced RVLM neuron hyperpolarization. Histological examinations revealed that Klotho, Na+/K+-ATPase, FGF23, and FGFR1 were present in RVLM neurons and that Klotho was localized in the same neurons as FGFR1. These results suggest that Klotho and FG23 regulate the activity of RVLM neurons. Klotho may reduce the activity of RVLM neurons via stimulating Na+/K+-ATPase on those neurons while FGF23 may activate those neurons via FGFR1.

Responses of neurons in the rostral ventrolateral medulla of conscious cats to anticipated and passive movements.

The vestibular system contributes to regulating sympathetic nerve activity and blood pressure. Initial studies in decerebrate animals showed that neurons in the rostral ventrolateral medulla (RVLM) respond to small-amplitude (<10°) rotations of the body, as in other brain areas that process vestibular signals, although such movements do not affect blood distribution in the body. However, a subsequent experiment in conscious animals showed that few RVLM neurons respond to small-amplitude movements. This study tested the hypothesis that RVLM neurons in conscious animals respond to signals from the vestibular otolith organs elicited by large-amplitude static tilts. The activity of approximately one-third of RVLM neurons whose firing rate was related to the cardiac cycle, and thus likely received baroreceptor inputs, was modulated by vestibular inputs elicited by 40° head-up tilts in conscious cats, but not during 10° sinusoidal rotations in the pitch plane that affected the activity of neurons in brain regions providing inputs to the RVLM. These data suggest the existence of brain circuitry that suppresses vestibular influences on the activity of RVLM neurons and the sympathetic nervous system unless these inputs are physiologically warranted. We also determined that RVLM neurons failed to respond to a light cue signaling the movement, suggesting that feedforward cardiovascular responses do not occur before passive movements that require cardiovascular adjustments.

GABA and glycine neurons from the ventral medullary region inhibit hypoglossal motoneurons.

Obstructive sleep apnea (OSA) is a common disorder characterized by repetitive sleep-related losses of upper airway patency that occur most frequently during rapid eye movement (REM) sleep. Hypoglossal motoneurons play a key role in regulating upper airway muscle tone and patency during sleep. REM sleep activates GABA and glycine neurons in the ventral medulla (VM) to induce cortical desynchronization and skeletal muscle atonia during REM sleep; however, the role of this brain region in modulating hypoglossal motor activity is unknown. We combined optogenetic and chemogenetic approaches with in-vitro and in-vivo electrophysiology, respectfully, in GAD2-Cre mice of both sexes to test the hypothesis that VM GABA/glycine neurons control the activity of hypoglossal motoneurons and tongue muscles. Here, we show that there is a pathway originating from GABA/glycine neurons in the VM that monosynaptically inhibits brainstem hypoglossal motoneurons innervating both tongue protruder genioglossus (GMNs) and retractor (RMNs) muscles. Optogenetic activation of ChR2-expressing fibers induced a greater postsynaptic inhibition in RMNs than in GMNs. In-vivo chemogenetic activation of VM GABA/glycine neurons produced an inhibitory effect on tongue electromyographic (EMG) activity, decreasing both the amplitude and duration of inspiratory-related EMG bursts without any change in respiratory rate. These results indicate that activation of GABA/glycine neurons from the VM inhibits tongue muscles via a direct pathway to both GMNs and RMNs. This inhibition may play a role in REM sleep associated upper airway obstructions that occur in patients with OSA.

A mouse model of RBD: inducing synucleinopathy in GABA/glycine neurons in the ventral medulla

A mouse model of RBD: inducing synucleinopathy in GABA/glycine neurons in the ventral medulla

Subregional differences in GABAA receptor subunit expression in the rostral ventrolateral medulla of sedentary versus physically active rats.

Neurons in the rostral ventrolateral medulla (RVLM) regulate blood pressure through direct projections to spinal sympathetic preganglionic neurons. Only some RVLM neurons are active under resting conditions due to significant, tonic inhibition by gamma-aminobutyric acid (GABA). Withdrawal of GABAA receptor-mediated inhibition of the RVLM increases sympathetic outflow and blood pressure substantially, providing a mechanism by which the RVLM could contribute chronically to cardiovascular disease (CVD). Here, we tested the hypothesis that sedentary conditions, a major risk factor for CVD, increase GABAA receptors in RVLM, including its rostral extension (RVLMRE ), both of which contain bulbospinal catecholamine (C1) and non-C1 neurons. We examined GABAA receptor subunits GABAAα1 and GABAAα2 in the RVLM/RVLMRE of sedentary or physically active (10-12 weeks of wheel running) rats. Western blot analyses indicated that sedentary rats had lower expression of GABAAα1 and GABAAα2 subunits in RVLM but only GABAAα2 was lower in the RVLMRE of sedentary rats. Sedentary rats had significantly reduced expression of the chloride transporter, KCC2, suggesting less effective GABA-mediated inhibition compared to active rats. Retrograde tracing plus triple-label immunofluorescence identified fewer bulbospinal non-C1 neurons immunoreactive for GABAAα1 but a higher percentage of bulbospinal C1 neurons immunoreactive for GABAAα1 in sedentary animals. Sedentary conditions did not significantly affect the number of bulbospinal C1 or non-C1 neurons immunoreactive for GABAAα2 . These results suggest a complex interplay between GABAA receptor expression by spinally projecting C1 and non-C1 neurons and sedentary versus physically active conditions. They also provide plausible mechanisms for both enhanced sympathoexcitatory and sympathoinhibitory responses following sedentary conditions.

GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing.

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.

Sequential activation of multiple persistent inward currents induces staircase currents in serotonergic neurons of medulla in ePet-EYFP mice.

Persistent inward currents (PICs) are widely reported in rodent spinal neurons. A distinctive pattern observed recently is staircase-like PICs induced by voltage ramp in serotonergic neurons of mouse medulla. The mechanism underlying this pattern of PICs is unclear. Combining electrophysiological, pharmacological, and computational approaches, we investigated the staircase PICs in serotonergic neurons of medulla in ePet-EYFP transgenic mice (postnatal days 1-7). Staircase PICs induced by 10-s voltage biramps were observed in 70% of serotonergic neurons (n = 73). Staircase PICs activated at -48.8 ± 5 mV and consisted of two components, with the first PIC of 45.8 ± 51 pA and the second PIC of 197.3 ± 126 pA (n = 51). Staircase PICs were also composed of low-voltage-activated sodium PIC (Na-PIC; onset -46.2 ± 5 mV, n = 34), high-voltage-activated calcium PIC (Ca-PIC; onset -29.3 ± 6 mV, n = 23), and high-voltage-activated tetrodotoxin (TTX)- and dihydropyridine-resistant sodium PIC (TDR-PIC; onset -16.8 ± 4 mV, n = 28). Serotonergic neurons expressing Na-PIC, Ca-PIC, and TDR-PIC were evenly distributed in medulla. Bath application of 1-2 μM TTX blocked the first PIC and decreased the second PIC by 36% (n = 23, P < 0.05). Nimodipine (25 μM) reduced the second PIC by 38% (n = 34, P < 0.001) without altering the first PIC. TTX and nimodipine removed the first PIC and reduced the second PIC by 59% (n = 28, P < 0.01). A modeling study mimicked the staircase PICs and verified experimental conclusions that sequential activation of Na-PIC, Ca-PIC, and TDR-PIC in order of voltage thresholds induced staircase PICs in serotonergic neurons. Further experimental results suggested that the multiple components of staircase PICs play functional roles in regulating excitability of serotonergic neurons in medulla.NEW & NOTEWORTHY Staircase persistent inward currents (PICs) are mediated by activation of L-type calcium channels in dendrites of mouse spinal motoneurons. A novel mechanism is explored in this study. Here we report that the staircase PICs are mediated by sequentially activating sodium and calcium PICs in serotonergic neurons of mouse medulla.

Effects of age on feeding response: Focus on the rostral C1 neuron and its glucoregulatory proteins.

Older people are likely to develop anorexia of aging. Rostral C1 (rC1) catecholaminergic neurons in rostral ventrolateral medulla (RVLM) are recently discovered its role in food intake control. It is well established that these neurons regulate cardiovascular function. This study aims to determine the effect of age on the function of rostral C1 (rC1) neurons in mediating feeding response. Male Sprague Dawley rats at 3-months (n = 22) and 24-months (n = 22) old were used and further divided into two subgroups; 1) treatment group with 2-deoxy-d-glucose (2DG) and 2) vehicle group. Feeding hormones such as cholecystokinin (CCK), ghrelin and leptin were analysed using enzyme-linked immunosorbent assay (ELISA). Rat brain was carefully dissected to obtain the brainstem RVLM region. Further analysis was carried out to determine the level of proteins and genes in RVLM that were associated with feeding pathway. Protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK) and neuropeptide Y Y5 receptor (NPY5R) were determined by western blot. Expression of TH, AMPK and NPY genes were determined by real-time PCR. This study showed that blood glucose level was elevated in young and old rats following 2DG administration. Plasma CCK-8 concentration was higher in the aged rats at basal and increased with 2DG administration in young rats, but the leptin and ghrelin showed no changes. Old rats showed higher TH and lower AMPK mRNA levels. Glucoprivation decreased AMPK mRNA level in young rats and decreased TH mRNA in old rats. Aged rC1 neurons showed higher NPY5R protein level. Following glucoprivation, rC1 neurons produced distinct molecular changes across age in which, in young rats, AMPK phosphorylation level was increased and in old rats, TH phosphorylation level was increased. These findings suggest that glucose-counterregulatory responses by rC1 neurons at least, contribute to the ability of young and old rats in coping glucoprivation. Age-induced molecular changes within rC1 neurons may attenuate the glucoprivic responses. This situation may explain the impairment of feeding response in the elderly.

Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells.

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

Slit neuronal secretion coordinates optic lobe morphogenesis in Drosophila

The complexity of the nervous system requires the coordination of multiple cellular processes during development. Among them, we find boundary formation, axon guidance, cell migration and cell segregation. Understanding how different cell populations such as glial cells, developing neurons and neural stem cells contribute to the formation of boundaries and morphogenesis in the nervous system is a critical question in neurobiology. Slit is an evolutionary conserved protein essential for the development of the nervous system. For signaling, Slit has to bind to its cognate receptor Robo, a single-pass transmembrane protein. Although the Slit/Robo signaling pathway is well known for its involvement in axon guidance, it has also been associated to boundary formation in the Drosophila visual system. In the optic lobe, Slit is expressed in glial cells, positioned at the boundaries between developing neuropils, and in neurons of the medulla ganglia. Although it has been assumed that glial cells provide Slit to the system, the contribution of the neuronal expression has not been tested. Here, we show that, contrary to what was previously thought, Slit protein provided by medulla neurons is also required for boundary formation and morphogenesis of the optic lobe. Furthermore, tissue specific rescue using modified versions of Slit demonstrates that this protein acts at long range and does not require processing by extracellular proteases. Our data shed new light on our understanding of the cellular mechanisms involved in Slit function in the fly visual system morphogenesis.

Differential responses of neurons in the rat caudal ventrolateral medulla to visceral and somatic noxious stimuli and their alterations in colitis

Visceral and somatic types of pain have been reported to manifest crucial differences not only in the experience, but also in their peripheral and central processing. However, the precise neuronal mechanisms that responsible for the modality-specific transmission of pain signals, especially at the supraspinal level, remain unclear. Very little is known also about the potential involvement of such mechanisms in the development of viscero-somatic hyperalgesia. Therefore, in the present study performed on urethane-anesthetized adult male Wistar rats we examined responses of neurons in the caudal ventrolateral medulla (CVLM)—the first site for supraspinal processing of both internal and external pain signals—to visceral (colorectal distension, CRD) and somatic (squeezing of the tail) noxious stimulations and evaluated alterations in response properties of these cells after the induction of colitis. It has been found out that the CVLM of healthy control rats, along with harboring of cells excited by both stimulations (23.7%), contained neurons that were activated by either visceral (31.9%) or somatic noxious stimuli (44.4%). In inflamed animals, the percentages of the visceral and somatic nociceptive cells were decreased (to 18.3% and 34.3%, correspondingly) and the number of bimodal neurons was increased (up to 47.4%); these alterations were associated with substantially enhanced responses of both the modality-specific and convergent CVLM neurons not only to CRD, but also to squeezing of the tail. Under these conditions, visceral and somatic pain stimuli induced similar changes in arterial blood pressure and respiratory rate, whereas in the absence of intestinal inflammation noxious CRD and tail stimulation evoked predominantly divergent autonomic reactions. The data obtained can benefit to a deeper understanding of the neuronal mechanisms that underlie differential supraspinal processing of visceral and somatic noxious stimuli and can potentially contribute to the realization of specific cardiovascular and respiratory accompaniments inherent to a particular type of pain. Therewith, results of the study elucidate colitis-induced alterations in these mechanisms, which may be responsible for the combined development of visceral hypersensitivity and somatic hyperalgesia.

On the presence and functional significance of sympathetic premotor neurons with collateralized spinal axons in the rat.

Spinally-projecting neurons of the rostral ventrolateral medulla (RVLM) determine sympathetic outflow to different territories of the body. Previous studies suggest the existence of RVLM neurons with distinct functional classes, such as neurons that target sympathetic nerves bound for functionally-similar tissue types (e.g. muscle vasculature). The existence of RVLM neurons with more general actions had not been critically tested. Using viral tracing, we show that a significant minority of RVLM neurons send axon collaterals to disparate spinal segments (T2 and T10 ). Furthermore, optogenetic activation of sympathetic premotor neurons projecting to lumbar spinal segments also produced activation of sympathetic nerves from rostral spinal segments that innervate functionally diverse tissues (heart and forelimb muscle). These findings suggest the existence of individual RVLM neurons for which the axons branch to drive sympathetic preganglionic neurons of more than one functional class and may be able to produce global changes in sympathetic activity. We investigate the extent of spinal axon collateralization of rat rostral ventrolateral medulla (RVLM) sympathetic premotor neurons and its functional consequences. In anatomical tracing experiments, two recombinant herpes viral vectors with retrograde tropism and expressing different fluorophores were injected into the intermediolateral column at upper thoracic and lower thoracic levels. Histological analysis revealed that ∼21% of RVLM bulbospinal neurons were retrogradely labelled by both vectors, indicating substantial axonal collateralization to disparate spinal segments. In functional experiments, another virus with retrograde tropism, a canine adenovirus expressing Cre recombinase, was injected into the left intermediolateral horn around the thoracolumbar junction, whereas a Cre-dependent viral vector encoding Channelrhodopsin2 under LoxP control was injected into the ipsilateral RVLM. In subsequent terminal experiments, blue laser light (473nm×20ms pulses at 10mW) was used to activate RVLM neurons that had been transduced by both vectors. Stimulus-locked activation, at appropriate latencies, was recorded in the following pairs of sympathetic nerves: forelimb and hindlimb muscle sympathetic fibres, as well as cardiac and either hindlimb muscle or lumbar sympathetic nerves. The latter result demonstrates that axon collaterals of lumbar-projecting RVLM neurons project to, and excite, both functionally similar (forelimb and hindlimb muscle) and functionally dissimilar (lumbar and cardiac) preganglionic neurons. Taken together, these findings show that the axons of a significant proportion of RVLM neurons collateralise widely within the spinal cord, and that they may excite preganglionic neurons of more than one functional class.

Response of Neurons in the Rostral Ventrolateral Medulla (RVLM) to Anticipated and Passive Movements

It is well‐established that the rostral ventrolateral medulla (RVLM) plays a predominant role in regulating the activity of sympathetic outflow to the cardiovascular system. We and others have previously shown that in addition to baroreceptor inputs, RVLM neurons respond to signals from the vestibular system. However, most recordings of RVLM unit activity have occurred in anesthetized or decerebrate preparations, limiting the assessment of cognitive effects on neuronal activity. In this study, felines were instrumented for chronic recordings of the electrocardiogram and/or carotid artery blood flow as well as activity of RVLM neurons during 40° head‐up tilts. RVLM units were identified through stereotaxic coordinates and having activity correlated with changes in blood pressure during the cardiac cycle. Tilts were preceded by a light cue, so animals anticipated the movement. We tested the hypothesis that head‐up tilts elicit an increase in the activity of RVLM neurons that is initiated between the light cue and the onset of movement. We also determined if there is a relationship between RVLM unit activity and heart rate, as would be expected if the changes in firing rate are dictated by baroreceptor signals. Head‐up tilts elicited an increase in firing rate for a third of RVLM units. However, for the majority of these units, there was not a significant change in firing rate following the light cue and prior to head‐up rotations. In addition, linear regression analyses showed that there was no appreciable relationship between unit firing rate and heart rate (R2 values ranged from 0.001–0.29, median of 0.03). These studies suggest that cognitive inputs do not produce feedforward alterations in excitability of RVLM neurons prior to perturbations that can alter blood pressure. In addition, they show that inputs to RVLM neurons are complex such that the firing rate of the units is not simply related to baroreceptor inputs.Support or Funding InformationNIH grant R01‐DC013788This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simultaneous Measurement of Hypothalamic Paraventricular Nucleus Neuronal and Sympathetic Nerve Activity in Freely Moving Rats

The hypothalamic paraventricular nucleus (PVN) plays important role in regulation of sympathetic nerve activity (SNA). Studies have reported that the PVN projects directly to the sympathetic preganglionic neurons of the spinal cord and pre‐sympathetic motor neurons of the rostral ventrolateral medulla. However, to our knowledge, simultaneous measurement of PVN neuronal activity (PVNNA) and SNA in freely moving animals has not been reported. Thus, the functional relationship between PVNNA and SNA remains unclear. The aim of the present study was to establish a method for simultaneous and continuous measurement of PVNNA and SNA in freely moving rats. Male Wistar rats were chronically instrumented with multiple electrodes (100‐μm stainless steel wire) for measurement of PVNNA, renal SNA (RSNA), electroencephalogram, electromyogram, and electrocardiogram, and with a catheter for measurement of arterial pressure (AP). PVNNA and RSNA were measured continuously in rats during daily activity and in response to air jet stress. We found that PVNNA changed in a behavioral‐dependent manner. Rapid eye movement sleep increased PVNNA, LSNA, and mean AP, but decreased RSNA and heart rate. In addition, PVN, RSNA, mean AP, and heart rate increased in response to air jet stress. These data suggest that the functional relationship between PVNNA, RSNA, and LSNA is altered depending on changes in physiological conditions.Support or Funding InformationJSPS GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Do Sedentary Conditions Affect the Number of C1 Neurons in Rat Rostral Ventrolateral Medulla?

Cardiovascular disease (CVD) is the leading cause of death in the United States. One of the primary risk factors for CVD is a lack of regular exercise. The brain controls the activity of the autonomic nervous system, which regulates many physiological functions; including blood pressure. The rostral ventrolateral medulla (RVLM) is an important region in the brainstem for blood pressure control because it contains a critical subset of adrenergic (C1) neurons that project to sympathetic preganglionic neurons in the spinal cord. C1 neurons from sedentary rats show more dendritic branching than in active rats, suggesting a novel mechanism by which inactivity could increase sympathetic activity and contribute to hypertension. However, it is also possible that the absolute number of C1 neurons changes along with the number of dendritic branches in sedentary rats. Therefore, the purpose of this study was to test the hypothesis that there are more C1 neurons in the RVLM of rats living under sedentary conditions compared to freely exercising rats. Aiming to understand the mechanisms by which inactivity leads to hypertension, we maintained four‐week old, male Sprague‐Dawley rats for 10–12 weeks under either physically active conditions (running wheels; 410±5.6 km total per rat) or sedentary conditions (no running wheels). Following perfusion and post‐fixation, sections of brainstem were first immunoperoxidase‐stained black for phenylethanolamine N‐methyltransferase (PNMT), a synthetic enzyme and marker of C1 neurons, and then brown for tyrosine hydroxylase (TH), the enzyme that all catecholamine neurons contain. PNMT+TH and TH only neurons in each rat were counted in the 6 sections containing the 600 μm caudal to the caudal pole of the facial nucleus (FN). Sedentary conditions did not produce a difference in the number, or rostrocaudal pattern, of C1 neurons compared to the active group nor an interaction between the two (two way RM ANOVA, p&gt;0.05 ea). Sedentary conditions also did not affect the low number of TH neurons (likely A1 neurons) in the RVLM compared to active rats (2±1 vs. 2±1, respectively). Because the number of RVLM C1 neurons is the same in active and sedentary rats, we can exclude the possibility that an increase in the number of C1 neurons is a cause of increased activation of vasomotor spinal neurons and that structural neuroplasticity in C1 neurons may underlie increased functional excitability of the RVLM in our other studies. Future functional neuroanatomical studies using Fos protein should show whether the overall activation of the population of C1 neurons differs between sedentary and active rats.Support or Funding Information(HL096787‐09)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(254) Functional and Anatomical Characterization of Descending Periaqueductal Gray (PAG) Projections and their Role in Pain Modulation

Endogenous analgesic pathways embody an alternative target for the development for chronic pain therapies. Previous studies have demonstrated the role of the ventrolateral periaqueductal gray (vlPAG) in descending pain modulation. It has been proposed that tonic GABAergic neurotransmission at the level of the vlPAG serves to inhibit efferent excitatory projections that mediate descending analgesia. Disinhibition of these projection neurons allows subsequent activation of rostral ventromedial medulla (RVM) neurons that inhibit nociception at the level of the spinal cord. However, lack of cell-type specificity in these studies has prevented the determination of the role of specific subsets of vlPAG neurons in analgesia. We have utilized recently developed genetic tools to selectively manipulate the activity of subclasses of vlPAG neurons to identify the circuit components critical for analgesia. Our data suggest that increasing the activity of glutamatergic vlPAG neurons or decreasing the activity of GABAergic vlPAG neurons results in increases in sensory thresholds in uninjured animals, while ameliorating hyperalgesia associated with chronic pain states. To further characterize descending pain circuitry at the level of the vlPAG, we decided to focus on efferent projections to the RVM. We hypothesize that these projections are partially responsible for the observed analgesia in our previous study. Population-specific anatomical tracing revealed that most of these projections are glutamatergic and originate from the ventrolateral columns of the PAG. Optogenetic stimulation of these PAG-RVM neurons results in elevation of thermal thresholds in uninjured animals and seems to reverse thermal hyperalgesia associated with inflammatory states. However, this is not the case in a model of neuropathic pain. In brief, our results provide direct experimental evidence supporting the GABA disinhibition hypothesis, highlighting the role of descending glutamatergic neurotransmission at the level of the PAG-RVM as a key component of endogenous analgesic pathways. This work is supported by NINDS F31NS103472 to JGGR.

Plasticity in the Link between Pain-Transmitting and Pain-Modulating Systems in Acute and Persistent Inflammation.

There is strong evidence that spinoparabrachial neurons in the superficial dorsal horn contribute to persistent pain states, and that the lateral parabrachial complex (PB) conveys relevant nociceptive information to higher structures. The role of PB itself in hyperalgesia and how it recruits descending facilitation has nevertheless received significantly less attention. The current study is a first step toward delineating the functional dynamics of PB and its link to descending control in acute and persistent inflammatory pain. In lightly anesthetized rats, we recorded behavioral withdrawal evoked by mechanical stimulation of the hindpaw and, simultaneously, the activity of identified pain-modulating neurons, "ON-cells" and "OFF-cells," in the rostral ventromedial medulla (RVM). This was done before and after the inactivation of PB, contralateral or ipsilateral to an inflamed paw [1 h, 1 d, or 5-6 d after intraplantar injection of Complete Freund's Adjuvant (CFA)]. The inactivation of contralateral, but not ipsilateral, PB interfered with nociceptive input to RVM under basal conditions, as well as in acute inflammation. By contrast, blocking ipsilateral, but not contralateral, PB in established inflammation interfered with behavioral hyperalgesia and ON-cell and OFF-cell responses. The lesioning of contralateral PB before CFA injection prevented this recruitment of ipsilateral PB in persistent inflammation. These experiments show that contralateral PB is required to initiate hyperalgesia, which is then maintained by ipsilateral PB, most likely in both cases via the engagement of pain-modulating neurons of the RVM.SIGNIFICANCE STATEMENT The lateral parabrachial complex (PB) relays nociceptive information to brain circuits that are important for the transmission and modulation of pain, but its specific role in persistent pain and engagement of descending control mechanisms has received relatively little attention. We show here that PB contralateral and ipsilateral to an inflammatory insult demonstrate different functions as inflammation persists, likely by engaging pain-facilitating neurons of the rostral ventromedial medulla. While the contralateral PB, the target of the major spinoparabrachial pathway, relays acute nociceptive information, the ipsilateral PB is recruited or unmasked in persistent inflammation to maintain hyperalgesia. These data point to plasticity in the PB itself or its direct and indirect connections with pain-modulating systems as central to the development and maintenance of persistent pain.

Upregulation of AT1 Receptor Mediates a Pressor Effect Through ROS-SAPK/JNK Signaling in Glutamatergic Neurons of Rostral Ventrolateral Medulla in Rats With Stress-Induced Hypertension.

The present study examined whether angiotensin II (Ang II) mediates the pressor effect through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS)-mitogen-activated protein kinase (MAPK) signaling in the glutamatergic neurons of the rostral ventrolateral medulla (RVLM) in stress-induced hypertensive rats (SIHR). The SIHR model was established using electric foot-shocks combined with noises for 15 days. We observed that Ang II type 1 receptor (AT1R) and the glutamatergic neurons co-localized in the RVLM of SIHR. Furthermore, glutamate levels in the intermediolateral column of the spinal cord were higher in SIHR than in controls. Microinjection of Ang II into the RVLM of SIHR activated stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), extracellular signal-regulated protein kinase (ERK) 1/2, and p38MAPK. Compared with controls, the activation of SAPK/JNK, ERK1/2, p38MAPK, and ROS in the RVLM were higher in SIHR, an effect that was blocked by an NADPH oxidase inhibitor (apocynin) and an AT1R antagonist (candesartan). RVLM microinjection of apocynin or a SAPK/JNK inhibitor (SP600125), but not an ERK1/2 inhibitor (U0126) or a p38MAPK inhibitor (SB203580), decreased AT1R mRNA and mean arterial blood pressure (MABP) in SIHR. The increase of AT1R protein expression and MABP was inhibited by intracerebroventricular infusion (ICV), for 14 days, of SP600125, but not U0126 or SB203580 in SIHR. We conclude that Ang II modulates the pressor effect through AT1R-dependent ROS-SAPK/JNK signaling in glutamatergic neurons in the RVLM of SIHR.

Glycinergic neurotransmission in the rostral ventrolateral medulla controls the time course of baroreflex-mediated sympathoinhibition.

To maintain appropriate blood flow to various tissues of the body under a variety of physiological states, autonomic nervous system reflexes regulate regional sympathetic nerve activity and arterial blood pressure. Our data obtained in anaesthetized rats revealed that glycine released in the rostral ventrolateral medulla (RVLM) plays a critical role in maintaining arterial baroreflex sympathoinhibition. Manipulation of brainstem nuclei with known inputs to the RVLM (nucleus tractus solitarius and caudal VLM) unmasked tonic glycinergic inhibition in the RVLM. Whole-cell, patch clamp recordings demonstrate that both GABA and glycine inhibit RVLM neurons. Potentiation of neurotransmitter release from the active synaptic inputs in the RVLM produced saturation of GABAergic inhibition and emergence of glycinergic inhibition. Our data suggest that GABA controls threshold excitability, wherreas glycine increases the strength of inhibition under conditions of increased synaptic activity within the RVLM. The arterial baroreflex is a rapid negative-feedback system that compensates changes in blood pressure by adjusting the output of presympathetic neurons in the rostral ventrolateral medulla (RVLM). GABAergic projections from the caudal VLM (CVLM) provide a primary inhibitory input to presympathetic RVLM neurons. Although glycine-dependent regulation of RVLM neurons has been proposed, its role in determining RVLM excitability is ill-defined. The present study aimed to determine the physiological role of glycinergic neurotransmission in baroreflex function, identify the mechanisms for glycine release, and evaluate co-inhibition of RVLM neurons by GABA and glycine. Microinjection of the glycine receptor antagonist strychnine (4mm, 100nL) into the RVLM decreased the duration of baroreflex-mediated inhibition of renal sympathetic nerve activity (control= 12± 1min; RVLM-strychnine= 5.1± 1min), suggesting that RVLM glycine plays a critical role in regulating the time course of sympathoinhibition. Blockade of output from the nucleus tractus solitarius and/or disinhibition of the CVLM unmasked tonic glycinergic inhibition of the RVLM. To evaluate cellular mechanisms, RVLM neurons were retrogradely labelled (prior injection of pseudorabies virus PRV-152) and whole-cell, patch clamp recordings were obtained in brainstem slices. Under steady-state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of the overall inhibition. By contrast, stimulation of synaptic inputs in the RVLM decreased GABAergic inhibition to 53%; and increased glycinergic inhibition to 47%. Thus, under conditions of increased synaptic activity in the RVLM, glycinergic inhibition is recruited to strengthen sympathoinhibition.