Medullary Neurons Research Articles

Trigeminal Aδ- and C-afferent supply of lamina I neurons in the trigeminocervical complex.

Nociceptive trigeminal afferents innervating craniofacial area, eg, facial skin and cranial meninges, project to a broad region in the medullary and upper cervical dorsal horn designated as the trigeminocervical complex. Lamina I neurons in the trigeminocervical complex integrate and relay peripheral inputs, thus playing a key role in both cranial nociception and primary headache syndromes. Because of the technically challenging nature of recording, the long-range trigeminal afferent inputs to the medullary and cervical lamina I neurons were not intensively studied so far. Therefore, we have developed an ex vivo brainstem-cervical cord preparation with attached trigeminal nerve for the visually guided whole-cell recordings from the medullary and cervical lamina I neurons. Two-thirds of recorded neurons generated intrinsic rhythmic discharges. The stimulation of the trigeminal nerve produced a complex effect; it interrupted the rhythmic discharge for hundreds of milliseconds but, if the neuron was silenced by a hyperpolarizing current injection, could elicit a discharge. The monosynaptic inputs from the trigeminal Aδ, high-threshold Aδ, low-threshold C, and C afferents were recorded in the medullary neurons, as well as in the cervical neurons located in the segments C1 to C2 and, to a lesser degree, in C3 to C4. This pattern of supply was consistent with our labelling experiments showing extensive cervical projections of trigeminal afferents. Excitatory inputs were mediated, although not exclusively, through AMPA/kainate and NMDA receptors, whereas inhibitory inputs through both GABA and glycine receptors. In conclusion, the trigeminocervical lamina I neurons receive a complex pattern of long-range monosynaptic and polysynaptic inputs from a variety of the trigeminal nociceptive afferents.

Brainstem cholinergic pathways diminish cardiovascular and neuroinflammatory actions of endotoxemia in rats: Role of NFκB/α7/α4β2AChRs signaling

Nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. Here, we test the hypothesis that brainstem antiinflammatory pathways of α7/α4β2 nicotinic acetylcholine receptors (nAChRs) modulate endotoxic cardiovascular derangements. Pharmacologic and molecular studies were performed to determine the influence of nicotine or selective α7/α4β2-nAChR ligands on cardiovascular derangements and brainstem neuroinflammation caused by endotoxemia in conscious rats. The i.v. administration of nicotine (50 μg/kg) abolished the lipopolysaccharide (LPS, 10 mg/kg i.v.)-evoked: (i) falls in blood pressure and spectral measure of cardiac sympathovagal balance (ratio of the low-frequency to high-frequency power, LF/HF), (ii) elevations in immunohistochemical protein expressions of NFκB and α4β2-nAChR in medullary neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), and (iii) decreases in medullary α7-nAChR protein expression. These favorable nicotine influences were replicated in rats treated intracisternally (i.c.) with PHA-543613 (selective α7-nAChR agonist) or 5-iodo-A-85380 (5IA, selective α4β2-nAChRs agonist). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the LPS depression of reflex bradycardic, but not tachycardic, activity. Moreover, the counteraction by nicotine of LPS hypotension was mostly inhibited after treatment with i.c. methyllycaconitine (MLA, α7-nAChR antagonist) in contrast to a smaller effect for dihydro-β-erythroidine (DHβE, α4β2-nAChR antagonist), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of brainstem α7, and to a lesser extent α4β2, receptors in the nicotine counteraction of detrimental cardiovascular and neuroinflammatory consequences of endotoxemia.

The Effects of Amphetamine and Methamphetamine on the Release of Norepinephrine, Dopamine and Acetylcholine From the Brainstem Reticular Formation.

Amphetamine (AMPH) and methamphetamine (METH) are widely abused psychostimulants, which produce a variety of psychomotor, autonomic and neurotoxic effects. The behavioral and neurotoxic effects of both compounds (from now on defined as AMPHs) stem from a fair molecular and anatomical specificity for catecholamine-containing neurons, which are placed in the brainstem reticular formation (RF). In fact, the structural cross-affinity joined with the presence of shared molecular targets between AMPHs and catecholamine provides the basis for a quite selective recruitment of brainstem catecholamine neurons following AMPHs administration. A great amount of investigations, commentary manuscripts and books reported a pivotal role of mesencephalic dopamine (DA)-containing neurons in producing behavioral and neurotoxic effects of AMPHs. Instead, the present review article focuses on catecholamine reticular neurons of the low brainstem. In fact, these nuclei add on DA mesencephalic cells to mediate the effects of AMPHs. Among these, we also include two pontine cholinergic nuclei. Finally, we discuss the conundrum of a mixed neuronal population, which extends from the pons to the periaqueductal gray (PAG). In this way, a number of reticular nuclei beyond classic DA mesencephalic cells are considered to extend the scenario underlying the neurobiology of AMPHs abuse. The mechanistic approach followed here to describe the action of AMPHs within the RF is rooted on the fine anatomy of this region of the brainstem. This is exemplified by a few medullary catecholamine neurons, which play a pivotal role compared with the bulk of peripheral sympathetic neurons in sustaining most of the cardiovascular effects induced by AMPHs.

Your input is a breath of fresh air! A chemosensory microcircuit of medullary raphe and RTN neurons.

Breathing is our first act upon birth and the last action we complete before death. The first to last breath taken, is in fact, how we define someone’s life. Since it was first reported that the blood concentration of CO2istightly controlled, and provides the dominant drive to breathe, the search for the cells that regulate it began. It took almost 60 years for the identification of the first central chemosensitive areas, regions within the brain that respond to specific chemical stimuli (such as CO2or its proxy H+), found at the ventrolateral surface of the medulla (VLM). Since then the debate over which cells in these areas are responsible for detectingCO2and signalling its fluctuations to the respiratory oscillators, has been extensive and heated. Chemosensitive cells are thought to have cell bodies located in, or close to, the VLM with dendrites in close apposition to blood vessels to better detect changes in blood gases. Several candidates fulfil this criteria, including the retrotrapezoid nucleus (RTN) and medullary raphe.

Central Terminals of Trigeminal Nerves that Innervate the Nose and Nasal Passages Colocalize with Medullary Dorsal Horn Neurons Activated during Voluntary Diving in Rats

Underwater submersion in mammals initiates the diving response, an autonomic cardiorespiratory reflex that includes apnea, a parasympathetically‐mediated bradycardia, and a sympathetically‐mediated increase in total peripheral resistance. The initial synapse of this reflex occurs within the ventral medullary dorsal horn (MDH) of the spinal trigeminal nucleus caudalis. The anterior ethmoidal and nasopalatine nerves, trigeminal nerves that innervate the internal nasal passages, also send their central terminations to the ventral MDH. The objective of this research was to detect colocalization of secondary MDH neurons activated by voluntary diving with the central terminations of the trigeminal nerves innervating the internal nasal passages. MDH activated neurons were identified by Fos protein production, while central terminals of the trigeminal nerves were located by the transganglionic tracer Wheat Germ Agglutinin (WGA). Sprague‐Dawley rats (N=12) were trained to voluntarily dive through a 5 m underwater Plexiglas maze. After training, 6 rats then received WGA injections into the anterior ethmoidal nerve, while 6 rats received WGA injections into the nasopalatine nerve. Four days post‐WGA injection, 3 rats from each nerve injection group performed 12 dive trials over 1 hour to activate brainstem neurons and initiate Fos production; 3 rats from each nerve injection group were non‐diving controls. Brainstem tissue from all rats was immunohistochemically processed to visualize both Fos and WGA, and was inspected using a Nikon C1 confocal microscope and Nikon Eclipse Ti2 inverted fluorescent microscope. Results indicate repetitive voluntary diving increases the number of Fos‐positive neurons within the ventral tip of the superficial MDH and adjacent paratrigeminal nucleus. WGA results show central termination sites from both the anterior ethmoidal and nasopalatine nerves within this same MDH location. Additionally, MDH Fos‐positive neurons from diving animals show extensive colocalization with WGA injected into both the anterior ethmoidal nerve and nasopalatine nerve compared to non‐diving control rats. We conclude both the anterior ethmoidal and nasopalatine nerves can provide afferent information necessary to activate MDH neurons during initiation of the mammalian diving response.Support or Funding InformationSupported by MWU Intramural funding (PFM) and CGS MBS program (MM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Sympathoexcitatory Pathway between The Deep Superior Colliculus and The Rostral Ventromedial Medulla

IntroductionThe superior colliculus plays key roles in the immediate processing of threatening sensory stimuli and generates rapid behavioural responses that are critical for survival. We have previously found that disinhibition of the deep superior colliculus (dSC) unmasks coordinated respiratory, sympathetic and somatomotor outputs that are independent of processing in higher centres. We postulate that these effects are a result of direct innervation of autonomic and respiratory medullary neurons based on the results of neuroanatomical tracing studies that identified dSC synaptic contacts on spinally projecting neurons within the rostral ventromedial medulla (RVMM). In the present study we investigate the physiological significance of this relay and compare effects of dSC stimulation to activation of dSC‐RVMM terminals.MethodsWe used an AAV vector to express Channelrhodopsin2 (ChR2) in the dSC and chronically implanted a fibre optic cannula into the dSC. After transgene expression electrophysiology experiments were conducted under urethane anesthesia with rats instrumented to record blood pressure, diaphragmatic EMG and splanchnic sympathetic nerve activity (SNA). One subset of experiments then positioned the optrode in the RVMM to deliver light to the terminal projections from the dSC. For the other subset of experiments, extracellular recordings were made of spinally projecting RVMM neurons.ResultsdSC photoactivation evoked an increase in respiratory frequency (35±8.7%, P=<0.01, N=7) and heart rate (11±2.6 bpm, P=<0.01, N=7), and a modest change in blood pressure when compared to controls. Stimulus‐triggered averaging of SNA revealed short‐latency excitatory potentials in most cases. Photoactivation of ChR2‐expressing dSC‐RVMM terminals evoked similar effects on cardiorespiratory outputs as dSC stimulation, with an increase in respiratory frequency (14 ±9.2%, P<0.001, N=6) and blood pressure (13 ±2 mmHg P<0.01, N=7), as well as qualitatively similar sympathoexcitation. In preliminary experiments that recorded extracellular action potentials in bulbospinal RVMM neurons we recorded excitatory effects of dSC stimulation in 6/8 spontaneously active neurons.ConclusionWe conclude that optogenetic activation of dSC neurons that project to the RVMM can drive cardiorespiratory effects that are consistent with our previous findings and that dSC stimulation evokes excitatory effects in some RVMM bulbospinal neurons. This confirms our hypothesis of the existence of a previously uncharacterized excitatory pathway between the dSC and RVMM. Ongoing experiments will functionally characterize RVMM neurons that receive dSC input.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct dampening effects of progesterone on the activity of nucleus tractus solitarii neurons in rat pups.

What is the central question of the study? Progesterone is considered a respiratory stimulant drug, but its effect on medullary respiratory neurons are poorly documented. We investigated whether progesterone alters spontaneous activity of neurons in the nucleus of the solitary tract (NTS). What is the main finding and its importance? In NTS neurons, progesterone decreases the action potential firing frequency in response to current injections and the amplitude of excitatory postsynaptic currents. Based on the established neuroprotective effect of progesterone against excitotoxicity resulting from insults, this inhibitory effect is likely to reflect inhibition of ion fluxes. These results are important because they further our understanding of the mechanisms underlying the diversity of respiratory effects of progesterone. Progesterone is known to stimulate breathing, but its actions on the respiratory control system have received limited attention. We addressed this issue at the cellular level by testing the hypothesis that progesterone augments excitatory currents at the level of the nucleus tractus solitarii (NTS). Medullary slices from juvenile male rats (14-17days of age) containing the commissural region of the NTS (NTScom) were incubated with progesterone (1μm) or vehicle (0.004% DMSO) for 60min. We performed whole-cell voltage-clamp recordings of spontaneous excitatory postsynaptic currents (EPSCs) in the NTScom and determined membrane properties by applying depolarizing current steps. In comparison to vehicle-treated cells, progesterone exposure attenuates the firing frequency response to current injection and reduces the EPSC amplitude without modifying the EPSC frequency or the basal membrane properties. These data do not support our hypothesis, because they indicate that incubation with progesterone attenuates intrinsic action potential generation and inhibits excitatory synaptic inputs in the NTS. Given that these results are more in line with the protective effect of progesterone against excitotoxicity resulting from various insults, we propose that progesterone acts via inhibition of ionic flux.

Gigantocellular Neurons Awaken the Brain from Deep Pharmacologically-Induced Coma

Attempts at neuromodulation using deep brain stimulation (DBS) have targeted a number of loci in brain arousal pathways such as the thalamus, mesencephalon and cervical cord. However, recovery following single location stimulation remains modest, especially in comatose patients. Although some of these pathways can trigger awakening from sleep or light planes of anesthesia upon activation are insufficient to promote arousal from a deeper arousal state like coma. Interpretation of these results suggests that to fully emerge from a deep arousal state, coordination of multiple pathways is required. The mechanism through which this coordination is achieved by the brain is poorly understood. Considering that medullary reticular neurons project to arousal- modulating areas throughout the brain, integrate a broad range of sensory and autonomic inputs, and fire in response to salient stimuli in close association with the initiation of behaviors, we modulated their activity during a pharmacologic induced coma (PIC). Our results showed that pharmacologic or optogenetic activation of an area located at the nucleus gigantocellularis (NGC)- elicited robust cortical, autonomic and motor arousal during a state of PIC. To understand how this small subpopulation of neurons exerts such widespread activational effects on arousal, we analyzed immunolabeling of c-Fos-a well-established marker of neuronal activation-following pharmacologic activation of NGC neurons. C-Fos labeling was sparse and largely limited to structures known to participate in arousal including rhombenchephalon (locus coeruleus and parabrachial nucleus); mesencephalon (periaqueductal gray and ventral tegmental area); diencephalon (intralaminar thalamic nuclei as well as posterior and anterior hypothalamus). These findings suggest that NGC is able to recruit these pathways to trigger arousal from a dense PIC. Results of this study will uncover novel mechanisms leading to new approaches for emerging treatments for patients under coma state.

Circulatory control of phrenic motor plasticity

Acute intermittent hypoxia (AIH) elicits distinct mechanisms of phrenic motor plasticity initiated by brainstem neural network activation versus local (spinal) tissue hypoxia. With moderate AIH (mAIH), hypoxemia activates the carotid body chemoreceptors and (subsequently) brainstem neural networks associated with the peripheral chemoreflex, including medullary raphe serotonergic neurons. Serotonin release and receptor activation in the phrenic motor nucleus then elicits phrenic long-term facilitation (pLTF). This mechanism is independent of tissue hypoxia, since electrical carotid sinus nerve stimulation elicits similar serotonin-dependent pLTF. In striking contrast, severe AIH (sAIH) evokes a spinal adenosine-dependent, serotonin-independent mechanism of pLTF. Spinal tissue hypoxia per se is the likely cause of sAIH-induced pLTF, since local tissue hypoxia elicits extracellular adenosine accumulation. Thus, any physiological condition exacerbating spinal tissue hypoxia is expected to shift the balance towards adenosinergic pLTF. However, since these mechanisms compete for dominance due to mutual cross-talk inhibition, the transition from serotonin to adenosine dominant pLTF is rather abrupt. Any factor that compromises spinal cord circulation will limit oxygen availability in spinal cord tissue, favoring a shift in the balance towards adenosinergic mechanisms. Such shifts may arise experimentally from treatments such as carotid denervation, or spontaneous hypotension or anemia. Many neurological disorders, such as spinal cord injury or stroke compromise local circulatory control, potentially modulating tissue oxygen, adenosine levels and, thus, phrenic motor plasticity. In this brief review, we discuss the concept that local (spinal) circulatory control and/or oxygen delivery regulates the relative contributions of distinct pathways to phrenic motor plasticity.

Firing properties of ventral medullary respiratory neurons in sino-aortic denervated rats.

What is the central question of this study? After sino-aortic denervation (SAD), rats present normal levels of mean arterial pressure (MAP), high MAP variability and changes in breathing. However, mechanisms involved in SAD-induced respiratory changes and their impact on the modulation of sympathetic activity remain unclear. Herein, we characterized the firing frequency of medullary respiratory neurons after SAD. What is the main finding and its importance? Sino-aortic denervation-induced prolonged inspiration was associated with a reduced interburst frequency of pre-inspiratory/inspiratory neurons and an increased long-term variability of late inspiratory neurons, but no changes were observed in the ramp-inspiratory and post-inspiratory neurons. This imbalance in the respiratory network might contribute to the modulation of sympathetic activity after SAD. In previous studies, we documented that after sino-aortic denervation (SAD) in rats there are significant changes in the breathing pattern, but no significant changes in sympathetic activity and mean arterial pressure compared with sham-operated rats. However, the neural mechanisms involved in the respiratory changes after SAD and the extent to which they might contribute to the observed normal sympathetic activity and mean arterial pressure remain unclear. Here, we hypothesized that after SAD, rats present with changes in the firing frequency of the ventral medullary inspiratory and post-inspiratory neurons. To test this hypothesis, male Wistar rats underwent SAD or sham surgery and 3days later were surgically prepared for an in situ experiment. The duration of inspiration significantly increased in SAD rats. During inspiration, the total firing frequency of ramp-inspiratory, pre-inspiratory/inspiratory and late-inspiratory neurons was not different between groups. During post-inspiration, the total firing frequency of post-inspiratory neurons was also not different between groups. Furthermore, the data demonstrate a reduced interburst frequency of pre-inspiratory/inspiratory neurons and an increased long-term variability of late-inspiratory neurons in SAD compared with sham-operated rats. These findings indicate that the SAD-induced prolongation of inspiration was not accompanied by alterations in the total firing frequency of the ventral medullary respiratory neurons, but it was associated with changes in the long-term variability of late-inspiratory neurons. We suggest that the timing imbalance in the respiratory network in SAD rats might contribute to the modulation of presympathetic neurons after removal of baroreceptor afferents.

Mecp2 Disruption in Rats Causes Reshaping in Firing Activity and Patterns of Brainstem Respiratory Neurons

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations in the MECP2 gene, have breathing abnormalities manifested as periodical hypoventilation with compensatory hyperventilation, which are attributable to a high incidence of sudden death. Similar breathing abnormalities have been found in animal models with Mecp2 disruptions. Although RTT-type hypoventilation is believed to be due to depressed central inspiratory activity, whether this is true remains unknown. Here we show evidence for reshaping in firing activity and patterns of medullary respiratory neurons in RTT-type hypoventilation without evident depression in inspiratory neuronal activity. Experiments were performed in decerebrate rats in vivo. In Mecp2-null rats, abnormalities in breathing patterns were apparent in both decerebrate rats and awake animals, suggesting that RTT-type breathing abnormalities take place in the brainstem without forebrain input. In comparison to their wild-type counterparts, both inspiratory and expiratory neurons in Mecp2-null rats extended their firing duration, and fired more action potentials during each burst. No changes in inspiratory or expiratory neuronal distributions were found. Most inspiratory neurons started firing in the middle of expiration and changed their firing pattern to a phase-spanning type. The proportion of post-inspiratory neurons was reduced in the Mecp2-null rats. With the increased firing activity of both inspiratory and expiratory neurons in null rats, phrenic discharges shifted to a slow and deep breathing pattern. Thus, the RTT-type hypoventilation appears to result from reshaping of firing activity of both inspiratory and expiratory neurons without evident depression in central inspiratory activity.

Insulin-responsive autonomic neurons in rat medulla oblongata.

Low blood glucose activates brainstem adrenergic and cholinergic neurons, driving adrenaline secretion from the adrenal medulla and glucagon release from the pancreas. Despite their roles in maintaining glucose homeostasis, the distributions of insulin-responsive adrenergic and cholinergic neurons in the medulla are unknown. We fasted rats overnight and gave them insulin (10 U/kg i.p.) or saline after 2 weeks of handling. Blood samples were collected before injection and before perfusion at 90 min. We immunoperoxidase-stained transverse sections of perfused medulla to show Fos plus either phenylethanolamine N-methyltransferase (PNMT) or choline acetyltransferase (ChAT). Insulin injection lowered blood glucose from 4.9 ± 0.3 mmol/L to 1.7 ± 0.2 mmol/L (mean ± SEM; n = 6); saline injection had no effect. In insulin-treated rats, many PNMT-immunoreactive C1 neurons had Fos-immunoreactive nuclei, with the proportion of activated neurons being highest in the caudal part of the C1 column. In the rostral ventrolateral medulla, 33.3% ± 1.4% (n = 8) of C1 neurons were Fos-positive. Insulin also induced Fos in 47.2% ± 2.0% (n = 5) of dorsal medullary C3 neurons and in some C2 neurons. In the dorsal motor nucleus of the vagus (DMV), insulin evoked Fos in many ChAT-positive neurons. Activated neurons were concentrated in the medial and middle regions of the DMV beneath and just rostral to the area postrema. In control rats, very few C1, C2, or C3 neurons and no DMV neurons were Fos-positive. The high numbers of PNMT-immunoreactive and ChAT-immunoreactive neurons that express Fos after insulin treatment reinforce the importance of these neurons in the central response to a decrease in glucose bioavailability.

Stress-Induced Alterations of Norepinephrine Release in the Bed Nucleus of the Stria Terminalis of Mice.

Stress can drive adaptive changes to maintain survival during threatening stimuli. Chronic stress exposure, however, may result in pathological adaptations. A key neurotransmitter involved in stress signaling is norepinephrine. Previous studies show that acute stress elevates norepinephrine levels in the bed nucleus of the stria terminalis (BNST), a critical node regulating anxiety and upstream of stress responses. Here, we use mice expressing channelrhodopsin in norepinephrine neurons to selectively activate terminals in the BNST, and measure norepinephrine release with optogenetics-assisted fast-scan cyclic voltammetry (FSCV). We demonstrate that while corticosterone habituates to chronic restraint stress, cFos activation of medullary norepinephrine neurons shows equivalent activation under both acute and chronic stress conditions. Mice exposed to a single restraint session show an identical optically stimulated norepinephrine release profile compared to that of unexposed mice. Mice experiencing 5 days of restraint stress, however, show elevated norepinephrine release across multiple stimulation parameters, and reduced sensitivity to the α2-adrenergic receptor (AR) antagonist idazoxan. These data are the first to examine norepinephrine release in the BNST to tonic and phasic stimulation frequencies, and confirm that repeated stress alters autoreceptor sensitivity.

Optogenetic manipulation of medullary neurons in the locust optic lobe.

The locust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts ( Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered excitatory postsynaptic potentials and firing of a postsynaptic, looming-sensitive neuron, the lobula giant movement detector. These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects. NEW & NOTEWORTHY Using Semliki Forest virus, we efficiently delivered channelrhodopsin into neurons of the locust optic lobe. We demonstrate that laser illumination increases the firing of the medullary neurons expressing channelrhodopsin and elicits excitatory postsynaptic potentials and spiking in an identified postsynaptic target neuron, the lobula giant movement detector neuron. This technique allows the manipulation of neuronal activity in locust neural circuits using optogenetics.

Involvement of median preoptic nucleus and medullary noradrenergic neurons in cardiovascular and sympathetic responses of hemorrhagic rats

The infusion of hypertonic saline solution (HSS) is known to be beneficial to the treatment of hypovolemic hemorrhage (HH). The central mechanism of HSS-induced cardiovascular and autonomic recovery of animals subjected to HH remains unclear. Hence, the present study evaluated the involvement of median preoptic nucleus (MnPO) and medullary noradrenergic neurons (A1 and A2) in HSS-induced cardiovascular and sympathetic responses in hemorrhagic rats. The wistar rats were subjected to specific lesion of noradrenergic neurons through the nanoinjections of anti-DβH-saporin into caudal ventrolateral medulla (A1 neurons) and nucleus of the solitary tract (A2 neurons). After recovery, mean arterial pressure (MAP) and renal sympathetic nervous activity were recorded. The HH was performed through blood withdrawal until a MAP of 60 mmHg was attained. In sham rats, HSS infusion (3M NaCl) reestablished MAP without change in HH-induced sympathoinhibition. The muscimol (agonist of GABAA receptor) was nanoinjected in MnPO during HH and MnPO inhibition abolished the recovery of MAP and HSS-induced sympathoinhibition. Simultaneous lesions of A1 and A2 abolished MAP restoration and sympathoinhibition after HSS infusion. These results suggest that the recovery of MAP and HSS-induced sympathoinhibition in hemorrhaged rats depend on intact neural projections from A1 and A2 to MnPO.

Colitis-induced alterations in response properties of visceral nociceptive neurons in the rat caudal medulla oblongata and their modulation by 5-HT3 receptor blockade

There is considerable clinical and experimental evidence that intestinal inflammation is associated with altered visceral nociceptive processing in the spinal cord and brain, but the underlying neuronal mechanisms, especially acting at the supraspinal level, remain unclear. Considering that the caudal ventrolateral medulla (CVLM) and the nucleus tractus solitarius (NTS) are the first sites for supraspinal processing of visceral pain signals, in the present study we evaluated the experimental colitis-induced changes in response properties of CVLM and NTS medullary neurons to noxious colorectal distension (CRD) in urethane-anesthetized adult male Wistar rats. To determine if gut inflammation alters the 5-HT3 receptor-dependent modulation of visceral pain-related CVLM and NTS cells, we examined the effects of intravenously administered selective 5-HT3 antagonist granisetron on ongoing and CRD-evoked activity of CVLM and NTS neurons in healthy control and colitic animals. In the absence of colonic pathology, the CVLM neurons were more excited by noxious CRD that the NTS cells, which demonstrated a greater tendency to be inhibited by the stimulation. The difference was eliminated after the development of colitis due to the increase in the proportion of CRD-excited neurons in both medullary regions associated with enhanced magnitude of the neuronal nociceptive responses. Intravenous granisetron (1 or 2 mg/kg) produced the dose-dependent suppression of the ongoing and evoked firing of CRD-excited cells within both the CVLM and NTS in normal conditions as well as was able to substantially reduce excitability of the caudal medullary neurons in the presence of colonic inflammation, arguing for the potential efficacy of the 5-HT3 receptor blockade with granisetron against both acute and inflammatory abdominal pain. Taken together, the data obtained can contribute to a deeper understanding of supraspinal serotonergic mechanisms responsible for the persistence of visceral hypersensitivity and hyperalgesia triggered by colonic inflammation.

Neurons in the dorsomedial medulla contribute to swallow pattern generation: Evidence of inspiratory activity during swallow.

Active contraction of the diaphragm and other inspiratory pump muscles during swallow create a negative thoracic pressure to improve the movement of the bolus (food/liquid) into the esophagus. We tested the hypothesis that dorsomedial medullary inspiratory neurons, including the nucleus tractus solitarius (NTS, pre-motor to the phrenic) would be active during swallow induced by oral water infusion. We recorded neurons in the NTS and medial reticular formation in anesthetized spontaneously breathing cats, and induced swallow by injection of water into the oropharynx. Our results indicate that: 1) a majority of inspiratory cells in the dorsomedial medulla are active during swallow, 2) expiratory neurons are present in the medial reticular formation (deeper to the NTS) in unparalyzed cats and a majority of these cells decreased firing frequency during swallow. Our findings suggest that the dorsomedial medulla is a source of inspiratory motor drive during swallow and that a novel population of breathing-modulated neurons that also are modulated during swallowing exist in the medial reticular formation in unparalyzed animals.

Inactivation of Serotonergic Neurons in the Rostral Medullary Raphé Attenuates Stress-Induced Tachypnea and Tachycardia in Mice.

The medullary raphé nuclei are involved in controlling cardiovascular, respiratory, and thermoregulatory functions, as well as mediating stress-induced tachycardia and hyperthermia. Although the serotonergic system of the medullary raphé has been suggested as the responsible entity, specific evidence has been insufficient. In the present study, we tested this possibility by utilizing an optogenetic approach. We used genetically modified mice [tryptophan hydroxylase 2 (Tph2); archaerhodopsin-T (ArchT) mice] in which ArchT, a green light-driven neuronal silencer, was selectively expressed in serotonergic neurons under the regulation of Tph2 promoters. We first confirmed that an intruder stress selectively activated medullary, but not dorsal or median raphé serotonergic neurons. This activation was suppressed by photo-illumination via a pre-implanted optical fiber, as evidenced by the decrease of a cellular activation marker protein in the neurons. Next, we measured electro cardiogram (ECG), respiration, body temperature (BT), and locomotor activity in freely moving mice during intruder and cage-drop stress tests, with and without photo-illumination. In the intruder test, photo inactivation of the medullary serotonergic neurons significantly attenuated tachycardia (362 ± 58 vs. 564 ± 65 bpm.min, n = 19, p = 0.002) and tachypnea (94 ± 82 vs. 361 ± 138 cpm.min, n = 9, p = 0.026), but not hyperthermia (1.0 ± 0.1 vs. 1.0 ± 0.1°C.min, n = 19, p = 0.926) or hyperlocomotion (17 ± 4 vs. 22 ± 4, arbitrary, n = 19, p = 0.089). Similar results were obtained from cage-drop stress testing. Finally, photo-illumination did not affect the basal parameters of the resting condition. We conclude that a subpopulation of serotonergic neurons in the medullary raphé specifically mediate stress-induced tachypnea and tachycardia, which have little involvement in the basal determination of respiratory frequency (Res) and heart rate (HR), specifically mediate stress-induced tachycardia and tachypnea.

Inputs to medullary respiratory neurons from a pontine subregion that controls breathing frequency

Neurons in a subregion of the medial parabrachial (PB) complex control expiratory duration (TE) and the inspiratory on-switch. To better understanding the underlying mechanisms, this study aimed to determine the types of medullary neurons in the rhythmogenic preBötzinger/Bötzinger Complex (preBötC/BötC) and adjacent areas that receive synaptic inputs from the PB subregion and whether these inputs are excitatory or inhibitory in nature. Highly localized electrical stimuli in the PB subregion combined with multi-electrode recordings from respiratory neurons and phrenic nerve activities were used to generate stimulus-to-spike event histograms to detect correlations in decerebrate, vagotomized dogs during isocapnic hyperoxia. Short-time scale correlations were found in 237/442 or ∼54% of the ventral respiratory column (VRC) neurons. Inhibition of E-neurons was ∼2.5X greater than for I-neurons, while Pre-I and I-neurons were excited. These findings indicate that the control of TE and the inspiratory on-switch by the PB subregion are mediated by a marked inhibition of BötC E-neurons combined with an excitation of I-neurons, especially pre-I neurons.

Calcium-induced calcium release activates spontaneous miniature outward currents in newt medullary reticular formation neurons.

Neurons in the medullary reticular formation are involved in the control of postural and locomotor behaviors in all vertebrates. Reticulospinal neurons in this brain region provide one of the major descending projections to the spinal cord. Although neurons in the newt medullary reticular formation have been extensively studied using in vivo extracellular recordings, little is known of their intrinsic biophysical properties or of the underlying circuitry of this region. Using whole cell patch-clamp recordings in brain slices containing the rostromedial reticular formation from adult male newts, we observed spontaneous miniature outward currents (SMOCs) in ~2/3 of neurons. Although SMOCs superficially resembled inhibitory postsynaptic currents (IPSCs), they had slower risetimes and decay times than spontaneous IPSCs. SMOCs required intracellular Ca2+ release from ryanodine receptors and were also dependent on the influx of extracellular Ca2+. SMOCs were unaffected by apamin but were partially blocked by iberiotoxin and charybdotoxin, indicating that SMOCs were mediated by big-conductance Ca2+-activated K+ channels. Application of the sarco/endoplasmic Ca2+ ATPase inhibitor cyclopiazonic acid blocked the generation of SMOCs and also increased neural excitability. Neurons with SMOCs had significantly broader action potentials, slower membrane time constants, and higher input resistance than neurons without SMOCs. Thus, SMOCs may serve as a mechanism to regulate action potential threshold in a majority of neurons within the newt medullary reticular formation. NEW & NOTEWORTHY The medullary reticular formation exerts a powerful influence on sensorimotor integration and subsequent motor behavior, yet little is known about the neurons involved. In this study, we identify a transient potassium current that regulates action potential threshold in a majority of medullary reticular neurons.