Noradrenergic, serotonergic and GABAergic antagonists injected together into the XII nucleus abolish the REM sleep-like depression of hypoglossal motoneuronal activity

Recently, we reported that the suppression of hypoglossal (XII) motoneuronal activity that occurs during the carbachol-induced, rapid eye movement (REM) sleep-like state is abolished by the microinjection into the XII nucleus of a drug mix that antagonizes aminergic excitation and amino acid-mediated inhibition (prazosin, methysergide, bicuculline and strychnine). We now assess the role of glycinergic inhibition in the depression of XII motoneuronal activity and estimate the distribution of the antagonists around the XII nucleus at the time when they are effective. Towards the first goal, REM sleep-like episodes were elicited in urethane-anesthetized rats by 10 nl carbachol microinjections into the dorsomedial pons prior to, and at different times after, combined microinjections into the XII nucleus of only three antagonists (strychnine omitted). As in our previous study, the carbachol-induced depression of XII activity was abolished during tests performed 42-88 min after the antagonists, whereas other characteristic effects of carbachol (appearance of hippocampal theta, cortical activation, decreased respiratory rate) remained intact. The depressant effect of carbachol on XII motoneurons partially recovered after 2.5 h. Towards the second goal, using a drug diffusion model, we determined that the tissue concentrations of the antagonists at the time when they were effective were within the range of their selective actions, and the drugs acted within 0.9-1.4 mm from the injection sites, thus within a space containing XII motoneurons and their dendrites. We conclude that antagonism of alpha-adrenergic, serotonergic, and GABA(A) receptors are sufficient to abolish the REM sleep-like atonia of XII motoneurons.     

Hypoglossal premotor neurons with rhythmical inspiratory-related activity in the cat: localization and projection to the phrenic nucleus

Localization and projection to the phrenic (PH) nucleus were studied in a sample of premotor neurons that directly projected to hypoglossal motoneurons (XII Mns) and showed respiratory-related patterns of activity. The experiments were carried out in cats, under pentobarbital anesthesia. In the first part of the study, the retrograde double-labeling technique was used to reveal the existence of neurons projecting to both the XII and the PH nuclei. Injection of a fluorescent dye (fast blue, FB) into the XII nucleus and another (nuclear yellow, NY) into the PH nucleus retrogradely labeled, with either FB or NY, medullary reticular neurons mainly in the regions ventrolateral to the nucleus of the tractus solitarius (vl-NTS), ventrolateral to the hypoglossal nucleus (vl-XII), and dorsomedial to the nucleus ambiguus (dm-AMB) bilaterally. In addition, some neurons in these regions were labeled with both FB and NY. In the second part of the study, unitary activity was recorded extracellularly from medullary respiratory neurons. In the regions vl-NTS, vl-XII, and dm-AMB, inspiratory neurons were found which antidromically responded to stimulation of the XII nucleus. Some of them also responded antidromically to stimulation of the PH nucleus. Averaging of rectified and integrated XII and PH nerve discharges by spontaneous spikes of single inspiratory neurons in the vl-NTS and dm-AMB regions revealed a facilitation in either XII nerve discharge or both XII and PH nerve discharges after a short latency of monosynaptic range. It is concluded that in the vl-NTS and dm-AMB regions there are inspiratory neurons that are excitatory premotor neurons projecting to XII Mns showing the respiratory-related activity. Some of them have excitatory synaptic connections to XII and PH Mns via bifurcating axons.     

Carbachol injections into the ventral pontine reticular formation activate locus coeruleus cells in urethane-anesthetized rats

STUDY OBJECTIVES: Two pontine reticular regions are implicated in cholinergic triggering of rapid eye movement (REM) sleep: the dorsomedial tegmental region and the ventral nucleus pontis oralis. We previously determined that, in urethane-anesthetized rats, microinjections of a cholinergic agonist, carbachol, into the dorsal region produce REM sleep-like effects comprising cortical activation, hippocampal theta rhythm, suppression of hypoglossal (XII) nerve activity, and silencing of pontine noradrenergic neurons. Our goal was to determine whether carbachol injections into the ventral nucleus pontis oralis elicits comparable effects. DESIGN: Recording of cortical electroencephalogram, hippocampal activity, XII nerve activity, and discharge of noradrenergic cells of the locus coeruleus. SETTING: Basic neurophysiologic research laboratory. PARTICIPANTS AND INTERVENTIONS: Urethane-anesthetized, paralyzed, and artificially ventilated or nonparalyzed and spontaneously breathing rats with microinjections of carbachol (10 nL, 10 mM) into the ventral nucleus pontis oralis. MEASUREMENTS AND RESULTS: In artificially ventilated rats, carbachol injections repeatedly elicited cortical activation and hippocampal theta rhythm. Concomitantly, the activity of locus coeruleus neurons increased from 2.0 per second +/- 0.4 (SE) to 2.6 per second +/- 0.4 (P < .05, n = 8), as did XII nerve activity (by 42.5% +/- 8.8%; P < .01). In spontaneously breathing animals, carbachol similarly activated the cortical electroencephalogram and hippocampal activity, whereas XII nerve activity was reduced by 6.7% +/- 2.5% (P < .05) together with increased ventilation, as indicated by reduced end-expiratory CO2. CONCLUSION: Carbachol injections into the ventral nucleus pontis oralis activate, rather than silence, noradrenergic locus coeruleus neurons. This is not compatible with the state of REM sleep.     

Respiratory pre-motor control of hypoglossal motoneurons in the rat

The goal of this study was to determine the origin and transmission pathway of respiratory drive to hypoglossal motoneurons. First we recorded intracellularly from 28 antidromically activated inspiratory hypoglossal motoneurons (resting membrane potential, −50±3 mV), and found that injection of chloride ions had no discernible effect on the shape of their membrane potential trajectories. We concluded that the membrane potential trajectories of these hypoglossal motoneurons were determined primarily by inspiratory excitation. To determine the origin of this excitation we cross-correlated the extracellular discharge of medullary inspiratory neurons, including those in the hypoglossal motor nucleus, with the hypoglossal nerve discharge. We found 27 inspiratory neurons within the hypoglossal motor nucleus that were not antidromically activated from the ipsilateral hypoglossal nerve; their cross-correlograms featured either central peaks (1.7±0.2 ms) alone (n=14; 39%), or central peaks (1.3±0.2 ms) followed by troughs (1.3±0.1 ms) at short latencies (1.1±0.4 ms) (n=13; 36%), and suggest that these neurons are hypoglossal interneurons. We recorded from 238 inspiratory neurons throughout the rest of the medulla; the cross-correlograms of 19 neurons (8%), located mostly in the lateral tegmental field, displayed narrow half-amplitude peaks (1.0±0.1 ms) at short latencies (0.9±0.1 ms), which we interpreted as evidence for monosynaptic excitation of hypoglossal motoneurons.

We conclude that the respiratory control of hypoglossal motoneurons originates from inspiratory premotor neurons scattered throughout the lateral tegmental field and interneurons within the hypoglossal motor nucleus.     

Monosynaptic excitation of medullary inspiratory neurons by bulbospinal inspiratory neurons of the ventral respiratory group in the cat

Summary In Nembutal-anesthetized, immobilized, and artificially ventilated cats, we studied the connectivity of medullary collaterals of bulbospinal inspiratory (BS-I) neurons in the ventral respiratory group (VRG). BS-I neurons which projected to the contralateral spinal cord were isolated extracellularly and intracellular recordings were made from the respiratory neurons in the contralateral VRG. The intracellular membrane potentials were averaged using extracellular spikes of the BS-I neurons as triggers (spike-triggered averaging method). Fast-rising and short-lasting depolarizing potentials locked to the triggering spikes were obtained and shown to be unitary EPSPs induced monosynaptically by the medullary collaterals of BS-I neurons. A total of 137 pairs were analyzed and unitary EPSPs were found in 11 pairs. Four BS-I neurons and 7 inspiratory vagal motoneurons received EPSPs from the medullary collaterals of BS-I neurons. These findings suggest that 1) BS-I neurons in the VRG drive medullary motoneurons of accessory respiratory muscles and phrenic or intercostal motoneurons simultaneously, 2) BS-I neurons on both sides synchronize via the excitatory connections, and 3) the augmenting firing pattern of BS-I neurons might partly be produced by this reexcitatory connection within the population of BS-I neurons.     

Inspiratory-phase short time scale synchrony in the brainstem slice is generated downstream of the pre-Bötzinger complex

Respiratory neurons are synchronized on a long time scale to generate inspiratory and expiratory-phase activities that are critical for respiration. Long time scale synchrony within the respiratory network occurs on a time scale of more than hundreds of milliseconds to seconds. During inspiration, neurons are synchronized on a short time scale to produce synchronous oscillations, which shape the pattern of inspiratory motor output. This latter form of synchrony within the respiratory network spans a shorter time range of tens of milliseconds. In the neonatal mouse rhythmically active medullary slice preparation, we recorded bilateral inspiratory activity from hypoglossal (XII) rootlets to study where in the slice synchronous oscillations are generated. Based on previous work that proposed the origin of these oscillations, we tested the pre-B?tzinger complex (PreB?tC) and the XII motor nucleus. Unilateral excitation of the PreB?tC, via local application of a perfusate containing high K(+), increased mean inspiratory burst frequency bilaterally (296+/-66%; n=10, P<0.01), but had no effect on the relative power of oscillations. In contrast, unilateral excitation of the XII nucleus increased both mean peak integrated activity bilaterally (ipsilateral: 41+/-10%, P<0.01; contralateral: 17+/-7%; P<0.05, n=10) and oscillation power in the ipsilateral (50+/-17%, n=7, P<0.05), but not in the contralateral rootlet. Cross-correlation analysis of control inspiratory activity recorded from the left and right XII rootlets produced cross-correlation histograms with significant peaks centered around a time lag of zero and showed no subsidiary harmonic peaks. Coherence analysis of left and right XII rootlet recordings demonstrated that oscillations are only weakly coherent. Together, the findings from local application experiments and cross-correlation and coherence analyses indicate that short time scale synchronous oscillations recorded in the slice are likely generated in or immediately upstream of the XII motor nucleus.     

Inhibition of pontine noradrenergic A7 cells reduces hypoglossal nerve activity in rats

Noradrenergic (NE) excitatory drive maintains activity of hypoglossal (XII) motoneurons during wakefulness. In predisposed persons, sleep-related decrements of NE cell activity may contribute to hypotonia of upper airway muscles innervated by XII motoneurons. The goal of this study was to determine whether NE neurons of the pontine A7 group, an anatomically identified source of NE projections to the XII nucleus, provide significant, endogenous NE excitatory drive to XII motoneurons. In anesthetized rats, we microinjected clonidine (0.75 mM, 20-40 nl), an alpha(2)-adrenergic receptor agonist that inhibits pontine NE cells, aiming at the A7 region. Nine injections were placed within 0.4 mm from the A7 group identified using tyrosine hydroxylase immunohistochemistry: they reduced XII nerve activity by 31.3+/-2.8% (standard error) and decreased the central respiratory rate by 6%. Another 21 injections, including eight placed near NE cells of the sub-coeruleus region, were made at distances over 0.5 mm from the A7 group and they did not alter either XII nerve activity or respiratory rate. In control experiments, clonidine injections into the A7 group preceded by injections of an alpha(2)-receptor antagonist, RS-79948, did not change XII nerve activity. Four experiments with unilateral clonidine injections into the A7 region and with Fos immunohistochemistry used as a marker of cell activity revealed that the percentage of Fos-positive A7 cells was significantly reduced on the injected side. There was also a significant positive correlation between Fos expression in A7 cells and XII nerve activity. Thus, decrements of NE excitatory drive from the A7 group may significantly reduce XII nerve activity. In obstructive sleep apnea patients, in whom the muscles innervated by XII motoneurons act as upper airway dilators, this may contribute to sleep-related respiratory disorders.     

Activation of XII motoneurons and premotor neurons during various oropharyngeal behaviors

Neural control of tongue muscles plays a crucial role in a broad range of oropharyngeal behaviors. Tongue movements must be rapidly and accurately adjusted in response to the demands of multiple complex motor tasks including licking/mastication, swallowing, vocalization, breathing and protective reflexes such as coughing. Yet, central mechanisms responsible for motor and premotor control of hypoglossal (XII) activity during these behaviors are still largely unknown. The aim of this article is to review the functional organization of the XII motor nucleus with particular emphasis on breathing, coughing and swallowing. Anatomical localization of XII premotor neurons is also considered. We discuss results concerned with multifunctional activity of medullary and pontine populations of XII premotor neurons, representing a single network that can be reconfigured to produce different oromotor response patterns. In this context, we introduce new data on swallowing-related activity of XII (and trigeminal) motoneurons, and finally suggest a prominent role for the pontine K?lliker-Fuse nucleus in the control of inspiratory-related activity of XII motoneurons supplying tongue protrusor and retrusor muscles.     

Effects of graded focal cold block in the solitary and para-ambigual regions of the medulla in the cat

Unilateral focal cold blocks in the region of the nucleus tractus solitarius and the dorsal respiratory group of neurons, DRG, of anaesthetized cats consistently caused apneustic-type breathing. There was no concomitant change in the initial rate of rise of inspiratory activity. The apneustic prolongation of inspiratory duration, TI, was most pronounced in, but was not confined to, the DRG. The apneustic effects were more marked after vagotomy. In cats with intact vagus nerves being given artificial ventilation, focal cooling at certain sites of the DRG region could produce 'unlocking' of the respiratory rhythm from that of the respiratory pump. At other sites in this region, focal cooling could selectively block the effects of the inspiration-facilitating reflex induced by deflation without blocking the inspiration-inhibiting Hering-Breuer reflex. Unilateral focal cold blocks in the region of the intermediate part of the ventral respiratory group of neurons, VRG, generally caused depression of the rate of rise of inspiratory activity, but almost never apneustic effects. All effects of unilateral focal cooling both in the DRG and VRG were bilaterally symmetrical. No systematic differences between the effects on phrenic and external intercostal inspiratory activity were found in response to focal cooling either of the DRG or VRG suggesting that differential control of phrenic and external intercostal motoneurons is not exerted mainly at the level of these medullary structures. The results suggest that the DRG and VRG areas exert somewhat different effects on the respiratory pattern: DRG appears to be more concerned with integration of vagal and other inputs contributing to the inspiratory off-switch mechanisms which, however, are not confined only to the DRG. The VRG inspiratory mechanisms, on the other hand, appear to be more involved in the gain control of the inspiratory output intensity.     

The role of dorsal respiratory group neurons studied with cross-correlation in the decerebrate rat

We examined the role of dorsal respiratory group (DRG) inspiratory neurons as transmitters of respiratory drive to phrenic and intercostal motoneurons and as relays of afferent information to ventral respiratory group (VRG) bulbospinal, inspiratory neurons. Attempts to antidromically activate 76 DRG neurons from the spinal cord at the C7 segment resulted in only 4 (5.3%) successes (3 contralateral, 1 ipsilateral). Cross-correlating DRG neuron discharge with that of the ipsilateral (56) and contralateral (20) phrenic nerve detected common activation peaks in 2 and 3 cases respectively, with no evidence for monosynaptic connections. Cross-correlating DRG neuron discharge with that of bulbospinal, inspiratory VRG neurons found some evidence for interaction. Peaks in 7 of 73 (10%) cross-correlation histograms were attributed to a monosynaptic excitation of DRG neurons by VRG neurons, although a common activation cannot be ruled out; troughs, some with an accompanying peak, in 9 (12.3%) histograms were interpreted as a combined excitation of the DRG neuron and delayed inhibition of the VRG neuron. In addition, 2 cross-correlation histograms showed peaks with latencies and half-amplitude widths consistent with a disynaptic excitation of a DRG neuron by a bulbospinal inspiratory VRG neuron. Cross-correlating the discharge of 57 pairs of DRG inspiratory neurons (6 contralateral) detected common activation peaks in 7 (12.3%) cases (none contralateral) and one case interpreted as evidence for a disynaptic excitation. These findings suggest that the role of the DRG inspiratory neurons in rats differs from that in cats, primarily because they do not act to transmit respiratory rhythmic drive directly to phrenic and intercostal motoneurons. The results offer some support for an excitation of DRG neurons by VRG inspiratory neurons, but no support for a role of DRG inspiratory neurons as mediators of afferent information transfer to VRG bulbospinal inspiratory neurons.     

Effects of hypocapnia on respiratory timing and inspiratory activities of the superior laryngeal, hypoglossal, and phrenic nerves in the vagotomized rat

Effects of acute hypocapnia on respiratory timing (inspiratory and expiratory times (TI, TE) ) and on inspiratory activities of the efferent superior laryngeal (Xs1), hypoglossal (XII), and phrenic (Phr) nerves were studied in artificially ventilated vagotomized, and anesthetized rats. Hyperventilation induced a decrease in respiratory frequency exclusively due to prolongation of TE and resulted in expiratory apnea. Inspiratory activities of three nerves decreased with reduction in CO2 concentration of end-tidal gas (FETCO2), and disappeared simultaneously at a threshold FETCO2 for apnea. The decrease in the peak inspiratory activity by hypocapnia was larger in the XII than in the Phr or Xs1 nerve (XII greater than Phr greater than Xs1). The results suggest that the CO2 stimulus (mainly via a central chemosensor) plays an important role in the process of terminating expiration or of expiratory-inspiratory phase switching and that the responses of the XII or Xs1 motoneurons to variation in CO2 stimulus differ from that of the Phr motoneurons (or of the Phr driving medullary neurons). A possible functional significance of these observations is discussed.     

Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Bötzinger complex in the cat

Summary Axonal projections and synaptic connectivity of expiratory B?tzinger neurons with an augmenting firing pattern (Bot-Aug neurons) to neurons in the ipsilateral ventral respiratory group (VRG) were studied in anaesthetized cats. Antidromic mapping revealed extensive axonal arborizations of Bot-Aug neurons (24 of 45) to the rostral or caudal VRG, with some having arbors in both regions. Of 234 pairs of neurons studied with intracellular recording and spike-triggered averaging, monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked in 49/221 VRG neurons by 38/98 Bot-Aug neurons. The highest incidence of monosynaptic inhibition was found in inspiratory bulbospinal neurons (10 of 23 tested). Evidence was also found for monosynaptic inhibition, by a separate group of Bot-Aug neurons, of expiratory bulbospinal neurons (12/58), while excitatory postsynaptic potentials (EPSPs) were identified in another two of these neurons. In addition, monosynaptic IPSPs were recorded from 13 of 53 identified laryngeal motoneurons, and from 14 of 100 respiratory propriobulbar neurons. Presumptive disynaptic IPSPs were recorded from 11 of the 221 VRG neurons. We conclude that Bot-Aug neurons exert widespread inhibition on all major neuron categories in the ipsilateral VRG, and should be regarded as an important element in shaping the spatiotemporal output pattern of both respiratory motoneurons and premotor neurons.     

Hypoglossal nerve response to 5-HT3 drugs injected into the XII nucleus and vena cava in the rat.

Systemically administered ondansetron, a 5-HT3 receptor antagonist, reduces obstructive sleep-disordered breathing (OSDB) events in the English bulldog. The neural mechanisms through which ondansetron acts are unknown. 5-HT3 receptor immunoreactivity and mRNA have been detected in the vicinity of upper airway dilator motoneurons (UAWDMn's), suggesting that this receptor may contribute to the state-dependent modulation of UADMn's. To characterize 5-HT3 receptor activity within a representative UAWD nucleus, we performed acute microinjections of selective 5-HT3 drugs, 1-(m-Chlorophenyl)-biguanide HCl, an agonist, and ondansetron, an antagonist, into a major population of UADMn's, the hypoglossal nucleus (XII), in anesthetized, paralyzed and mechanically-ventilated rats. The 5-HT3-selective drugs neither altered the baseline XII nerve activity nor the excitatory effect of 5-HT microinjected into the XII. In contrast, systemic-administration of ondansetron (3 mg/kg) produced a significant increase in the inspiratory modulation of XII nerve activity (to 195.8%+/-19.3 of control, p<0.001). Together, these data suggest that 5-HT3 receptors within the XII nucleus do not mediate 5-HT effects on XII motoneurons, rather antagonism of 5-HT3 receptors outside the XII nucleus can increase respiratory drive to XII motoneurons. These results highlight the importance of understanding serotonergic effects on respiratory drive outside the UAWD motor nuclei as we search for 5-HT drug therapies for OSDB.     

Multimodal medullary neurons and correlational linkages of the respiratory network.

This study addresses the hypothesis that multiple sensory systems, each capable of reflexly altering breathing, jointly influence neurons of the brain stem respiratory network. Carotid chemoreceptors, baroreceptors, and foot pad nociceptors were stimulated sequentially in 33 Dial-urethan-anesthetized or decerebrate vagotomized adult cats. Neuronal impulses were monitored with microelectrode arrays in the rostral and caudal ventral respiratory group (VRG), nucleus tractus solitarius (NTS), and n. raphe obscurus. Efferent phrenic nerve activity was recorded. Spike trains of 889 neurons were analyzed with cycle-triggered histograms and tested for respiratory-modulated firing rates. Responses to stimulus protocols were assessed with peristimulus time and cumulative sum histograms. Cross-correlation analysis was used to test for nonrandom temporal relationships between spike trains. Spike-triggered averages of efferent phrenic activity and antidromic stimulation methods provided evidence for functional associations of bulbar neurons with phrenic motoneurons. Spike train cross-correlograms were calculated for 6,471 pairs of neurons. Significant correlogram features were detected for 425 pairs, including 189 primary central peaks or troughs, 156 offset peaks or troughs, and 80 pairs with multiple peaks and troughs. The results provide evidence that correlational medullary assemblies include neurons with overlapping memberships in groups responsive to different sets of sensory modalities. The data suggest and support several hypotheses concerning cooperative relationships that modulate the respiratory motor pattern. 1) Neurons responsive to a single tested modality promote or limit changes in firing rate of multimodal target neurons. 2) Multimodal neurons contribute to changes in firing rate of neurons responsive to a single tested modality. 3) Multimodal neurons may promote responses during stimulation of one modality and "limit" changes in firing rates during stimulation of another sensory modality. 4) Caudal VRG inspiratory neurons have inhibitory connections that provide negative feedback regulation of inspiratory drive and phase duration.     

Control of abdominal muscles by brain stem respiratory neurons in the cat

Control of abdominal musculature by brain stem respiratory neurons was studied in decerebrate unanesthetized cats by determining 1) which brain stem respiratory neurons could be antidromically activated from the lumbar cord, from which the abdominal muscles receive part of their innervation, and 2) if lumbar-projecting respiratory neurons make monosynaptic connections with abdominal motoneurons. A total of 462 respiratory neurons, located between caudal C2 and the retrofacial nucleus (B?tzinger complex), were tested for antidromic activation from the upper lumbar cord. Fifty-eight percent of expiratory (E) neurons (70/121) in the caudal ventral respiratory group (VRG) between the obex and rostral C1 were antidromically activated from contralateral L1. Eight of these neurons were activated at low thresholds from lamina VIII and IX in the L1-2 gray matter. One-third (14/41) of the E neurons that projected to L1 could also be activated from L4-5. Almost all antidromic E neurons had an augmenting firing pattern. Ten scattered inspiratory (I) neurons projected to L1 but could not be activated from L4-5. No neurons that fired during both E and I phases (phase-spanning neurons) were antidromically activated from the lumbar cord. In order to test for possible monosynaptic connections between descending E neurons and abdominal motoneurons, cross-correlations were obtained between 27 VRG E neurons, which were antidromically activated from caudal L2 and contralateral L1 and L2 abdominal nerve activity (47 neuron-nerve combinations). Only two neurons showed a correlation with one of the two nerves tested. Although there is a large projection to the lumbar cord from expiratory neurons in the ventral respiratory group caudal to the obex, cross-correlation analyses suggest that strong monosynaptic connections between these neurons and abdominal motoneurons are scarce.     

Differential responses of respiratory nuclei to anoxia in rhythmic brain stem slices of mice

The response of the neonatal respiratory system to hypoxia is characterized by an initial increase in ventilation, which is followed within a few minutes by a depression of ventilation below baseline levels. We used the transverse medullary slice of newborn mice as a model system for central respiratory control to investigate the effects of short-lasting periods of anoxia. Extracellular population activity was simultaneously recorded from the ventral respiratory group (VRG) and the hypoglossus (XII) nucleus (a respiration-related motor output nucleus). During anoxia, respiratory frequency was modulated in a biphasic manner and phase-locked in both the VRG and the XII. The amplitude of phasic respiratory bursts was increased only in the XII and not in the VRG. This increase in XII burst amplitude commenced approximately 1 min after the anoxic onset concomitant with a transient increase in tonic activity. The burst amplitude remained elevated throughout the entire 5 min of anoxia. Inspiratory burst amplitude in the VRG, in contrary, remained constant or even decreased during anoxia. These findings represent the first simultaneous extracellular cell population recordings of two respiratory nuclei. They provide important data indicating that rhythm generation is altered in the VRG without a concomitant alteration in the VRG burst amplitude, whereas the burst amplitude is modulated only in the XII nucleus. This has important implications because it suggests that rhythm generation and motor pattern generation are regulated separately within the respiratory network.     

Intercostal and abdominal muscle afferent influence on caudal medullary expiratory neurons that drive abdominal muscles

Summary Our objective was to determine if caudal ventral respiratory group (VRG) expiratory (E) neurons that drive abdominal expiratory motoneurons in the lumbar cord respond to intercostal and lumbar nerve afferent stimulation. Results showed that 92% of medullary E-neurons that were antidromically activated from the upper lumbar cord reduced their activity in response to stimulation of external and internal intercostal and lumbar nerve afferents. We conclude that afferent information from intercostal and abdominal muscle tendon organs has an inhibitory effect on caudal VRG E-neurons that drive abdominal expiratory motoneurons.This study was supported by National Heart, Lung, and Blood Institute grant RO1-HL-17715     

Chemical activation of caudal medullary expiratory neurones alters the pattern of breathing in the cat.

1. The purpose of this work was to ascertain whether the activation of caudal expiratory neurones located in the caudal part of the ventral respiratory group (VRG) may affect the pattern of breathing via medullary axon collaterals. 2. We used microinjections of DL-homocysteic acid (DLH) to activate this population of neurones in pentobarbitone-anaesthetized, vagotomized, paralysed and artificially ventilated cats. Both phrenic and abdominal nerve activities were monitored; extracellular recordings from medullary and upper cervical cord respiratory neurones were performed. 3. DLH (160 mM) microinjected (10-30 nl for a total of 1.6-4.8 nmol) into the caudal VRG, into sites where expiratory activity was encountered, provoked an intense and sustained activation of the expiratory motor output associated with a corresponding period of silence in phrenic nerve activity. During the progressive decline of the activation of abdominal motoneurones, rhythmic inspiratory activity resumed, displaying a decrease in frequency and a marked reduction or the complete suppression of postinspiratory activity as its most consistent features. 4. Medullary and upper cervical cord inspiratory neurones exhibited inhibitory responses consistent with those observed in phrenic nerve activity, while expiratory neurones in the caudal VRG on the side contralateral to the injection showed excitation patterns similar to those of abdominal motoneurones. On the other hand, in correspondence to expiratory motor output activation, expiratory neurones of the Bötzinger complex displayed tonic discharges whose intensity was markedly lower than the peak level of control breaths. 5. Bilateral lignocaine blockades of neural transmission at C2-C3 affecting the expiratory and, to a varying extent, the inspiratory bulbospinal pathways as well as spinal cord transections at C2-C3 or C1-C2, did not suppress the inhibitory effect on inspiratory neurones of either the ipsi- or contralateral VRG in response to DLH microinjections into the caudal VRG. 6. The results show that neurones within the column of caudal VRG expiratory neurones promote inhibitory effects on phrenic nerve activity and resetting of the respiratory rhythm. We suggest that these effects are mediated by medullary bulbospinal expiratory neurones, which may, therefore, have a function in the control of breathing through medullary axon collaterals.     

Genioglossus premotoneurons and the negative pressure reflex in rats

Reflex increases in genioglossus (GG) muscle activity in response to negative pharyngeal pressure are important for maintenance of upper airway patency in humans. However, little is known of the central circuitry that mediates this negative pressure reflex (NPR). We used two approaches to determine which GG premotoneurons relay negative pressure-related information to the hypoglossal motor nucleus. First, to identify GG premotoneurons, we injected pseudorabies virus (PRV152) into the GG muscle. We found that medullary GG premotoneurons were concentrated mainly in the reticular formation adjacent to the hypoglossal motor nucleus. Second, in order to determine whether these perihypoglossal neurons were involved in the NPR, we quantified GG EMG responses to negative pressure applied to the isolated upper airway in anaesthetized rats before and after microinjection of muscimol (9 nl; 0.25 m m ), a GABA-A receptor agonist, into the perihypoglossal premotor field. Pressures as low as −4 cmH₂O increased inspiratory phase-related GG activity. The NPR was abolished following bilateral injections of muscimol into the perihypoglossal premotor field at and up to 500 μm rostral to the obex. Muscimol in this location also increased the amplitude of basal, unstimulated phasic GG activity. By contrast, inhibition of neurons caudal to the obex decreased phasic GG activity but had no impact on the NPR. These results suggest that perihypoglossal GG premotoneurons near the obex mediate the NPR and those caudal to the obex are important mediators of respiratory-related GG activity but are not involved in the NPR.     

Serotonergic modulation of respiratory motoneurons and interneurons in brainstem slices of perinatal rats

Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-B?tzinger complex (pre-B?tC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (approximately 11 mV) and tonic spike discharge in XII motoneurons, whereas pre-B?tC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-B?tC exclusively increased respiratory frequency by 30-220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-B?tC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT2 receptor agonist -methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-B?tC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT2 receptors of pre-B?tC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT1A receptors.