The purpose of the present study was to determine the effect on breathing in the awake state of carotid body denervation (CBD) over 1-2 wk after denervation. Studies were completed on adult goats repeatedly before and 1) for 15 days after bilateral CBD (n = 8), 2) for 7 days after unilateral CBD (n = 5), and 3) for 15 days after sham CBD (n = 3). Absence of ventilatory stimulation when NaCN was injected directly into a common carotid artery confirmed CBD. There was a significant (P < 0.01) hypoventilation during the breathing of room air after unilateral and bilateral CBD. The maximum PaCO2 increase (8 Torr for unilateral and 11 Torr for bilateral) occurred approximately 4 days after CBD. This maximum was transient because by 7 (unilateral) to 15 (bilateral) days after CBD, PaCO2 was only 3-4 Torr above control. CO2 sensitivity was attenuated from control by 60% on day 4 after bilateral CBD and by 35% on day 4 after unilateral CBD. This attenuation was transient, because CO2 sensitivity returned to control temporally similar to the return of PaCO2 during the breathing of room air. During mild and moderate treadmill exercise 1-8 days after bilateral CBD, PaCO2 was unchanged from its elevated level at rest, but, 10-15 days after CBD, PaCO2 decreased slightly from rest during exercise. These data indicate that 1) carotid afferents are an important determinant of rest and exercise breathing and ventilatory CO2 sensitivity, and 2) apparent plasticity within the ventilatory control system eventually provides compensation for chronic loss of these afferents.

.-In awake goats, 29% bilateral destruction of neurokinin-1 receptorexpressing neurons in the pre-Bötzinger complex (pre-BötzC) area with saporin conjugated to substance P results in transient disruptions of the normal pattern of eupneic respiratory muscle activation (Wenninger JM, Pan LG, Klum L, Leekley T, Bastastic J, Hodges MR, Feroah T, Davis S, and Forster HV. J Appl Physiol 97: 1620 -1628, 2004). Therefore, the purpose of these studies was to determine whether large or total lesioning in the pre-BötzC area of goats would eliminate phasic diaphragm activity and the eupneic breathing pattern. In awake goats that already had 29% bilateral destruction of neurokinin-1 receptor-expressing neurons in the pre-BötzC area, bilateral ibotenic acid (10 l, 50 mM) injection into the pre-BötzC area resulted in a tachypneic hyperpnea that reached a maximum (132 Ϯ 10.1 breaths/min) ϳ30 -90 min after bilateral injection. Thereafter, breathing frequency declined, central apneas resulted in arterial hypoxemia (arterial PO 2 ϳ40 Torr) and hypercapnia (arterial PCO2 ϳ60 Torr), and, 11 Ϯ 3 min after the peak tachypnea, respiratory failure was followed by cardiac arrest in three airway-intact goats. However, after the peak tachypnea in four tracheostomized goats, mechanical ventilation was initiated to maintain arterial blood gases at control levels, during which there was no phasic diaphragm or abdominal muscle activity. When briefly removed from the ventilator (ϳ90 s), these goats became hypoxemic and hypercapnic. During this time, minimal, passive inspiratory flow resulted from phasic abdominal muscle activity. We estimate that 70% of the neurons within the pre-BötzC area were lesioned in these goats. We conclude that, in the awake state, the pre-BötzC is critical for generating a diaphragm, eupneic respiratory rhythm, and that, in the absence of the pre-BötzC, spontaneous breathing reflects the activity of an expiratory rhythm generator. respiratory rhythm generator; terminal apnea; inspiratory and preinspiratory neurons SMITH ET AL. (21) DEMONSTRATED in the in vitro neonatal rat brain stem preparation that elimination of the pre-Bötzinger complex (pre-BötzC) caused cessation of respiratory rhythm. Since then, results from many in vitro studies support the pre-BötzC as the site or "kernel" of respiratory rhythm generation (9,19,20,21). Furthermore, in in vivo studies on anesthetized cats and rats, injection of the glutamate receptor agonist DL-homocysteic acid into the pre-BötzC area increases tonic and/or phasic phrenic nerve output, whereas injections into other proximal or distal nuclei do not increase respiratory rhythm (1,15,22), thus providing a physiological definition of the preBötzC. In addition, in vivo studies in anesthetized or decerebrate cats or rats demonstrate that lesioning of the pre-BötzC results in transient (24) or irreversible (7, 10, 18) elimination of eupneic respiratory activity. Further demonstrating the importance of the pre-BötzC in control of breathing was a study showing that Ͼ80% destruction o...

Our aim was to determine the effects of focal acidification in the raphe obscurus (RO) and raphe pallidus (RP) on ventilation and other physiological variables in both the awake and sleep states in adult goats. Through chronically implanted microtubules, 1) a focal acidosis was created by microdialysis of mock cerebrospinal fluid (mCSF), equilibrated with various levels of CO2, and 2) medullary extracellular fluid (ECF) pH was measured by using a custom-made pH electrode. Focal acidosis in the RO or RP, by dialyzing either 25 or 80% CO2 (mCSF pH approximately 6.8 or 6.3), increased (P < 0.05) inspiratory flow by 8 and 12%, respectively, while the animals were awake during the day, but not at night while they were awake or in non-rapid eye movement sleep. While the animals were awake during the day, there were also increases in heart rate and blood pressure (P< 0.05) but no significant change in metabolic rate or arterial Pco2. Dialysis with mCSF equilibrated with 25 or 80% CO2 reduced ECF pH by the same amount (25%) or three times more (80%) than when inspired CO2 was increased to 7%. During CO2 inhalation, the reduction in ECF pH was only 50% of the reduction in arterial pH. Finally, dialysis in vivo only decreased ECF pH by 19.1% of the change during dialysis in an in vitro system. We conclude that 1) the physiological responses to focal acidosis in the RO and RP are consistent with the existence of chemoreceptors in these nuclei, and 2) local pH buffering mechanisms act to minimize changes in brain pH during systemic induced acidosis and microdialysis focal acidosis and that these mechanisms could be as or more important to pH regulation than the small changes in inspiratory flow during a focal acidosis.

Muere C, Neumueller S, Miller J, Olesiak S, Hodges MR, Pan L, Forster HV. Atropine microdialysis within or near the pre-Bötzinger Complex increases breathing frequency more during wakefulness than during NREM sleep. J Appl Physiol 114: 694-704, 2013. First published December 27, 2012 doi:10.1152/japplphysiol.00634.2012.-Normal activity of neurons within the medullary ventral respiratory column (VRC) in or near the pre-Bötzinger Complex (preBötC) is dependent on the balance of inhibitory and excitatory neuromodulators acting at their respective receptors. The role of cholinergic neuromodulation during awake and sleep states is unknown. Accordingly, our objective herein was to test the hypotheses that attenuation of cholinergic modulation of VRC/ preBötC neurons in vivo with atropine would: 1) decrease breathing frequency more while awake than during non-rapid-eye-movement (NREM) sleep and 2) increase other excitatory neuromodulators. To test these hypotheses, we unilaterally dialyzed mock cerebrospinal fluid (mCSF) or 50 mM atropine in mCSF in or near the preBötC region of adult goats during the awake (n ϭ 9) and NREM sleep (n ϭ 7) states. Breathing was monitored, and effluent dialysate was collected for analysis of multiple neurochemicals. Compared with dialysis of mCSF alone, atropine increased (P Ͻ 0.05) breathing frequency while awake during the day [ϩ10 breaths (br)/min] and at night (ϩ9 br/min) and, to a lesser extent, during NREM sleep (ϩ5 br/min). Atropine increased (P Ͻ 0.05) effluent concentrations of serotonin (5-HT), substance P (SP), and glycine during the day and at night. When atropine was dialyzed in one preBötC and mCSF in the contralateral preBötC, 5-HT and SP increased only at the site of atropine dialysis. We conclude: 1) attenuation of a single neuromodulator results in local changes in other neuromodulators that affect ventilatory control, 2) effects of perturbations of cholinergic neuromodulation on breathing are state-dependent, and 3) interpretation of perturbations in vivo requires consideration of direct and indirect effects.neuromodulators; control of breathing; acetylcholine; non-rapid-eyemovement sleep THE NEUROMODULATOR ACETYLCHOLINE (ACh) is involved in a variety of central and peripheral neural circuits, including the control of breathing and sleep (7,12,16,19,25,39). The effects of ACh are mediated through either nicotinic or muscarinic receptors coupled to second messenger pathways (6, 10, 13, 42). There are five known muscarinic ACh receptor (mAChR) subtypes (M 1 to M 5 ) expressed in the brain stem, differing in expression profiles and in effects on breathing (2,9,11,13,33,34). For example, the pontine respiratory group (PRG) nuclei express M 1 -M 3 mAChRs (34). The predominant effect of cholinergic modulation of PRG neurons within the Kölliker-Fuse Nucleus (KFN) appears to be excitatory, since reverse dialysis of the nonselective mAChR antagonist atropine reduced minute ventilation (V I ) and breathing frequency in both the awake state and during non-rapid-eye-movement (NREM...

To gain insight into why there are chemoreceptors at widespread sites in the brain, mircrotubules were chronically implanted at two or three sites in the medullary raphe nuclei of adult goats (n = 7). After >2 wk, microdialysis (MD) probes were inserted into the microtubules to create focal acidosis (FA) in the awake state using mock cerebral spinal fluid (mCSF) equilibrated with 6.4% (pH = 7.3), 50% (pH = 6.5), or 80% CO(2) (pH = 6.3), where MD with 50 and 80% CO(2) reduces tissue pH by 0.1 and 0.18 pH unit, respectively. There were no changes in all measured variables with MD with 6.4% at single or multiple raphe sites (P > 0.05). During FA at single raphe sites, only 80% CO(2) elicited physiological changes as inspiratory flow was 16.9% above (P < 0.05) control. However, FA with 50 and 80% CO(2) at multiple sites increased (P < 0.05) inspiratory flow by 18.4 and 30.1%, respectively, where 80% CO(2) also increased (P < 0.05) tidal volume, heart rate, CO(2) production, and O(2) consumption. FA with 80% CO(2) at multiple raphe sites also led to hyperventilation (-2 mmHg), indicating that FA had effects on breathing independent of an increased metabolic rate. We believe these findings suggest that the large ventilatory response to a global respiratory brain acidosis reflects the cumulative effect of stimulation at widespread chemoreceptor sites rather than a large stimulation at a single site. Additionally, focal acidification of raphe chemoreceptors appears to activate an established thermogenic response needed to offset the increased heat loss associated with the CO(2) hyperpnea.

No abstract

. Transient attenuation of CO 2 sensitivity after neurotoxic lesions in the medullary raphe area of awake goats. J Appl Physiol 97: 2236 -2247, 2004. First published August 20, 2004; doi:10.1152/japplphysiol.00584.2004The major objective of this study was to gain insight into whether under physiological conditions medullary raphe area neurons influence breathing through CO 2/H ϩ chemoreceptors and/or through a postulated, nonchemoreceptor modulatory influence. Microtubules were chronically implanted into the raphe of adult goats (n ϭ 13), and breathing at rest (awake and asleep), breathing during exercise, as well as CO 2 sensitivity were assessed repeatedly before and after sequential injections of the neurotoxins saporin conjugated to substance P [SP-SAP; neurokinin-1 receptor (NK1R) specific] and ibotenic acid (IA; nonspecific glutamate receptor excitotoxin). In all goats, microtubule implantation alone resulted in altered breathing periods, manifested as central or obstructive apneas, and fractionated breathing. The frequency and characteristics of the altered breathing periods were not subsequently affected by injections of the neurotoxins (P Ͼ 0.05). Three to seven days after SP-SAP or subsequent IA injection, CO 2 sensitivity was reduced (P Ͻ 0.05) by 23.8 and 26.8%, respectively, but CO 2 sensitivity returned to preinjection control values Ͼ7 days postinjection. However, there was no hypoventilation at rest (awake, non-rapid eye movement sleep, or rapid eye movement sleep) or during exercise after these injections (P Ͼ 0.05). The neurotoxin injections resulted in neuronal death greater than three times that with microtubule implantation alone and reduced (P Ͻ 0.05) both tryptophan hydroxylase-expressing (36%) and NK1R-expressing (35%) neurons at the site of injection. We conclude that both NK1R-and glutamate receptor-expressing neurons in the medullary raphe nuclei influence CO 2 sensitivity apparently through CO 2/H-expressing chemoreception, but the altered breathing periods appear unrelated to CO 2 chemoreception and thus are likely due to non-chemoreceptor-related neuromodulation of ventilatory control mechanisms. central chemoreception; control of breathing THE MEDULLARY RAPHE NUCLEI project to and supposedly modulate several central nervous system regions, including the pre-Bötzinger complex; the hypoglossal, phrenic, and spinal motoneurons; the nucleus ambiguus; the nucleus of the solitary tract; and various pontine nuclei (3,4,8,16,(37)(38)(39). The neuronal population in the raphe is heterogeneous, containing neurokinin-1 receptor-expressing (NK1R) and serotonergic neurons (28). Serotonergic neurons corelease thyrotropin-releasing hormone and substance P (SP), which are all capable of modulating ventilation (5, 7, 14, 15, 19 -21). Therefore, these raphe neurons are capable of influencing breathing through multiple neuromodulators acting at multiple levels, including the rhythm-and pattern-generating neurons and motoneurons. The primary site of raphe neuromodulation during physiological condition...

In awake rats, >80% bilateral reduction of neurokinin-1 receptor (NK1R)-expressing neurons in the pre-Bötzinger complex (pre-BötzC) resulted in hypoventilation and an "ataxic" breathing pattern (Gray PA, Rekling JC, Bocchiaro CM, Feldman JL, Science 286: 1566-1568, 1999). Accordingly, the present study was designed to gain further insight into the role of the pre-BötzC area NK1R-expressing neurons in the control of breathing during physiological conditions. Microtubules were chronically implanted bilaterally into the medulla of adult goats. After recovery from surgery, the neurotoxin saporin conjugated to substance P, specific for NK1R-expressing neurons, was bilaterally injected (50 pM in 10 microl) into the pre-BötzC area during the awake state (n = 8). In unoperated goats, 34 +/- 0.01% of the pre-BötzC area neurons are immunoreactive for the NK1R, but, in goats after bilateral injection of SP-SAP into the pre-BötzC area, NK1R immunoreactivity was reduced to 22.5 +/- 2.5% (29% decrease, P < 0.01). Ten to fourteen days after the injection, the frequency of abnormal breathing periods was sixfold greater than before injection (107.8 +/- 21.8/h, P < 0.001). Fifty-six percent of these periods were breaths of varying duration and volume with an altered respiratory muscle activation pattern, whereas the remaining were rapid, complete breaths with coordinated inspiratory-expiratory cycles. The rate of occurrence and characteristics of abnormal breathing periods were not altered during a CO2 inhalation-induced hyperpnea. Pathological breathing patterns were eliminated during non-rapid eye movement sleep in seven of eight goats, but they frequently occurred on arousal from non-rapid eye movement sleep. We conclude that a moderate reduction in pre-BötzC NK1R-expressing neurons results in state-dependent transient changes in respiratory rhythm and/or eupneic respiratory muscle activation patterns.