The mechanisms which contribute to the time‐dependent recovery of resting ventilation and the ventilatory CO2 chemoreflex after carotid body denervation (CBD) are poorly understood. The initiation of neuronal plasticity often involves changes in Ca2+ permeability. Therefore, we tested the hypothesis that during recovery from the acute effects of CBD in goats, there is an increase in Ca2+ permeable GluA1R and GluN1R and/or a decrease in Ca2+ impermeable GluA2R expression at brainstem sites that contribute to respiratory control. We tested this hypothesis via optical densitometric (OD) quantification of medullary neurons immunostained with antibodies targeting GluA1R, GluA2R, and GluN1R at 5‐13 days or 30 days after bilateral CBD. CBD resulted in hypoventilation and a reduced CO2 chemoreflex, which both recovered significantly over 30 days post‐CBD. At 5‐13 days post‐CBD, GluA1 OD was unchanged from control goats, but GluA2 and GluN1 OD were reduced 15‐30% within 6 of medullary respiratory nuclei measured. However, at 30 days post‐CBD GluA1 OD remained unchanged, but GluA2 and GluN1 OD returned to control levels. We conclude that a shift toward Ca2+ permeable glutamate receptors 5‐13 days post‐CBD may initiate the recovery of ventilation after CBD. Furthermore, at 30 days post‐CBD the partial recovery of ventilation is maintained by relatively normal glutamate receptor expression.Grant Funding Source: Supported by National Heart, Lung, and Blood Institute Grants HL25739 and HL007852 and by VA

It has been hypothesized that the pFRG/RTN acts as an integrator of multiple excitatory respiratory inputs [J Appl Physiol 78 (1):247–257, 1995], and/or an expiratory rhythm generator [J Physiol 570.2: 407–420, 2006]. Here, we studied the effects on ventilation and respiratory muscle activity of ibotenic acid (IA) pFRG/RTN lesions in adult goats, where the lesion sites were targeted by surface‐cooling induced apnea under anesthesia. Upon recovery, all goats hypoventilated (PaCO2 8–15 mmHg above control) for several days during eupnea, with inspiratory and expiratory muscle activity present throughout. During this period, there was a decreased CO2 sensitivity and an attenuation of the diaphragm response to CO2, but a concomitant compensatory increase in abdominal muscle activity. More than 3 weeks post‐lesioning, all goats demonstrated a near‐complete recovery, similar to the time‐dependent plasticity observed in carotid body denervated goats [J Appl Physiol 85: 1299–1306, 1998]. Postmortem histology indicated a deficit of Phox2b stained neurons ventral and lateral to the facial nucleus, which likely includes the pFRG/RTN. These data suggest pFRG/RTN neurons contribute to eupneic ventilatory drive, CO2 sensitivity and inspiratory muscle activity, but do not support the hypothesis that the pFRG/RTN acts as an expiratory rhythm generator. Supported by NIH HL25739 and Veterans Administration

Both carotid and intracranial chemoreceptors are critical to a normal ventilatory CO2-H+ chemosensitivity. At low levels of hypercapnia, the carotid contribution is probably greater than the central contribution but, at high levels, the intracranial chemoreceptors are dominant. The carotid chemoreceptors are also critical to maintaining a stable and normal eupneic PaCO2, but lesion-induced attenuation of intracranial CO2-H+ chemosensitivity does not consistently alter eupneic PaCO2. A major unanswered question is why do intracranial chemoreceptors in carotid body denervation (CBD) animals tolerate an acidosis during eupnea which prior to CBD elicits a marked increase in breathing.

Dialyzing mock cerebral spinal fluid (mCSF) equilibrated with 25% CO2 in the retrotrapezoid nucleus and the nucleus of the solitary tract of the awake rat increased inspiratory flow (VI) and tidal volume (VT) by 24 and 11%. In the awake goat, we found that dialyzing 25 and 80% CO2 at one site in the raphe increased VI and VT 8–10%, while dialyzing 50% in the rostral part of the cerebellar fastigial nucleus increased VI and VT 14–16%. The pre-Botzinger complex (PBC), a hypothesized respiratory rhythm generator, increased phrenic nerve activity after an acetazolamide-induced acidosis in the anesthetized cat. Thus, we tested the hypothesis that focal microdialysis (MD) of CO2/H+ in the PBC during wakfulness would stimulate breathing. Stainless steel microtubules were bilaterally implanted into the PBC of adult goats. Unilateral MD of mCSF equilibrated with 6.4% CO2 did not affect VI, VT or f. Unilateral MD of 25 or 50% CO2 did not affect VI or VT, but did significantly increase f by a maximum of 10% (P<0.05, n=12). Bilateral MD of 6.4 and 25% CO2 did not significantly affect VI, VT, or f. However, bilateral MD of 50% significantly increased f by a maximum of 8% (n<0.05, n=5), although VI and VT was unaffected. The findings that FA in the PBC of the awake goat affects only respiratory frequency lends support to the PBC’s role in respiratory rhythm generation. Thus, our data suggests that the PBC may not only be instrinsically rhythmogenic, but chemosensitive as well. (Supported by NIH 25739 and the Veterans Administration)

The purpose herein is to report several examples in awake mammals of a dissociation between CO2-H+ ventilatory chemosensitivity and room air VI. We found in piglets that eupneic PaCO2 increased gradually from 26.5 ± 0.5 to 34.4 ± 1.2 between postnatal days (PN) 3–4 and PN 19–21 but CO2 sensitivity (expressed as VI 5% CO2/ VI room air) was unchanged between PN 3–4 and PN 19–21. In Sprague Dawley rats, we found that CO2 sensitivity was minimal between PN 0 and PN 14, but then it increased dramatically between PN 14 and PN 21, whereas room air VI /body weight decreased gradually from PN 0 to PN21. Finally, we found that in adult goats, neurotoxic lesioning of the rostral ventrolateral medulla, medullary raphe, and cerebellar fastigial nucleus (CFN) decreased CO2 sensitivity, but only increased eupneic PaCO2 by 2.0 ± 1.1, 0.2 ± 0.8, and 2.9 ± 0.3% respectively. Moreover, the CFN lesions doubled the day-to-day variability in CO2 sensitivity but did not alter variability of eupneic PaCO2. We conclude that under the conditions listed above, CO2-H+ ventilatory chemosensitivity is not a critical determinant of eupneic VI. (Supported by NIH HL-25739 and Department of Veterans Affairs.)

Controversies regarding the role of pontine nuclei in the control of breathing and paucity of studies during physiologic conditions have prompted us to chronically implant microtubules (two unilateral and two bilateral) into the rostral pons of 4 goats. All goats recovered uneventfully and 3 weeks thereafter resting PaCO2 and CO2 sensitivity were near normal. Reverse microdialysis (MD) of atropine during the day (25, 50 or 100mM) or at night (25 or 50mM) affected absolute pulmonary ventilation (VI) in 3 of the 4 goats, as compared to MD with mock cerebral spinal fluid. In 2 goats, during the day, MD on one side and bilateral MD decreased VI, breathing frequency (f) and tidal volume (VT) but MD on the other side increased VI. In these goats, MD at night either had no effect or stabilized VI and f. In the other two goats, unilateral MD had no effect or increased VI during the day. In one of these goats, injection of atropine (5mM, 500nl or 1μl) increased VI, f and VT compared to mock injection. In all 4 goats, daytime MD did not stabilize VI, f or VT. Neither injection (5mM, 500nl or 1μl) nor MD (50mM) of atropine had any effect on CO2 sensitivity. These findings suggest that atropine delivered to the rostral pons acutely modulates ventilation in awake and asleep goats. Supported by the Department of Veterans Affairs and NIH HL25739.

Our objective in this study was to test the hypothesis that focal acidosis (FA) in the cerebellar fastigial nucleus (CFN) of awake goats arising from global brain acidosis induced by increasing inspired CO2 will increase breathing. FA was created by reverse microdialysis of mock cerebral spinal fluid, equilibrated with 6.4, 25, 50, or 80% CO2 through chronically implanted microtubules (cannula). Dialysis with 6.4% CO2 had no significant effects on any physiological parameters. However, microdialysis at higher levels of CO2 increased pulmonary ventilation (V(I)) in one group of studies and decreased V(I) in a second group and the difference between the groups was significant (t = 9.16, P < 0.001). In one group of studies (n = 8), FA with 50 and 80% CO2 significantly increased (P < 0.05) Vi by 16 and 12%, respectively, and significantly increased (P < 0.05) heart rate by 13 and 9%, respectively. In contrast, in another group of studies (n = 6), FA with 25 and 50% CO2 significantly decreased (P < 0.05) Vi by 7 and 10%, respectively. In this group oxygen consumption was decreased during dialysis with 80% CO2. On the basis of histology, we estimate that the increased and decreased responses were associated with FA primarily in the rCFN and cCFN, respectively. We conclude that there are CO2/H+-sensitive neurons in the CFN that do not uniformly affect breathing. In addition, the significant changes in heart rate and oxygen consumption during FA indicate that the CFN can also influence non-respiratory-related control systems.

Controversies regarding the role of pontine nuclei in the control of breathing and paucity of studies during physiologic conditions have prompted us to chronically implant microtubules into the pons of 2 goats, one caudally (bilateral) and one rostrally (unilateral). Both goats recovered uneventfully and 3 weeks thereafter resting PaCO2 and CO2 sensitivity were near normal. However, both goats had abnormal post-inspiratory diaphragm activity following implantation. Injection of atropine (500nL, 5mM) during the day decreased CO2 sensitivity without altering resting PaCO2. Dialysis of atropine (50mM) at night disrupted activation patterns of respiratory muscles and depressed and destabilized breathing which was state independent. Injection of ibotenic acid during the day also acutely destabilized breathing and disrupted coordination of respiratory muscles, transiently altered PaCO2 and CO2 sensitivity, and chronically altered REM generating mechanisms at night. Subsequent histological analysis of the pons of both goats implicates the medial parabrachial nucleus in the caudally implanted goat and the pedunculopontine tegmental nucleus in the rostrally implanted goat. These findings suggest that chronically instrumented goats are a viable model to further elucidate the role of pontine nuclei in the control of breathing. Supported by NIH HL-25739 and by the Department of Veterans Affairs.

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.

The pharyngeal constrictors have been hypothesized to play an important role in the regulation of upper airway (UAW) patency in patients with sleep apnea. However, little research has focused on the activation and control of muscles that determine the lateral and posterior wall of the retropalatal airway dimensions. Our aim was to investigate the effects of slow wave sleep (SWS) and rapid eye movement (REM) sleep on the activation of pharyngeal constrictor (thyropharyngeus; TP) and dilator (stylopharyngeus; SP) muscles during eupneic breathing and induced central apneas. In nine goats, we found that eupneic TP and SP activity progressively decreased from awake to SWS (57 and 56%, respectively; P<0.01) and further in REM (25.6 and 19.9%, respectively; P<0.01). In contrast, diaphragm activity decreased equally during SWS and REM (89.3 and 87.7%, respectively; P<0.01) compared to awake. Following induced apneas while SP activity was eliminated in every state, maximal TP activity was highest in awake state (318.6% of control; P<0.02), less in SWS (157.6%; P<0.02), and nearly absent in REM (117.3%; P>0.02). During the recovery from an induced apnea when diaphragm activity was at 95% of its' control, awake TP activity remained significantly elevated and SP reduced (P>0.02) while TP activity during SWS was elevated and SP had returned to control level. During REM, TP and SP activity were not different from their reduced controls (P>0.02). The data supports our hypotheses that SWS and REM sleep causes a reduction in the eupneic TP and SP activity, as well as a reduction in TP response to induced apneas. However, the relative imbalance in TP vs SP activity during the recovery from an apnea (awake and SWS) suggest that an imbalance of active neuromuscular forces may contribute to upper airway narrowing in mixed apneas, but not in central apnea during sleep.