Sleep state dependence of ventilatory long-term facilitation following acute intermittent hypoxia in Lewis rats.

Ventilatory long-term facilitation (vLTF) is a form of respiratory plasticity induced by acute intermittent hypoxia (AIH). Although vLTF has been reported in unanesthetized animals, little is known concerning the effects of vigilance state on vLTF expression. We hypothesized that AIH-induced vLTF is preferentially expressed in sleeping vs. awake male Lewis rats. Vigilance state was assessed in unanesthetized rats with chronically implanted EEG and nuchal EMG electrodes, while tidal volume, frequency, minute ventilation (Ve), and CO(2) production were measured via plethysmography, before, during, and after AIH (five 5-min episodes of 10.5% O(2) separated by 5-min normoxic intervals), acute sustained hypoxia (25 min of 10.5% O(2)), or a sham protocol without hypoxia. Vigilance state was classified as quiet wakefulness (QW), light and deep non-rapid eye movement (NREM) sleep (l-NREM and d-NREM sleep, respectively), or rapid eye movement sleep. Ventilatory variables were normalized to pretreatment baseline values in the same vigilance state. During d-NREM sleep, vLTF was observed as a progressive increase in Ve post-AIH (27 + or - 5% average, 30-60 min post-AIH). In association, Ve/Vco(2) (36 + or - 2%), tidal volume (14 + or - 2%), and frequency (7 + or - 2%) were increased 30-60 min post-AIH during d-NREM sleep. vLTF was significant but less robust during l-NREM sleep, was minimal during QW, and was not observed following acute sustained hypoxia or sham protocols in any vigilance state. Thus, vLTF is state-dependent and pattern-sensitive in unanesthetized Lewis rats, with the greatest effects during d-NREM sleep. Although the physiological significance of vLTF is not clear, its greatest significance to ventilatory control is most likely during sleep.

Hypoxic and hypercapnic ventilatory responses in aging male vs. aging female rats.

It is clear that sex hormones impact ventilation. While the effects of the menstrual cycle, pregnancy, testosterone, and progesterone on resting ventilation have been well documented, effects of sex hormones on the hypoxic (HVR) and hypercapnic ventilatory responses (HCVR) are inconclusive. In addition, in no study have systemic sex steroid hormone levels been measured. Age and sex differences in long-term facilitation in response to episodic hypoxia were found in anesthetized rats. The purpose of the present study was to assess the effects of sex and age [young, 3-4 mo; middle age, 12-13 mo; and old, >20 mo] on the HVR and the HCVR of awake rats relative to systemic hormone levels. Based on findings from long-term facilitation studies, we hypothesized that the HVR would be influenced by both sex and age. We found no age-related changes in the HVR or HCVR. However, female rats have a greater HVR than male rats at old age, and at middle age female rats have a greater HCVR than male rats. Additionally, we found no correlation between the minute ventilation/oxygen consumption and the progesterone-to-estrogen ratio during hypoxia or hypercapnia. However, changes in ventilatory responses with age were not similar between the sexes. Thus it is critical to take sex, age, estrous cycle stage, and systemic hormone levels into consideration when conducting and reporting studies on respiratory control.

Small reduction of neurokinin-1 receptor-expressing neurons in the pre-Bötzinger complex area induces abnormal breathing periods in awake goats.

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.

Large lesions in the pre-Bötzinger complex area eliminate eupneic respiratory rhythm in awake goats.

In awake goats, 29% bilateral destruction of neurokinin-1 receptor-expressing 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 microl, 50 mM) injection into the pre-BötzC area resulted in a tachypneic hyperpnea that reached a maximum (132 +/- 10.1 breaths/min) approximately 30-90 min after bilateral injection. Thereafter, breathing frequency declined, central apneas resulted in arterial hypoxemia (arterial Po2 approximately 40 Torr) and hypercapnia (arterial Pco2 approximately 60 Torr), and, 11 +/- 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 (approximately 90 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.

Effects on breathing in awake and sleeping goats of focal acidosis in the medullary raphe.

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.

Multiple rostral medullary nuclei can influence breathing in awake goats.

The purpose of this study was to determine the effect on breathing of neuronal dysfunction in the retrotrapezoid (RTN), facial (FN), gigantocellularis reticularis (RGN), or vestibular (VN) nuclei of adult awake goats. Microtubules were chronically implanted to induce neuronal dysfunction by microinjection of an excitatory amino acid (EAA) receptor antagonist or a neurotoxin. The EAA receptor antagonist had minimal effect on eupneic breathing, but 8--10 days after injection of the neurotoxin, 7 of 10 goats hypoventilated (arterial PCO(2) increased 3.2 +/- 0.7 Torr). Overall there were no significant (P > 0.10) effects of the EAA receptor antagonist on CO(2) sensitivity. However, for all nuclei, > or =66% of the antagonist injections altered CO(2) sensitivity by more than the normal 12.7 +/- 1.6% day-to-day variation. These changes were not uniform, inasmuch as the antagonist increased (RTN, n = 2; FN, n = 7; RGN, n = 6; VN, n = 1) or decreased (RTN, n = 2; RGN, n = 3; VN, n = 2) CO(2) sensitivity. Ten days after injection of the neurotoxin into the FN (n = 3) or RGN (n = 5), CO(2) sensitivity was also reduced. Neuronal dysfunction also did not have a uniform effect on the exercise arterial PCO(2) response, and there was no correlation between effects on CO(2) sensitivity and the exercise hyperpnea. We conclude that there is a heterogeneous population of neurons in these rostral medullary nuclei (or adjacent tissue) that can affect breathing in the awake state, possibly through chemoreception or chemoreceptor-related mechanisms.