Ventilatory sensitivity to hypercapnia is greater in Dahl salt‐sensitive (SS) rats than in Brown Norway (BN) inbred rats (J Appl Physiol). Since pH‐sensitive potassium ion (K+) channels are located within various chemosensitive nuclei including the nucleus of the solitary tract (NTS), and are postulated to contribute to CO2− H+ detection/signaling in chemosensitive neurons, we tested the hypothesis that there are more pH‐sensitive Kv 1.4 channel‐immunoreactive (ir) neurons within the NTS of SS than within the NTS of BN rats. The medullary sections (25 μm) containing the NTS from male and female BN and SS rats were stained with cresyl violet or with antibodies targeting Kv 1.4. Kv 1.4 channel‐ir neurons were quantified and compared with the total neurons in the region. The average number of Kv 1.4 channel‐ir neurons per hemisection was significantly (P < 0.05) greater in both male and female SS (n = 6 males and 6 females) than in the BN (n = 6 males and 6 females) rats. In addition, the percentage of Nissl stained neurons that express Kv 1.4 was statistically greater in SS male versus BN male rats. That trend however, was not observed in female SS and BN rats (P > 0.05). Our findings further support the hypothesis that a greater number of K+ channel‐ir neurons in the NTS of SS may contribute to their relatively greater sensitivity to CO2. (Supported by the Department of Veterans Affairs and NIH HL25739)

Abrupt, bilateral destruction of the pre-Bötzinger Complex (preBötC) leads to terminal apnea in unanesthetized goats and rats. In contrast, respiratory rhythm and pattern and arterial blood gases in goats during wakefulness and sleep are normal after incremental (over a month) destruction of >90% of the preBötC. Here, we tested the hypothesis that the difference in effects between abrupt and incremental destruction of the preBötC are a result of time-dependent plasticity, which manifests as anatomic changes at sites within the respiratory network. Accordingly, we report data from histological analyses comparing the brainstems of control goats, and goats that had undergone bilateral, incremental, ibotenic acid (IA)-induced preBötC lesioning. A major focus was on the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the pontine respiratory group (PRG), which are sites thought to contribute to respiratory rhythmogenesis. We also studied the facial (FN), rostral nucleus ambiguus (NA), medullary raphé (MRN), hypoglossal (HN), and the dorsal motor vagal (DMV) nuclei. Neuronal counts, count region area (mm2), and neuronal densities were calculated using computer-assisted analyses and/or manual microscopy to compare control and preBötC-lesioned animals. We found that within the ventral and lateral medulla 2 mm rostral to the caudal pole of the FN (presumed pFRG/RTN), there were 25% and 65% more (P < 0.001) neurons, respectively, in preBötC-lesioned compared to control goats. Lesioned goats also showed 14% and 13% more (P < 0.001) neurons in the HN and medial parabrachialis nucleus, but 46%, 28%, 7%, and 17% fewer (P < 0.001) neurons in the FN, NA, DMV, and Kölliker-Fuse nuclei, respectively. In the remaining sites analyzed, there were no differences between groups. We conclude that anatomic changes at multiple sites within the respiratory network may contribute to the time-dependent plasticity in breathing following incremental and near-complete destruction of the preBötC.

The purpose of this retrospective study was to gain insight into the contribution of the dorsolateral pons to the coordination of swallowing and breathing in awake goats. In 4 goats, cannulas were chronically implanted bilaterally through the lateral (LPBN) and medial (MPBN) parabrachial nuclei just dorsal to the Kölliker–Fuse nucleus (KFN). After >2weeks recovery from this surgery, the goats were studied for 5½h on a control day, and on separate days after receiving 1 and 10μl injections of ibotenic acid (IA) separated by 1week. The frequency of swallows did not change during the control and 1μl IA studies, but after injection of 10μl IA, there was a transient 65% increase in frequency of swallows (P<0.05). Under control conditions swallows occurred throughout the respiratory cycle, where late-E swallows accounted for 67.6% of swallows. The distribution of swallow occurrence throughout the respiratory cycle was unaffected by IA injections. Consistent with the concept that swallowing is dominant over breathing, we found that swallows increased inspiratory (TI) and expiratory (TE) time and decreased tidal volume (VT) of the breath of the swallow (n) and/or the subsequent (n+1) breath. Injections of 10μl IA attenuated the normal increases in TI and TE and further attenuated VT of the n breath. Additionally, E and I swallows reset respiratory rhythm, but injection of 1 or 10μl IA progressively attenuated this resetting, suggesting a decreased dominance over respiratory motor output with increasing IA injections. Post mortem histological analysis revealed about 50% fewer (P<0.05) neurons remained in the KFN, LPBN, and MPBN in lesioned compared to control goats. We conclude that dorsolateral pontine nuclei have a modulatory role in a hypothesized holarchical neural network regulating swallowing and breathing particularly contributing to the normal dominance of swallowing over breathing in both rhythm and motor pattern generation.

We MD atropine, a muscarinic receptor antagonist, at sites in the rostral pons of awake and sleeping goats to test the hypothesis that cholinergic mechanisms have site‐ and state‐dependent effects on the control of breathing. Cannula were bilaterally implanted into either rostral pontine tegmental nuclei (n = 3), the lateral (n = 3) or medial (n = 4) parabrachial nuclei, or the KFN (n = 6). After >2 weeks recovery from surgery, the goats were studied during a 45 minute period of MD with mock cerebrospinal fluid, followed by 30 minutes of recovery and 45 minutes of MD with atropine. Unilateral and bilateral MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12 to 14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no affect on these variables but increased the variation of genioglossus muscle activity. Nighttime unilateral and bilateral MD of atropine into the KFN increased NREM sleep by 63% (p > 0.05) and 365% (p < 0.01), respectively. MD during the day or at night into the other 3 pontine sites had only minimal effects on any variable studied. Finally, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysate. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.

The objective of the present study was to test the hypothesis that, in the in vivo awake goat model, perturbation/lesion in the pontine respiratory group (PRG) would decrease the sensitivity to hypercapnia and hypoxia. The study reported herein was part of two larger studies in which cholinergic modulation in the PRG was attenuated by microdialysis of atropine and subsequently ibotenic acid injections neurotoxically lesioned the PRG. In 14 goats, cannula were bilaterally implanted into either the lateral ( n = 4) or medial ( n = 4) parabrachial nuclei or the Kölliker-Fuse nucleus (KFN, n = 6). Before and after cannula implantation, microdialysis of atropine, and injection of ibotenic acid, hypercapnic and hypoxic ventilatory sensitivities were assessed. Hypercapnic sensitivity was assessed by three 5-min periods at 3, 5, and 7% inspired CO2. In all groups of goats, CO2 sensitivity was unaffected ( P > 0.05) by any PRG perturbations/lesions. Hypoxic sensitivity was assessed with a 30-min period at 10.8% inspired O2. The response to hypoxia was typically triphasic, with a phase 1 increase in pulmonary ventilation, a phase 2 roll-off, and a phase 3 prolonged increase associated with shivering and increased metabolic rate and body temperature. In all groups of goats, the phase 1 of the hypoxic ventilatory responses was unaffected by any PRG perturbations/lesions, and there were no consistent effects on the phase 2 responses. However, in the KFN group of goats, the phase 3 ventilatory, shivering, metabolic rate, and temperature responses were markedly attenuated after the atropine dialysis studies, and the attenuation persisted after the ibotenic acid studies. These findings support an integrative or modulatory role for the KFN in the phase 3 responses to hypoxia.

To probe further the contributions of the rostral pons to eupneic respiratory rhythm and pattern, we tested the hypothesis that ibotenic acid (IA) injections in the pontine respiratory group (PRG) would disrupt eupneic respiratory rhythm and pattern in a site- and state-specific manner. In 15 goats, cannulas were bilaterally implanted into the rostral pontine tegmental nuclei (RPTN; n = 3), the lateral (LPBN; n = 4) or medial parabrachial nuclei (MPBN; n = 4), or the Kölliker-Fuse nucleus (KFN; n = 4). After recovery from surgery, 1- and 10-microl injections (1 wk apart) of IA were made bilaterally through the implanted cannulas during the day. Over the first 5 h after the injections, there were site-specific ventilatory effects, with increased (P < 0.05) breathing frequency in RPTN-injected goats, increased (P < 0.05) pulmonary ventilation (Vi) in LPBN-injected goats, no effect (P < 0.05) in MPBN-injected goats, and a biphasic Vi response (P < 0.05) in KFN-injected goats. This biphasic response consisted of a hyperpnea for 30 min, followed by a prolonged hypopnea and hypoventilation with marked apneas, apneusis-like breathing patterns, and/or shifts in the temporal relationships between inspiratory flow and diaphragm activity. In the awake state, 10-15 h after the 1-microl injections, the number of apneas was greater (P < 0.05) than during other studies at night. However, there were no incidences of terminal apneas. Breathing rhythm and pattern were normal 22 h after the injections. Subsequent histological analysis revealed that for goats with cannulas implanted into the KFN, there were nearly 50% fewer neurons (P < 0.05) in all three PRG subnuclei than in control goats. We conclude that in awake goats, 1) IA injections into the PRG have site-specific effects on breathing, and 2) the KFN contributes to eupneic respiratory pattern generation.

Ventilatory sensitivity to hypercapnia is greater in Dahl salt-sensitive (SS) rats than in Fawn Hooded hypertensive (FHH) and Brown Norway (BN) inbred rats. Since pH-sensitive potassium ion (K(+)) channels are postulated to contribute to the sensing and signaling of changes in CO(2)-H(+) in chemosensitive neurons, we tested the hypothesis that there are more pH-sensitive K(+) channel-immunoreactive (ir) neurons within the medullary raphé nuclei of the highly chemosensitive SS rats than in the other two strains. Medullary tissues from male and female BN, FHH, and SS rats were stained with cresyl violet or with antibodies targeting TASK-1, K(v)1.4, and Kir2.3 channels. K(+) channel-ir neurons were quantified and compared with the total neurons in the region. The total number of neurons in the medullary raphé 1) was greater in male FHH than the other male rats, 2) did not differ among the female rats, and 3) did not differ between sexes. The average number of K(+) channel-ir neurons per section was 30-60 neurons higher in the male SS than in the other rat strains. In contrast, for the females, the number of K(+) channel-ir neurons was greatest in the BN. We also found significant differences in the number of K(+) channel-ir neurons between sexes in SS (males > females) and BN (females > males) rats, but not the FHH strain. Our findings support the hypothesis for males but not for females, suggesting that both genetic background and sex are determinants of K(+) channel immunoreactivity of medullary raphé neurons, and that the expression of pH-sensitive K(+) channels in the medullary raphé does not correlate with the ventilatory sensitivity to hypercapnia.

Thirty seconds of cooling the RVLM induces apnea and reduced breathing in anesthetized and awake goats, respectively (J Appl Physiol 78: 247–257, 1995). We recently studied the effects on breathing in two goats of unilateral ibotenic acid (IA: 1 μl) induced RVLM lesions 1 mm below the medullary surface at a critical site identified by cooling under anesthesia. Upon recovery from anesthesia, both goats hyperventilated at rest for 5 days, but thereafter hypoventilated (PaCO2 8–10 mmHg above control) for several days due to decreased tidal volume and breathing frequency. In one goat CO2 sensitivity was attenuated nearly 50% during the period of hypoventilation, but the ventilatory responses to hypoxia and exercise, and breathing during NREM sleep were unaffected. In this goat resting PaCO2 and CO2 sensitivity returned to control about 15 days post‐lesion. Both goats were in excellent health throughout the post surgery period. Post‐mortem histology indicated the lesioned sites were in an area of high density neurokinin‐1 receptor expressing neurons, near or including the retrotrapezoid nucleus. These data suggest that neurons near the RVLM surface are necessary to maintain normal levels of eupneic breathing, and contribute to respiratory CO2 chemoreception.

We tested the hypothesis that IA injections into the PRG would disrupt eupneic respiratory rhythm and pattern. Cannula were bilaterally implanted in adult goats into either the lateral (LPBN, n = 4) or medial (MPBN, n = 4) parabrachial nuclei, or the Kölliker‐Fuse nucleus (KFN, n = 4). After recovery from surgery, 1 or 10μl injections of IA were made bilaterally through the implanted cannula during the awake state. Over the first 5 hours after the 10 μl injections, pulmonary ventilation was increased (P<0.05) in LPBN goats, there were no effects (P > 0.05) in MPBN goats, and there was a biphasic response (P < 0.05) in KFN goats. This biphasic response consisted of a tachypneic hyperpnea for 30 minutes followed by a prolonged hypopnea and hypoventilation with marked apneas and apneusis‐like breathing patterns. While awake and during NREM sleep 10–15 hours after the 1 μl and 10 μl injections respectively, there were above control number of apneas and augmented breaths in all groups. However, there were no incidences of terminal apneas. Breathing rhythm and pattern were normal 22 hours after the injections. Subsequent histological analysis showed <10%, 27%, and 62% reduction in number of neurons in the LPBN, MPBN and KFN goats, respectively. The data do not provide evidence that any of the lesioned sites are obligatory for respiratory rhythm generation, but suggest that the KFN contributes to eupneic pattern generation.

For many years, acetylcholine has been known to contribute to the control of breathing and sleep. To probe further the contributions of cholinergic rostral pontine systems in control of breathing, we designed this study to test the hypothesis that microdialysis (MD) of the muscarinic receptor antagonist atropine into the pontine respiratory group (PRG) would decrease breathing more in animals while awake than while in NREM sleep. In 16 goats, cannulas were bilaterally implanted into rostral pontine tegmental nuclei (n = 3), the lateral (n = 3) or medial (n = 4) parabrachial nuclei, or the Kölliker-Fuse nucleus (KFN; n = 6). After >2 wk of recovery from surgery, the goats were studied during a 45-min period of MD with mock cerebrospinal fluid (mCSF), followed by at least 30 min of recovery and a second 45-min period of MD with atropine. Unilateral and bilateral MD studies were completed during the day and at night. MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12-14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no effect on these variables. Unilateral and bilateral nighttime MD of atropine into the KFN increased levels of NREM sleep by 63 and 365%, respectively. MD during the day or at night into the other three pontine sites had minimal effects on any variable studied. Finally, compared with MD of mCSF, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysis fluid. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.

Bonis JM, Neumueller SE, Krause KL, Kiner T, Smith A, Marshall BD, Qian B, Pan LG, Forster HV. A role for the KöllikerFuse nucleus in cholinergic modulation of breathing at night during wakefulness and NREM sleep. J Appl Physiol 109: 159–170, 2010. First published April 29, 2010; doi:10.1152/japplphysiol.00933.2009.—For many years, acetylcholine has been known to contribute to the control of breathing and sleep. To probe further the contributions of cholinergic rostral pontine systems in control of breathing, we designed this study to test the hypothesis that microdialysis (MD) of the muscarinic receptor antagonist atropine into the pontine respiratory group (PRG) would decrease breathing more in animals while awake than while in NREM sleep. In 16 goats, cannulas were bilaterally implanted into rostral pontine tegmental nuclei (n 3), the lateral (n 3) or medial (n 4) parabrachial nuclei, or the Kölliker-Fuse nucleus (KFN; n 6). After 2 wk of recovery from surgery, the goats were studied during a 45-min period of MD with mock cerebrospinal fluid (mCSF), followed by at least 30 min of recovery and a second 45-min period of MD with atropine. Unilateral and bilateral MD studies were completed during the day and at night. MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12–14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no effect on these variables. Unilateral and bilateral nighttime MD of atropine into the KFN increased levels of NREM sleep by 63 and 365%, respectively. MD during the day or at night into the other three pontine sites had minimal effects on any variable studied. Finally, compared with MD of mCSF, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysis fluid. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.

Opioids are clinically important in the alleviation of pain. An undesirable side effect of opioids is depression of breathing. Data from isolated preparations suggest this effect is due to attenuation of discharge activity of neurons in the pre-Bötzinger complex (preBötzC), a medullary area with respiratory rhythmogenic properties. The purpose of this study was to examine how [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, affected breathing after injection into the presumed preBötzC of the adult awake goat. We hypothesized that DAMGO would cause breathing to decrease and become irregular when injected into the presumed preBötzC and the surrounding region of the conscious animal. We further hypothesized that ventilatory sensitivity to CO(2) and hypoxia would be blunted after the injection of DAMGO. Microtubules were bilaterally implanted into the presumed preBötzC of 10 adult female goats. After recovery from the surgery, DAMGO (0.5-10 mul, 1 nM-10 muM) was injected into the presumed preBötzC during the awake state. DAMGO had no effect on pulmonary ventilation [inspiratory minute ventilation (Vi)], respiratory rhythm and pattern, the activation pattern of inspiratory and expiratory muscles, or arterial blood gases during eupneic breathing conditions (P > 0.10). However, DAMGO attenuated (P < 0.05) the evoked increase in breathing frequency when inspired CO(2) was increased, and DAMGO attenuated the Vi response to reduction of inspired O(2) to 10.8% (P < 0.05). We conclude that our data do not provide support for the concept that in awake mammals opioid depression of breathing is due to a directed action of opioids on preBötzC neurons.

Ascalaphidae occur in Canada. Here, we document the presence of Ululodes quadrimaculata (Say) in southern Ontario, which was suspected by Kevan (1979).SPECIMEN MATERIAL. Ontario: ESSEX COUNTY: Harrow, ♀, VIII.1974 (W. Elliott; UG). LAMBTON COUNTY: Pinery Prov. Pk., ♂, 4.VII. 1977 (B. Marshall & W. Maddison; #770112b, ROM). MUSKOKA DISTRICT: Georgian Bay, Island #421, ♀, 5.VII. 1963 (J. P. Bogart; UG); Gravenhurst, ♀, 20.VIII. 1975 (D. J. Aspinall; UG); Muskoka, ♀, VIII.1957 (D. Barr; ROM).