Effects of sub-lethal doses of fentanyl on vital physiologic functions and withdrawal-like behaviors in adult goats.

Synthetic opioids like fentanyl have improved the standard of care for many patients in the clinical setting, but their abuse leads to tens of thousands of overdose deaths annually. The current opioid epidemic underscores a critical need for insights into the physiological effects of fentanyl on vital functions. High doses of opioids in small mammals cause opioid-induced respiratory depression (OIRD) leading to hypoventilation, hypoxemia, and hypercapnia. In addition, opioids can also increase the alveolar to arterial oxygen (A-a) gradient and airway dysfunction. However, little is known about the physiologic effects of sub-lethal doses of opioids in large mammals. Here we report the effects of a sub-lethal dose range of fentanyl (25-125 µg/kg; IV) on vital physiologic functions over 90 min (min) and withdrawal-like behaviors over the subsequent 4 h (h) in adult female goats (n = 13). Fentanyl induced decreases in breathing frequency in the first few min post-injection, but then led to a sustained increase in tidal volume, total ventilation, and blood pressure with a reduced heart rate for ≥90 min. These ventilatory changes resulted in time-dependent arterial hypocapnia and hypoxemia and an increased alveolar to arterial oxygen gradient ∼30 min post-injection indicative of impaired gas exchange in the lung. The predominant effects of fentanyl on breathing were stimulatory, underscored by an increased rate of rise of the diaphragm muscle activity and increased activation of upper airway, intercostal and abdominal muscles. Beginning 90 min post-injection we also quantified withdrawal-like behaviors over 4 h, demonstrating dose- and time-dependent increases in locomotor, biting, itching, and pawing behaviors. We conclude that fentanyl at sublethal doses induces multiple physiologic and behavior changes that emerge along different time courses suggesting multiple independent mechanisms underlying effects of opioids.

Mild and moderate chronic hypercapnia elicit distinct transcriptomic responses of immune function in cardiorespiratory nuclei.

Chronic hypercapnia (CH) is a hallmark of respiratory-related diseases, and the level of hypercapnia can acutely or progressively become more severe. Previously, we have shown time-dependent adaptations in steady-state physiology during mild (arterial Pco2 ∼55 mmHg) and moderate (∼60 mmHg) CH in adult goats, including transient (mild CH) or sustained (moderate CH) suppression of acute chemosensitivity suggesting limitations in adaptive respiratory control mechanisms as the level of CH increases. Changes in specific markers of glutamate receptor plasticity, interleukin-1ß, and serotonergic modulation within key nodes of cardiorespiratory control do not fully account for the physiological adaptations to CH. Here, we used an unbiased approach (bulk tissue RNA sequencing) to test the hypothesis that mild or moderate CH elicits distinct gene expression profiles in important brain stem regions of cardiorespiratory control, which may explain the contrasting responses to CH. Gene expression profiles from the brain regions validated the accuracy of tissue biopsy methodology. Differential gene expression analyses revealed greater effects of CH on brain stem sites compared with the medial prefrontal cortex. Mild CH elicited an upregulation of predominantly immune-related genes and predicted activation of immune-related pathways and functions. In contrast, moderate CH broadly led to downregulation of genes and predicted inactivation of cellular pathways related to the immune response and vascular function. These data suggest that mild CH leads to a steady-state activation of neuroinflammatory pathways within the brain stem, whereas moderate CH drives the opposite response. Transcriptional shifts in immune-related functions may underlie the cardiorespiratory network's capability to respond to acute, more severe hypercapnia when in a state of progressively increased CH.NEW & NOTEWORTHY Mild chronic hypercapnia (CH) broadly upregulated immune-related genes and a predicted activation of biological pathways related to immune cell activity and the overall immune response. In contrast, moderate CH primarily downregulated genes related to major histocompatibility complex signaling and vasculature function that led to a predicted inactivation of pathways involving the immune response and vascular endothelial function. The severity-dependent effect on immune responses suggests that neuroinflammation has an important role in CH and may be important in the maintenance of proper ventilatory responses to acute and chronic hypercapnia.

Chronic moderate hypercapnia suppresses ventilatory responses to acute CO₂ challenges.

Chronic hypercapnia (CH) is a hallmark of chronic lung disease, and CH increases the risk for acute-on-chronic exacerbations leading to greater hypoxemia/hypercapnia and poor health outcomes. However, the role of hypercapnia per se (duration and severity) in determining an individual's ability to tolerate further hypercapnic exacerbations is unknown. Our primary objective herein was to test the hypothesis that mild-to-moderate CH (arterial [Formula: see text] ∼50-70 mmHg) increases susceptibility to pathophysiological responses to severe acute CO₂ challenges. Three groups (GR) of adult female goats were studied during 14 days of exposure to room air (<i>GR 1</i>; control) or 6% inspired CO₂ (<i>GR 2</i>; mild CH), or 7 days of 6% inspired CO₂ followed by 7 days of 8% inspired CO₂ (<i>GR 3</i>; moderate CH). Consistent with previous reports, there were no changes in physiological parameters in <i>GR 1</i> (RA control), but mild CH (<i>GR 2</i>) increased steady-state ventilation and transiently suppressed CO₂/[H⁺] chemosensitivity. Further increasing InCO₂ from 6% to 8% (<i>GR 3</i>) transiently increased ventilation and arterial [H⁺]. Similar to mild CH, moderate CH increased ventilation to levels greater than predicted. However, in contrast to mild CH, acute ventilatory chemosensitivity was suppressed throughout the duration of moderate CH, and the arterial - mixed expired CO₂ gradient became negative. These data suggest that moderate CH limits physiological responses to acute severe exacerbations and provide evidence of recruitment of extrapulmonary systems (i.e., gastric CO₂ elimination) during times of moderate-severe hypercapnia.<b>NEW &amp; NOTEWORTHY</b> Moderate levels of chronic hypercapnia (CH; ∼70 mmHg) in healthy adult female goats elicited similar steady-state physiological adaptations compared with mild CH (∼55 mmHg). However, unlike mild CH, moderate CH chronically suppressed acute CO₂/[H⁺] chemosensitivity and reversed the arterial to mixed expired CO₂ gradient. These findings suggest that moderate CH suppresses vital mechanisms of ventilatory control and recruits additional physiological systems (i.e., gastric CO₂ release) to help buffer excess CO₂.

Physiological and neurochemical adaptations following abrupt termination of chronic hypercapnia in goats.

Chronic hypercapnia (CH) is a hallmark of respiratory diseases such as chronic obstructive pulmonary disease. In such patients, mechanical ventilation is often used to restore normal blood-gas homeostasis. However, little is known regarding physiological changes and neuroplasticity within physiological control networks after termination of CH. Utilizing our goat model of increased inspired CO2-induced CH, we determined whether termination of CH elicits time-dependent physiological and neurochemical changes within brain stem sites of physiological control. Thirty days of CH increased [Formula: see text] (+15 mmHg) and steady-state ventilation (SS V̇i; 283% of control). Within 24 h after terminating CH, SS V̇i, blood gases, arterial [H+], and most physiological measurements returned to control. However, the acute ventilatory chemoreflex (ΔV̇i/Δ[H+]) was greater than control, and measured SS V̇i exceeded ventilation predicted by arterial [H+] and ΔV̇i/Δ[H+]. Potentially contributing to these differences were increased excitatory neuromodulators serotonin and norepinephrine in the nucleus tractus solitarius, which contrasts with minimal changes observed at 24 h and 30 days of hypercapnia. Similarly, there were minimal changes found in markers of neuroinflammation and glutamate receptor-dependent neuroplasticity upon termination of CH, which were previously increased following 24 h of hypercapnia. Thus, following termination of CH: 1) ventilatory, renal, and other physiological functions rapidly return to control; 2) neuroplasticity within the ventilatory control network may contribute to the difference between measured vs. predicted ventilation and the elevation in the acute ventilatory [H+] chemoreflex; and 3) neuroplasticity is fundamentally distinct from acclimatization to CH.NEW & NOTEWORTHY In healthy adult goats, steady-state ventilation and most physiological measures return to control within 24 h after termination of chronic hypercapnia (CH). However, the acute [H+] chemoreflex is increased, and measured ventilation exceeds predicted ventilation. At 24 h of recovery, excitatory neuromodulators are above control, but other measured markers of neuroplasticity are unchanged from control. Our data suggest that CH elicits persistent physiological and neurochemical changes for up to 24 h after termination of CH.

Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Despite the prevalence of CO2 retention in human disease, little is known about the adaptive neurobiological effects of chronic hypercapnia. We have recently shown 30-d exposure to increased inspired CO2 (InCO2) leads to a steady-state ventilation that exceeds the level predicted by the sustained acidosis and the acute CO2/H+ chemoreflex, suggesting plasticity within respiratory control centers. Based on data showing brainstem changes in aminergic and inflammatory signaling during carotid body denervation-induced hypercapnia, we hypothesized chronic hypercapnia per se will lead to similar changes. We found that: 1) increased InCO2 increased IL-1ß in the medullary raphe (MR), ventral respiratory column, and cuneate nucleus after 24 h, but not after 30 d of hypercapnia; 2) the number of serotonergic and total neurons were reduced within the MR and ventrolateral medulla following 30 d of increased InCO2; 3) markers of tryptophan metabolism were altered following 24 h, but not 30 d of InCO2; and 4) there were few changes in brainstem amine levels following 24 h or 30 d of increased InCO2. We conclude that these changes may contribute to initiating or maintaining respiratory neuroplasticity during chronic hypercapnia but alone do not account for ventilatory acclimatization to chronic increased InCO2.-Burgraff, N. J., Neumueller, S. E., Buchholz, K. J., LeClaire, J., Hodges, M. R., Pan, L., Forster, H. V. Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Midbrain and cerebral inflammatory and glutamatergic adaptations during chronic hypercapnia in goats.

Cognitive impairment is associated with multiple human diseases that have in common chronic hypercapnia. However, the mechanisms leading to chronic hypercapnia-induced cognitive decline are not known. We have previously shown chronic hypercapnia through exposure to increased inspired CO2 (6% InCO2) in conscious goats caused an immediate (within hours) and sustained decline in cognitive performance during a shape discrimination test. Herein, within the same goats, we assessed markers of neuroinflammation and glutamate receptor expression/phosphorylation within CNS regions important for cognitive function following 24 hours (h) or 30 days (d) of chronic hypercapnia. Within 24 h, chronic hypercapnia increased expression of the inflammatory cytokine IL-1ß in the orbitofrontal cortex and medial prefrontal cortex, but at 30d IL-1ß levels were not different relative to time-matched goats exposed to room-air. Additionally, Iba1 expression (a marker of microglial activation) was unaltered by chronic hypercapnia in all regions tested. Finally, levels of the total and phosphorylated AMPA receptor subunit GluR2 were reduced within the hippocampus at both 24 h and 30 d of hypercapnia, and reduced following 30 d within the anterior insular cortex. These data suggest that chronic hypercapnia leads to CNS site-dependent acute inflammatory responses and shifts in select glutamate receptor expression/phosphorylation in brain regions contributing to cognitive function. Such changes may be indicative of alterations in glutamatergic receptor-mediated signaling and neuronal dysfunction that contribute to declines in cognitive function associated with human diseases defined or marked by chronic CO2 retention.

Glutamate receptor plasticity in brainstem respiratory nuclei following chronic hypercapnia in goats.

Patients that retain CO2 in respiratory diseases such as chronic obstructive pulmonary disease (COPD) have worse prognoses and higher mortality rates than those with equal impairment of lung function without hypercapnia. We recently characterized the time-dependent physiologic effects of chronic hypercapnia in goats, which suggested potential neuroplastic shifts in ventilatory control mechanisms. However, little is known about how chronic hypercapnia affects brainstem respiratory nuclei (BRN) that control multiple physiologic functions including breathing. Since many CNS neuroplastic mechanisms include changes in glutamate (AMPA (GluR) and NMDA (GluN)) receptor expression and/or phosphorylation state to modulate synaptic strength and network excitability, herein we tested the hypothesis that changes occur in glutamatergic signaling within BRN during chronically elevated inspired CO2 (InCO2 )-hypercapnia. Healthy goats were euthanized after either 24 h or 30 days of chronic exposure to 6% InCO2 or room air, and brainstems were rapidly extracted for western blot analyses to assess GluR and GluN receptor expression within BRN. Following 24-hr exposure to 6% InCO2 , GluR or GluN receptor expression were changed from control (P < 0.05) in the solitary complex (NTS & DMV),ventrolateral medulla (VLM), medullary raphe (MR), ventral respiratory column (VRC), hypoglossal motor nucleus (HMN), and retrotrapezoid nucleus (RTN). These neuroplastic changes were not found following 30 days of chronic hypercapnia. However, at 30 days of chronic hypercapnia, there was overall increased (P < 0.05) expression of glutamate receptors in the VRC and RTN. We conclude that time- and site-specific glutamate receptor neuroplasticity may contribute to the concomitant physiologic changes that occur during chronic hypercapnia.

Ventilatory and integrated physiological responses to chronic hypercapnia in goats.

KEY POINTS: Chronic hypercapnia per se has distinct effects on the mechanisms regulating steady-state ventilation and the CO2 /H+ chemoreflex. Chronic hypercapnia leads to sustained hyperpnoea that exceeds predicted ventilation based upon the CO2 /H+ chemoreflex. There is an integrative ventilatory, cardiovascular and metabolic physiological response to chronic hypercapnia. Chronic hypercapnia leads to deterioration of cognitive function. ABSTRACT: Respiratory diseases such as chronic obstructive pulmonary disease (COPD) often lead to chronic hypercapnia which may exacerbate progression of the disease, increase risk of mortality and contribute to comorbidities such as cognitive dysfunction. Determining the contribution of hypercapnia per se to adaptations in ventilation and cognitive dysfunction within this patient population is complicated by the presence of multiple comorbidities. Herein, we sought to determine the role of chronic hypercapnia per se on the temporal pattern of ventilation and the ventilatory CO2 /H+ chemoreflex by exposing healthy goats to either room air or an elevated inspired CO2 (InCO2 ) of 6% for 30 days. A second objective was to determine whether chronic hypercapnia per se contributes to cognitive dysfunction. During 30 days of exposure to 6% InCO2 , steady-state (SS) ventilation ( V̇I ) initially increased to 335% of control, and then within 1-5 days decreased and stabilized at ∼230% of control. There was an initial respiratory acidosis that was partially mitigated over time due to increased arterial [HCO3- ]. There was a transient decrease in the ventilatory CO2 /H+ chemoreflex, followed by return to pre-exposure levels. The SS V̇I during chronic hypercapnia was greater than predicted from the acute CO2 /H+ chemoreflex, suggesting separate mechanisms regulating SS V̇I and the chemoreflex. Finally, as assessed by a shape discrimination test, we found a sustained decrease in cognitive function during chronic hypercapnia. We conclude that chronic hypercapnia per se results in: (1) a disconnect between SS V̇I and the CO2 /H+ chemoreflex, and (2) deterioration of cognitive function.

Ventilation and neurochemical changes during µ-opioid receptor activation or blockade of excitatory receptors in the hypoglossal motor nucleus of goats.

Neuromodulator interdependence posits that changes in one or more neuromodulators are compensated by changes in other modulators to maintain stability in the respiratory control network. Herein, we studied compensatory neuromodulation in the hypoglossal motor nucleus (HMN) after chronic implantation of microtubules unilaterally ( n = 5) or bilaterally ( n = 5) into the HMN. After recovery, receptor agonists or antagonists in mock cerebrospinal fluid (mCSF) were dialyzed during the awake and non-rapid eye movement (NREM) sleep states. During day studies, dialysis of the µ-opioid inhibitory receptor agonist [d-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO; 100 µM) decreased pulmonary ventilation (V̇i), breathing frequency ( f), and genioglossus (GG) muscle activity but did not alter neuromodulators measured in the effluent mCSF. However, neither unilateral dialysis of a broad spectrum muscarinic receptor antagonist (atropine; 50 mM) nor unilateral or bilateral dialysis of a mixture of excitatory receptor antagonists altered V̇i or GG activity, but all of these did increase HMN serotonin (5-HT) levels. Finally, during night studies, DAMGO and excitatory receptor antagonist decreased ventilatory variables during NREM sleep but not during wakefulness. These findings contrast with previous dialysis studies in the ventral respiratory column (VRC) where unilateral DAMGO or atropine dialysis had no effects on breathing and bilateral DAMGO or unilateral atropine increased V̇i and f and decreased GABA or increased 5-HT, respectively. Thus we conclude that the mechanisms of compensatory neuromodulation are less robust in the HMN than in the VRC under physiological conditions in adult goats, possibly because of site differences in the underlying mechanisms governing neuromodulator release and consequently neuronal activity, and/or responsiveness of receptors to compensatory neuromodulators. NEW & NOTEWORTHY Activation of inhibitory µ-opioid receptors in the hypoglossal motor nucleus decreased ventilation under physiological conditions and did not affect neurochemicals in effluent dialyzed mock cerebral spinal fluid. These findings contrast with studies in the ventral respiratory column where unilateral [d-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO) had no effects on ventilation and bilateral DAMGO or unilateral atropine increased ventilation and decreased GABA or increased serotonin, respectively. Our data support the hypothesis that mechanisms that govern local compensatory neuromodulation within the brain stem are site specific under physiological conditions.

Effects on breathing of agonists to µ-opioid or GABAA receptors dialyzed into the ventral respiratory column of awake and sleeping goats.

Pulmonary ventilation (V̇I) in awake and sleeping goats does not change when antagonists to several excitatory G protein-coupled receptors are dialyzed unilaterally into the ventral respiratory column (VRC). Concomitant changes in excitatory neuromodulators in the effluent mock cerebral spinal fluid (mCSF) suggest neuromodulatory compensation. Herein, we studied neuromodulatory compensation during dialysis of agonists to inhibitory G protein-coupled or ionotropic receptors into the VRC. Microtubules were implanted into the VRC of goats for dialysis of mCSF mixed with agonists to either µ-opioid (DAMGO) or GABAA (muscimol) receptors. We found: (1) V̇I decreased during unilateral but increased during bilateral dialysis of DAMGO, (2) dialyses of DAMGO destabilized breathing, (3) unilateral dialysis of muscimol increased V̇I, and (4) dialysis of DAMGO decreased GABA in the effluent mCSF. We conclude: (1) neuromodulatory compensation can occur during altered inhibitory neuromodulator receptor activity, and (2) the mechanism of compensation differs between G protein-coupled excitatory and inhibitory receptors and between G protein-coupled and inotropic inhibitory receptors.

State-dependent and -independent effects of dialyzing excitatory neuromodulator receptor antagonists into the ventral respiratory column.

Unilateral dialysis of the broad-spectrum muscarinic receptor antagonist atropine (50 mM) into the ventral respiratory column [(VRC) including the pre-Bötzinger complex region] of awake goats increased pulmonary ventilation (V̇i) and breathing frequency (f), conceivably due to local compensatory increases in serotonin (5-HT) and substance P (SP) measured in effluent mock cerebral spinal fluid (mCSF). In contrast, unilateral dialysis of a triple cocktail of antagonists to muscarinic (atropine; 5 mM), neurokinin-1, and 5-HT receptors does not alter V̇i or f, but increases local SP. Herein, we tested hypotheses that 1) local compensatory 5-HT and SP responses to 50 mM atropine dialyzed into the VRC of goats will not differ between anesthetized and awake states; and 2) bilateral dialysis of the triple cocktail of antagonists into the VRC of awake goats will not alter V̇i or f, but will increase local excitatory neuromodulators. Through microtubules implanted into the VRC of goats, probes were inserted to dialyze mCSF alone (time control), 50 mM atropine, or the triple cocktail of antagonists. We found 1) equivalent increases in local 5-HT and SP with 50 mM atropine dialysis during wakefulness compared with isoflurane anesthesia, but V̇i and f only increased while awake; and 2) dialyses of the triple cocktail of antagonists increased V̇i, f, 5-HT, and SP (<0.05) during both day and night studies. We conclude that the mechanisms governing local neuromodulator levels are state independent, and that bilateral excitatory receptor blockade elicits an increase in breathing, presumably due to a local, (over)compensatory neuromodulator response.NEW & NOTEWORTHY The two major findings are as follows: 1) during unilateral dialysis of 50 mM atropine into the ventral respiratory column to block excitatory muscarinic receptor activity, a compensatory increase in other neuromodulators was state independent, but the ventilatory response appears to be state dependent; and 2) the hypothesis that absence of decreased V̇i and f during unilateral dialysis of excitatory receptor antagonists was due to compensation by the contralateral VRC was not supported by findings herein.

Dynamic Control of Optical Response in Layered Metal Chalcogenide Nanoplates.

Tunable optical transitions in ultrathin layered 2-dimensional (2D) materials unveil the electronic structures of materials and provide exciting prospects for potential applications in optics and photonics. Here, we present our realization of dynamic optical modulation of layered metal chalcogenide nanoplates using ionic liquid (IL) gating over a wide spectral range. The IL gating significantly increased the tuning range of the Fermi level and, as a result, substantially altered the optical transitions in the nanoplates. Using heavily n-doped Bi2Se3 nanoplates, we substantially modulated the light transmission through the ultrathin layer. A tunable, high-transmission spectral window in the visible to near-infrared region has been observed due to simultaneous shifts of both the plasma edge and absorption edge of the material. On the other hand, optical response of multilayer MoSe2 flakes gated by IL has shown enhanced transmission in both positive and negative biases, which is consistent with their ambipolar electrical behavior. The electrically controlled optical property tuning in metal chalcogenide material systems provides new opportunities for potential applications, such as wide spectral range optical modulators, optical filters, and electrically controlled smart windows with extremely low material consumption.

Combined unilateral blockade of cholinergic, peptidergic, and serotonergic receptors in the ventral respiratory column does not affect breathing in awake or sleeping goats.

Previous work in intact awake and sleeping goats has found that unilateral blockade of excitatory inputs in the ventral respiratory column (VRC) elicits changes in the concentrations of multiple neurochemicals, including serotonin (5-HT), substance P, glycine, and GABA, while increasing or having no effect on breathing. These findings are consistent with the concept of interdependence between neuromodulators, whereby attenuation of one modulator elicits compensatory changes in other modulators to maintain breathing. Because there is a large degree of redundancy and multiplicity of excitatory inputs to the VRC, we herein tested the hypothesis that combined unilateral blockade of muscarinic acetylcholine (mACh), neurokinin-1 (NK1, the receptor for substance P), and 5-HT2A receptors would elicit changes in multiple neurochemicals, but would not change breathing. We unilaterally reverse-dialyzed a cocktail of antagonists targeting these receptors into the VRC of intact adult goats. Breathing was continuously monitored while effluent fluid from dialysis was collected for quantification of neurochemicals. We found that neither double blockade of mACh and NK1 receptors, nor triple blockade of mACh, NK1, and 5-HT2A receptors significantly affected breathing (P ≥ 0.05) in goats that were awake or in non-rapid eye movement (NREM) sleep. However, both double and triple blockade increased the effluent concentration of substance P (P < 0.001) and decreased GABA concentrations. These findings support our hypothesis and, together with past data, suggest that both in wakefulness and NREM sleep, multiple neuromodulator systems collaborate to stabilize breathing when a deficit in one or multiple excitatory neuromodulators exists.

Blockade of neurokinin-1 receptors in the ventral respiratory column does not affect breathing but alters neurochemical release.

Substance P (SP) and its receptor, neurokinin-1 (NK1R), have been shown to be excitatory modulators of respiratory frequency and to stabilize breathing regularity. Studies in anesthetized mice suggest that tonic activation of NK1Rs is particularly important when other excitatory inputs to the pre-Bötzinger complex in the ventral respiratory column (VRC) are attenuated. Consistent with these findings, muscarinic receptor blockade in the VRC of intact goats elicits an increase in breathing frequency associated with increases in SP and serotonin concentrations, suggesting an involvement of these substances in neuromodulator compensation. To gain insight on the contribution to breathing of endogenous SP and NK1R activation, and how NK1R modulates the release of other neurochemicals, we individually dialyzed antagonists to NK1R (133, 267, 500 µM Spantide; 3 mM RP67580) throughout the VRC of awake and sleeping goats. We found that NK1R blockade with either Spantide at any dose or RP67580 had no effect on breathing or regularity. Both antagonists significantly (P < 0.001) increased SP, while RP67580 also increased serotonin and glycine and decreased thyrotropin-releasing hormone concentrations in the dialysate. Taken together, these data support the concept of neuromodulator interdependence, and we believe that the loss of excitatory input from NK1Rs was locally compensated by changes in other neurochemicals.

Evidence for respiratory neuromodulator interdependence after cholinergic disruption in the ventral respiratory column.

Reverse dialysis of the muscarinic receptor antagonist, atropine (ATR, 50 mM), into the pre-Bötzinger Complex region of the ventral respiratory column (VRC) of awake and sleeping goats increases breathing frequency and serotonin (5-HT), substance P (SP), glycine, and GABA concentrations in the effluent dialysate. Herein, we report data from goats in which we reverse dialyzed 5 mM ATR or specific antagonists of M2 or M3 muscarinic receptors into the VRC. The effects on frequency of all three antagonists were not significantly different from time control studies. 5 mM ATR and the M3 antagonist increased SP sevenfold less than 50 mM ATR. The antagonists had no effect on 5-HT, glycine, and/or GABA, suggesting that the increases in glycine and GABA with 50 mM ATR were secondary to the larger increases in 5-HT and/or SP. These data are suggestive of neuromodulator interdependence, whereby attenuation of one neuromodulator is compensated for by local changes in other neuromodulators to stabilize breathing.

Changes in glutamate receptor subunits within the medulla in goats after section of the carotid sinus nerves.

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. Herein we tested the hypothesis that there are time-dependent changes in the expression of specific AMPA, NMDA, and/or neurokinin-1 (NK1R) receptors within respiratory-related brain stem nuclei acutely or chronically after CBD in adult goats. Brain stem tissues were collected acutely (5 days) or chronically (30 days) after sham or bilateral CBD, immunostained with antibodies targeting AMPA (GluA1 or GluA2), NMDA (GluN1), or NK-1 receptors, and optical density (OD) compared. Physiological measurement confirmed categorization of each group and showed ventilatory effects consistent with bilateral CBD (Miller et al. J Appl Physiol 115: 1088-1098, 2013). Acutely after CBD, GluA1 OD was unchanged or slightly increased, but GluA2 and GluN1 OD were reduced 15-30% within the nucleus tractus solitarius (NTS) and in other medullary respiratory nuclei. Chronically after CBD, GluA1 was reduced (P < 0.05) within the caudal NTS and in other nuclei, but there was significant recovery of GluA2 and GluN1 OD. NK1 OD was not significantly different from control after CBD. We conclude that the initial decrease in GluA2 and GluN1 after CBD likely contributes to hypoventilation and the reduced CO2 chemoreflex. The partial recovery of ventilation and the CO2 chemoreflex after CBD parallel a time-dependent return of these receptors to near control levels but likely depend upon additional initiating and maintenance factors for neuroplasticity.

Contributions of the pre-Bötzinger complex and the Kölliker-fuse nuclei to respiratory rhythm and pattern generation in awake and sleeping goats.

We investigated in three groups of awake and sleeping goats whether there are differences in ventilatory responses after injections of Ibotenic acid (IA, glutamate receptor agonist and neurotoxin) into the pre-Bötzinger complex (preBötC), lateral parabrachial (LPBN), medial (MPBN) parabrachial, or Kölliker-Fuse nuclei (KFN). In one group, within minutes after bilateral injection of 10µl IA (50mM) into the preBötC, there was a 10-fold increase in breathing frequency, but 1.5h later, the goats succumbed to terminal apnea. These data are consistent with findings in reduced preparations that the preBötC is critical to sustaining normal breathing. In a second group, increasing volumes (0.5-10µl) of IA injected at weekly intervals into the preBötC elicited a near-dose-dependent tachypnea and irregular breathing that lasted at least 5h. There were apneas restricted to wakefulness, but none were terminal. Postmortem histology revealed that the preBötC was 90% destroyed, but there was a 25-40% above normal number of neurons in the presumed parafacial respiratory group that may have contributed to maintenance of arterial blood gas homeostasis. In a third group, bilateral injections (1 and 10µl) of IA into the LPBN, MPBN, or KFN did not significantly increase breathing in any group, and there were no terminal apneas. However, 3-5h after the injections into the KFN, breathing frequency was decreased and the three-phase eupneic breathing pattern was eliminated. Between 10 and 15h after the injections, the eupneic breathing pattern was not consistently restored to normal, breathing frequency remained attenuated, and there were apneas during wakefulness. Our findings during wakefulness and NREM sleep warrant concluding that (a) the preBötC is a primary site of respiratory rhythm generation; (b) the preBötC and the KFN are determinants of respiratory pattern generation; (c) after IA-induced lesions, there is time-dependent plasticity within the respiratory control network; and (d) ventilatory control mechanisms are state dependent.

Changes in neurochemicals within the ventrolateral medullary respiratory column in awake goats after carotid body denervation.

A current and major unanswered question is why the highly sensitive central CO2/H(+) chemoreceptors do not prevent hypoventilation-induced hypercapnia following carotid body denervation (CBD). Because perturbations involving the carotid bodies affect central neuromodulator and/or neurotransmitter levels within the respiratory network, we tested the hypothesis that after CBD there is an increase in inhibitory and/or a decrease in excitatory neurochemicals within the ventrolateral medullary column (VMC) in awake goats. Microtubules for chronic use were implanted bilaterally in the VMC within or near the pre-Bötzinger Complex (preBötC) through which mock cerebrospinal fluid (mCSF) was dialyzed. Effluent mCSF was collected and analyzed for neurochemical content. The goats hypoventilated (peak +22.3 ± 3.4 mmHg PaCO2) and exhibited a reduced CO2 chemoreflex (nadir, 34.8 ± 7.4% of control ΔVE/ΔPaCO2) after CBD with significant but limited recovery over 30 days post-CBD. After CBD, GABA and glycine were above pre-CBD levels (266 ± 29% and 189 ± 25% of pre-CBD; P < 0.05), and glutamine and dopamine were significantly below pre-CBD levels (P < 0.05). Serotonin, substance P, and epinephrine were variable but not significantly (P > 0.05) different from control after CBD. Analyses of brainstem tissues collected 30 days after CBD exhibited 1) a midline raphe-specific reduction (P < 0.05) in the percentage of tryptophan hydroxylase-expressing neurons, and 2) a reduction (P < 0.05) in serotonin transporter density in five medullary respiratory nuclei. We conclude that after CBD, an increase in inhibitory neurotransmitters and a decrease in excitatory neuromodulation within the VMC/preBötC likely contribute to the hypoventilation and attenuated ventilatory CO2 chemoreflex.

Atropine microdialysis within or near the pre-Botzinger Complex increases breathing frequency more during wakefulness than during NREM sleep.

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 = 9) and NREM sleep (n = 7) states. Breathing was monitored, and effluent dialysate was collected for analysis of multiple neurochemicals. Compared with dialysis of mCSF alone, atropine increased (P < 0.05) breathing frequency while awake during the day [+10 breaths (br)/min] and at night (+9 br/min) and, to a lesser extent, during NREM sleep (+5 br/min). Atropine increased (P < 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.

A comparison of test-ordering choices of college physicians and emergency physicians for young adults with abdominal pain: influences and preferences for CT use.

The objective of this study was to compare through questionnaires the test-ordering behavior of college health professionals and emergency physicians with respect to the choosing of computed tomography scans under two clinical scenarios-suspicion of appendicitis and nondescript abdominal pain. Surveys were sent to physician members of both the American College Health Association and the American College of Emergency Physicians. The recipients were asked if their initial workup would include a computed tomography (CT) scan for either clinical scenario. They were queried on their estimation of the importance of physical examination findings, practice standards, economic considerations, and interpersonal factors on the decision to obtain a CT. They were also asked if their decision to order a CT was related to physical exam findings, parental influence, established protocol, costs to student, insurance considerations, medical literature recommendations, and relationship with radiologist. For the first presentation, a clinical suspicion of appendicitis, there was little difference between the choices of the two groups. Seventy seven percent of the college health professionals would obtain one and 76% of the ER physicians would do the same. However, for the workup of nondescript pain, three times as many ER physicians as college health professionals would obtain a CT scan (34% vs 11%). Of the seven factors, the most important determinant for both groups of physicians was the results of physical exam and least important by far was the relationship to the radiologist.