Justin Miller

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.

Opioid-induced Respiratory Depression Is Only Partially Mediated by the preBötzinger Complex in Young and Adult Rabbits In Vivo

Background:The preBötzinger Complex (preBC) plays an important role in respiratory rhythm generation. This study was designed to determine whether the preBC mediated opioid-induced respiratory rate depression at clinically relevant opioid concentrations in vivo and whether this role was age dependent. Methods:Studies were performed in 22 young and 32 adult New Zealand White rabbits. Animals were anesthetized, mechanically ventilated, and decerebrated. The preBC was identified by the tachypneic response to injection of D,L-homocysteic acid. (1) The &mgr;-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO, 100 &mgr;M) was microinjected into the bilateral preBC and reversed with naloxone (1 mM) injection into the preBC. (2) Respiratory depression was achieved with intravenous remifentanil (0.08 to 0.5 &mgr;g kg−1 min−1). Naloxone (1 mM) was microinjected into the preBC in an attempt to reverse the respiratory depression. Results:(1) DAMGO injection depressed respiratory rate by 6 ± 8 breaths/min in young and adult rabbits (mean ± SD, P < 0.001). DAMGO shortened the inspiratory and lengthened the expiratory fraction of the respiratory cycle by 0.24 ± 0.2 in adult and young animals (P < 0.001). (2) During intravenous remifentanil infusion, local injection of naloxone into the preBC partially reversed the decrease in inspiratory fraction/increase in expiratory fraction in young and adult animals (0.14 ± 0.14, P < 0.001), but not the depression of respiratory rate (P = 0.19). PreBC injections did not affect respiratory drive. In adult rabbits, the contribution of non-preBC inputs to expiratory phase duration was larger than preBC inputs (3.5 [−5.2 to 1.1], median [25 to 75%], P = 0.04). Conclusions:Systemic opioid effects on respiratory phase timing can be partially reversed in the preBC without reversing the depression of respiratory rate.

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.

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.

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.

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.

A Subregion of the Parabrachial Nucleus Partially Mediates Respiratory Rate Depression from Intravenous Remifentanil in Young and Adult Rabbits

Background: The efficacy of opioid administration to reduce postoperative pain is limited by respiratory depression. We investigated whether clinically relevant opioid concentrations altered the respiratory pattern in the parabrachial nucleus, a pontine region contributing to respiratory pattern generation, and compared these effects with a medullary respiratory site, the pre-Bötzinger complex. Methods: Studies were performed in 40 young and 55 adult artificially ventilated, decerebrate rabbits. We identified an area in the parabrachial nucleus where &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid microinjections elicited tachypnea. Two protocols were performed in separate sets of animals. First, bilateral microinjections of the &mgr;-opioid receptor agonist [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin (100 &mgr;M) into the “tachypneic area” determined the effect of maximal &mgr;-opioid receptor activation. Second, respiratory rate was decreased with continuous IV infusions of remifentanil. The opioid antagonist naloxone (1 mM) was then microinjected bilaterally into the “tachypneic area” of the parabrachial nucleus to determine whether the respiratory rate depression could be locally reversed. Results: Average respiratory rate was 27 ± 10 breaths/min. First, [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin injections decreased respiratory rate by 62 ± 20% in young and 45 ± 26% in adult rabbits (both P < 0.001). Second, during IV remifentanil infusion, bilateral naloxone injections into the “tachypneic area” of the parabrachial nucleus reversed respiratory rate depression from 55 ± 9% to 20 ± 14% in young and from 46 ± 20% to 18 ± 27% in adult rabbits (both P < 0.001). The effects of bilateral [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin injection and IV remifentanil on respiratory phase duration in the “tachypneic area” of the parabrachial nucleus was significantly different from the pre-Bötzinger complex. Conclusions: The “tachypneic area” of the parabrachial nucleus is highly sensitive to &mgr;-opioid receptor activation and mediates part of the respiratory rate depression by clinically relevant administration of opioids.

Automatic Classification of Canine PRG Neuronal Discharge Patterns using K-means Clustering

Respiratory-related neurons in the parabrachial-Kölliker-Fuse (PB-KF) region of the pons play a key role in the control of breathing. The neuronal activities of these pontine respiratory group (PRG) neurons exhibit a variety of inspiratory (I), expiratory (E), phase spanning and non-respiratory related (NRM) discharge patterns. Due to the variety of patterns, it can be difficult to classify them into distinct subgroups according to their discharge contours. This report presents a method that automatically classifies neurons according to their discharge patterns and derives an average subgroup contour of each class. It is based on the K-means clustering technique and it is implemented via SigmaPlot User-Defined transform scripts. The discharge patterns of 135 canine PRG neurons were classified into seven distinct subgroups. Additional methods for choosing the optimal number of clusters are described. Analysis of the results suggests that the K-means clustering method offers a robust objective means of both automatically categorizing neuron patterns and establishing the underlying archetypical contours of subtypes based on the discharge patterns of group of neurons.

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.