Muere C, Neumueller S, Miller J, Olesiak S, Hodges MR, Pan L, Forster HV. Atropine microdialysis within or near the pre-Bötzinger Complex increases breathing frequency more during wakefulness than during NREM sleep. J Appl Physiol 114: 694-704, 2013. First published December 27, 2012 doi:10.1152/japplphysiol.00634.2012.-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 ϭ 9) and NREM sleep (n ϭ 7) states. Breathing was monitored, and effluent dialysate was collected for analysis of multiple neurochemicals. Compared with dialysis of mCSF alone, atropine increased (P Ͻ 0.05) breathing frequency while awake during the day [ϩ10 breaths (br)/min] and at night (ϩ9 br/min) and, to a lesser extent, during NREM sleep (ϩ5 br/min). Atropine increased (P Ͻ 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.neuromodulators; control of breathing; acetylcholine; non-rapid-eyemovement sleep THE NEUROMODULATOR ACETYLCHOLINE (ACh) is involved in a variety of central and peripheral neural circuits, including the control of breathing and sleep (7,12,16,19,25,39). The effects of ACh are mediated through either nicotinic or muscarinic receptors coupled to second messenger pathways (6, 10, 13, 42). There are five known muscarinic ACh receptor (mAChR) subtypes (M 1 to M 5 ) expressed in the brain stem, differing in expression profiles and in effects on breathing (2,9,11,13,33,34). For example, the pontine respiratory group (PRG) nuclei express M 1 -M 3 mAChRs (34). The predominant effect of cholinergic modulation of PRG neurons within the Kölliker-Fuse Nucleus (KFN) appears to be excitatory, since reverse dialysis of the nonselective mAChR antagonist atropine reduced minute ventilation (V I ) and breathing frequency in both the awake state and during non-rapid-eye-movement (NREM...

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.

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.

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 seven-fold 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.

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.