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

Abstract

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 ) 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) 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 ) 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) 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. 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.

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

“…This instability contrasts to studies (Langer et al, 2016; Muere et al, 2013, 2015a; Muere et al, 2015b; Muere et al, 2015c) in which dialyses of antagonists to multiple excitatory receptors in the VRC did not destabilize breathing. The destabilization with DAMGO could be due to time-dependent changes in the direct effect of opioid-induced depression and the indirect effect of potential compensatory mechanisms.…”

“…However, findings herein ( Figure 5 ) do not support this possibility as bilateral DAMGO dialysis increased ventilation. This finding is similar to bilateral dialysis of antagonists to excitatory receptors which also increased ventilation even though ventilation did not change during unilateral dialysis of these antagonists (Langer et al, 2016). Accordingly, during unilateral and bilateral dialysis, there likely are multiple factors affecting ventilation, which makes it difficult to measure a steady-state, maximal effect, or recovery effect.…”

“…Microdialysis of antagonists to excitatory, neuromodulatory, G protein-coupled receptors into the VRC of awake and sleeping goats (Langer et al, 2016; Muere et al, 2013, 2015a; Muere et al, 2015b; Muere et al, 2015c) support the concept of neuromodulator compensation, whereby decreases in excitatory receptor activity are compensated by increases in other (predominantly excitatory) neuromodulators to prevent a decrease in ventilation and breathing frequency (Doi and Ramirez, 2008, 2010). In other words, there is “compensation” defined as no change in ventilation with dialysis of excitatory antagonists and that this may be due to the observed concomitant increase in excitatory (SP) and decrease in inhibitory neurotransmitters (GABA).…”

“…We have no explanation for why the increased ventilation with bilateral DAMGO dialysis did not result in arterial hypocapnia even though metabolic rate did not change. Nevertheless, whatever the exact mechanism of the neurochemical response to perturbation of neuromodulator receptors, it does not appear unique to awake and NREM sleep states as Langer et al found increases in 5-HT and SP equal in awake and anesthetized goats during dialysis of 50 mM atropine (Langer et al, 2016). …”

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

“…The reduced GABA levels were concomitant with transient decreases in V I with unilateral DAMGO, whereas bilateral dialysis of 100 M DAMGO increased V I and f and increased the variability of V I (20), indicative of local neuromodulator compensation for the increased inhibition by the exogenously delivered opioid. Thus, collectively, the summarized results support the concept of neuromodulator interdependence in respiratory rhythm-and pattern-generating nuclei such as the preBötC of the VRC (7,8,20,21,(32)(33)(34)(35). The mechanism of compensation is unknown, but the changes in neuromodulators are local; thus compensation may occur within individual ionic currents of a single neuron.…”