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 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.

Keywords: breathing; neuromodulation; pre-Bötzinger complex; sleep; ventral respiratory column.

Fig. 1.

A: sections of the goat brain stem at three caudal to rostral distances from obex, depicting the microtubule placement in nine goats. Each goat is numbered (1–9), and the nos. for the left and right microtubule are located in the images, where the distal end of the microtubule was identified by postmortem histological analysis. The 0.5-mm-wide porous membrane of the dialysis probe extended 2 mm beyond the distal end of the microtubule; thus we inserted a 2-mm long and 1-mm wide gray rectangle for each number to represent a conservative estimate of the region of diffusion of the dialyzed substances. The circle in each section is the approximate VRC in goats rostral to obex. B: quantitation of the breathing frequency response to NMDA injections displayed as percent increase from control levels before injection. Note goats 2 and 3 had unilaterally placed microtubules rostral to the preBötC, and these goats had the lowest response to NMDA injections. Note also that the left-side microtubule placement in goat 6 was the only case in which the rectangle did not extend to the estimated location of the VRC, which suggests widespread diffusion of NMDA in particular, but also dialyzed substances. CN, cuneate nucleus; FTL, lateral tegmental field; HP, nucleus praepositus hypoglossi; INT, nucleus intercalatus; IO, inferior olivary complex; IV, trochlear nucleus; NA, nucleus ambiguus; NTS, nucleus tractus solitarii; nX, vagus nerve; nXII, hypoglossal nerve; P, pyramidal tract; R, raphe nucleus; RB, restiform body; RG, nucleus gigantocellularis reticularis; 5SP, spinal trigeminal nucleus; 5ST, spinal trigeminal tract; TS, tractus solitaries; V, vestibular nucleus; X, dorsal motor nucleus of the vagus; XII, hypoglossal nucleus.

Fig. 2.

During 180 min of bilateral dialysis during the day, the triple cocktail of antagonists to muscarinic, NK-1, and 5-HT_2A receptors (5.0 mM atropine, 500 µM spantide, 0.5 mM MDL) (●; n = 7) dialyzed between 60 and 120 min (indicated by horizontal solid line) significantly (P < 0.001) increased ventilation (V̇i; A) and breathing frequency (f; B), but did not alter (P = 0.567) tidal volume (Vt; C) compared with bilateral dialysis of mCSF alone (○, n = 7). Values are means ± SE. The F and P values shown are the interaction terms obtained from two-way repeated-measures ANOVA using time and dose as factors. *Values during and after triple cocktail dialysis that differed from hour 1 mCSF dialyses, as indicated by Holm-Sidak post hoc analysis.

Fig. 3.

During 180 min of bilateral dialysis during the day, the triple cocktail of antagonists to muscarinic, NK-1, and 5-HT_2A receptors (5.0 mM atropine, 500 µM spantide, 0.5 mM MDL) (●; n = 7) dialyzed between 60 and 120 min (indicated by horizontal solid line) significantly (P < 0.001) decreased inspiratory time (Ti; A) and expiratory time (Te; B) and increased respiratory drive (Vt/ Ti; C) during hours 2 and 3 compared with bilateral dialysis of mCSF alone (○, n = 7). Values are means ± SE. A two-way repeated-measures ANOVA using time and dose as factors was used to obtain the F and P values. *Values during and after triple cocktail dialysis that differed from hour 1 mCSF dialyses, as indicated by Holm-Sidak post hoc analysis.

Fig. 4.

Daytime bilateral dialysis of a triple cocktail of antagonists to muscarinic, NK1, and 5-HT_2A receptors (5.0 mM atropine, 500 µM spantide, 0.5 mM MDL) (●; n = 7) dialyzed between 60 and 120 min (indicated by horizontal solid line) significantly increased oxygen consumption (V̇o₂, P = 0.022; A) and body temperature (P = 0.02; B) compared with bilateral dialysis of mCSF alone (○, n = 7). Values are means ± SE. A two-way repeated-measures ANOVA (time and dose as factors) was used to determine if significant interactions occurred between treatments. *Values during and after triple cocktail dialysis that differed from hour 1 mCSF dialyses, as indicated by Holm-Sidak post hoc analysis.

Fig. 5.

There were no significant differences between day and night studies in the measured neurochemical responses to the triple cocktail dialysis, as indicated by a two-way repeated-measures ANOVA using time and dose as factors (F and P values presented at the top left of each panel). Daytime (solid bars) and nighttime (shaded bars) bilateral dialysis of a triple cocktail of antagonists to muscarinic, NK-1, and 5-HT_2A receptors (5.0 mM atropine, 500 µM spantide, 0.5 mM MDL) during hour 2 of dialysis significantly (P ≤ 0.033) increased levels of substance P (A) and 5-HT (B) in effluent mCSF during hour 2 and hour 3 of dialysis compared with dialysis of mCSF alone (open bars) (n = 7). C: glycine was significantly (P = 0.011) lower throughout the 3 h of mCSF dialysis compared with the 3 h when the triple cocktail was dialyzed in hour 2. GABA (D) and glutamine (E) were not significantly (P ≥ 0.379) altered by triple cocktail dialysis during the day or at night. Values are means ± SE. *Values during and after triple cocktail dialysis that differed from hour 1 mCSF dialyses, as indicated by Holm-Sidak post hoc analysis. Hr1–Hr3, hours 1–3.

Fig. 6.

A two-way repeated-measures ANOVA analysis of the effects of bilateral triple cocktail dialysis indicates that the ventilation (V̇i; A), breathing frequency (f; B), and tidal volume (Vt; C) responses did not differ between awake (●) and NREM sleep (○) (F and P values, top left of each panel). B: bilateral dialysis of a triple cocktail of antagonists to muscarinic, NK1, and 5-HT_2A receptors (5.0 mM atropine, 500 µM spantide, 0.5 mM MDL) (n = 5) (indicated by solid horizontal line) significantly (P ≤ 0.002) increased f at night during awake and NREM sleep states (one-way ANOVA). A: V̇i was significantly increased during NREM sleep (P = 0.007), but not in the night awake state. C: Vt was not significantly affected in either state. Values are means ± SE.

Fig. 7.

During the night studies, the coefficient of variation (CV) for f (P = 0.038; B) and Vt (P = 0.04; C) within 15-min periods was greater for the awake state (●) than for NREM slee (○), indicated by the interaction term of two-way ANOVA (F and P at top left). A: the CV of V̇i did not differ between awake and NREM sleep. A one-way ANOVA indicated that, in the awake state at night, the triple cocktail significantly increased the CV for V̇i, f, and Vt (P < 0.05), whereas for NREM sleep, the triple cocktail only significantly increased the CV for V̇i (P = 0.039) (P, right side). The horizontal solid line in A denotes that the triple cocktail was dialyzed between 60 and 120 min. Values are means ± SE.

Fig. 8.

Unilateral dialysis of 50 mM atropine during the day (n = 6; ●) (indicated by horizontal solid line) significantly (P < 0.001) increased ventilation (V̇i; A) and breathing frequency (f; B), but did not significantly (P = 0.578) alter tidal volume (Vt; C) compared with mCSF time control studies (○). The F and P values are the interaction terms from two-way repeated-measures ANOVA using dose and time as factors. Values are means ± SE. *Values during and after atropine dialysis that differed from hour 1 mCSF dialyses, as indicated by Holm-Sidak post hoc analysis.

Fig. 9.

There were no statistically significant differences between the awake (open bars) and anesthetized (solid bars) state in substance P (A), 5-HT (B), glycine (C), or GABA (D) levels, as indicated by the F and P values from the interaction term (top left in each panel) of the two-way repeated-measures ANOVA. E: glutamine was significantly (P = 0.034) greater during anesthesia studies. Values are means ± SE. *For awake and anesthesia, unilateral dialysis of 50 mM atropine significantly (P < 0.001) increased substance P (A) and 5-HT (B) in effluent dialysate during both hours 2 and 3 (obtained from Holm-Sidak post hoc analysis).

Fig. 10.

${\mathrm{Pa}}_{\mathrm{C}{\mathrm{O}}_2}$ did not change during unilateral dialysis of 50 mM atropine in anesthetized goats that were mechanically ventilated (●). However, ${\mathrm{Pa}}_{\mathrm{C}{\mathrm{O}}_2}$ increased in goats under anesthesia that were not mechanically ventilated (▲ and ■). Values are means ± SE.