Cholinergic neurotransmission in the preBötzinger Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm

Abstract

We investigated whether there is endogenous acetylcholine (ACh) release in the preBötzinger Complex (preBötC), a medullary region hypothesized to contain neurons generating respiratory rhythm, and how endogenous ACh modulates preBötCneuronal function and regulates respiratory pattern. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve roots (XIIn) and patch-clamped preBötC inspiratory neurons. Unilateral microinjection of physostigmine, an acetylcholinesterase inhibitor, into the preBötC increased the frequency of respiratory-related rhythmic activity from XIIn to 116+/-13% (mean+/-S.D.) of control. Ipsilateral physostigmine injection into the hypoglossal nucleus (XII nucleus) induced tonic activity, increased the amplitude and duration of the integrated inspiratory bursts of XIIn to 122+/-17% and 117+/-22% of control respectively; but did not alter frequency. In preBötC inspiratory neurons, bath application of physostigmine (10 microM) induced an inward current of 6.3+/-10.6 pA, increased the membrane noise, decreased the amplitude of phasic inspiratory drive current to 79+/-16% of control, increased the frequency of spontaneous excitatory postsynaptic currents to 163+/-103% and decreased the whole cell input resistance to 73+/-22% of control without affecting the threshold for generation of action potentials. Bath application of physostigmine concurrently induced tonic activity, increased the frequency, amplitude and duration of inspiratory bursts of XIIn motor output. Bath application of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 2 microM), a M3 muscarinic acetylcholine receptor (mAChR) selective antagonist, increased the input resistance of preBötC inspiratory neurons to 116+/-9% of control and blocked all of the effects of physostigmine except for the increase in respiratory frequency. Dihydro-beta-erythroidine (DH-beta-E; 0.2 microM), an alpha4beta2 nicotinic receptor (nAChR) selective antagonist, blocked all the effects of physostigmine except for the increase in inspiratory burst amplitude. In the presence of both 4-DAMP and DH-beta-E, physostigmine induced opposite effects, i.e. a decrease in frequency and amplitude of XIIn rhythmic activity. These results suggest that there is cholinergic neurotransmission in the preBötC which regulates respiratory frequency, and in XII nucleus which regulates tonic activity, and the amplitude and duration of inspiratory bursts of XIIn in neonatal rats. Physiologically relevant levels of ACh release, via mAChRs antagonized by 4-DAMP and nAChRs antagonized by DH-beta-E, modulate the excitability of inspiratory neurons and excitatory neurotransmission in the preBötC, consequently regulating respiratory rhythm.

Fig. 1

Pressure microinjection of Physo (150 μM, 10 nl) into the preBötC increased frequency of integrated rhythmic activity of hypoglossal nerve (∫XIIn) while injection into XII nu induced tonic activity, increased amplitude and duration of integrated inspiratory bursts of XIIn. (A) Traces 1–4 indicate injections into the ipsilateral preBötC (1), contralateral preBötC (2), contralateral XII nu (3) and ipsilateral XII nu (4). The injection pipettes were inserted 100–200 μm below the surface of the slice. “Control” traces were prior to, and “Physo” traces were at least 1 min after Physo injection. Arrowheads on trace 4 indicate tonic activity. Traces 1, 2, 3 and 4 were from different preparations. (B) Physo injection into the preBötC decreased respiratory cycle periods and variability of the periods. Arrow indicates the time of injection. The data were from the same preparation as trace 2 in panel A. (C) Physo injection into ipsilateral XII nu increased the amplitude of integrated inspiratory bursts of XIIn. Arrow indicates the time of injection. Summaries of the Physo effects on respiratory frequency (D), amplitude (E) and duration (F) of integrated inspiratory bursts of XIIn (mean±S.E.). Respiratory frequency was determined as the reciprocal of the average of 10 consecutive respiratory periods for each condition. The amplitude and duration were determined with averaged envelope of five consecutive inspiratory bursts for each condition. * Statistical significance of Physo injection vs. control condition (paired t-test). Numbers of preparations (n) for every experiment are indicated in the text of Results section.

Fig. 2

(A) Bath application of Physo (10 μM) depolarized preBötC inspiratory neurons in current-clamp mode. (B) Effects of Physo on an extended time scale; a and b correspond to the labeled times a and b in panel A. Amp, amplitude of integrated inspiratory bursts; Anta, 4-DAMP+DH-β-E; Ctrl, control; Frq, respiratory frequency; Im, membrane current; Input R, input resistance; Insp Amp, inspiratory drive current amplitude; Physo, physostigmine; sEPSC Amp, sEPSC amplitude; sEPSC Frq, sEPSC frequency; Vm, membrane voltage; XII nu, the hypoglossal nucleus; XIIn, the hypoglossal nerve roots; ∫XIIn, integrated XIIn activity

Fig. 3

Bath application of Physo (10 μM) induced a tonic inward current, increased membrane noise, decreased the amplitude of phasic inspiratory drive current, increased the frequency of sEPSCs and decreased the input resistance in preBötC inspiratory neurons. Physo also increased respiratory frequency, the amplitude and duration of integrated inspiratory bursts of hypoglossal nerve. (A) Im, membrane current of inspiratory neuron voltage-clamped at −65 mV. (B) Phasic inspiratory drive current of an inspiratory neuron voltage-clamped at −65 mV in Ctrl and Physo conditions. Each trace was an average of five consecutive inspiratory periods triggered by the upstroke of the integrated inspiratory bursts from XIIn and the Im trace was low-pass filtered at 20 Hz. (C) sEPSCs during expiratory periods on an extended time scale; a and b correspond to the times labeled a and b in panel A. (D) Summary of the effects of Physo on XIIn rhythmic activity and on inspiratory neurons (mean±S.E.). The parameters of XIIn rhythmic activity include Frq, Amp and duration of integrated inspiratory bursts. The parameters of inspiratory neurons include Insp Amp and duration, Input R, sEPSC Frq and sEPSC Amp. * Statistical significance during Physo application vs. pre-Physo control (paired t-test). Numbers of neurons (n) for every experiment are indicated in the text of Results section.

Fig. 4

Bath application of 4-DAMP (2 μM) partially blocked the effects of Physo (10 μM). Physo increased Frq in the presence of 4-DAMP. (A) Im: membrane current from a voltage-clamped (−65 mV) inspiratory neuron. (B) Phasic inspiratory drive current of an inspiratory neuron voltage-clamped at −65 mV in control (Ctrl), 4-DAMP and 4-DAMP+Physo conditions. Each trace was an average of five consecutive inspiratory periods triggered by the upstroke of the integrated inspiratory bursts from XIIn and the Im trace was low-pass filtered at 20 Hz. (C) Summary of the effects of 4-DAMP, 4-DAMP+Physo on XIIn rhythmic activity and on inspiratory neurons (mean±S.E.). * Statistical significance during 4-DAMP application vs. pre-drug control. ^† Statistical significance during Physo application in the presence of 4-DAMP vs. 4-DAMP only conditions (one-way repeated measures ANOVA followed by post hoc comparison analyses based on Tukey). Numbers of neurons (n) for every experiment are indicated in the text of Results section.

Fig. 5

Bath application of DH-β-E (0.2 μM) partially blocked the effects of Physo (10 μM). Physo increased the amplitude of integrated inspiratory bursts of XIIn in the presence of DH-β-E. (A) Im:membrane current from a voltage-clamped (−65 mV) inspiratory neurons. (B) Phasic inspiratory drive current of an inspiratory neuron voltage-clamped at −65 mV in control (Ctrl), DH-β-E and DH-β-E+Physo conditions. Each trace is an average of five consecutive inspiratory periods triggered by the upstroke of the integrated inspiratory bursts from XIIn and the Im trace was low-pass filtered at 20 Hz. (C) Summary of the effects of DH-β-E, DH-β-E+Physo on ∫XIIn and on inspiratory neuron. ^† Statistical significance during Physo application in the presence of DH-β-E vs. DH-β-E only conditions (one-way repeated measures ANOVA followed by post hoc comparison analyses based on Tukey). Numbers of neurons (n) for every experiment are indicated in the text of Results section.

Fig. 6

Bath application of 4-DAMP (2 μM) together with DH-β-E (0.2 μM) completely blocked the effects of Physo (10 μM). In the presence of antagonists 4-DAMP + DH-β-E (Anta), Physo decreased frequency and amplitude of respiratory-related rhythmic XIIn motor output. (A) Im: membrane current from a voltage-clamped (−65 mV) inspiratory neuron. (B) Phasic inspiratory drive current of an inspiratory neuron voltage-clamped at −65 mV in Ctrl, Anta and Anta +Physo conditions. Each trace is an average of five consecutive inspiratory periods triggered by the upstroke of the integrated inspiratory bursts from XIIn and the Im trace was low-pass filtered at 20 Hz. (C) Summary of the effects of Anta, Anta +Physo on XIIn rhythmic activity and on inspiratory neurons. ^† Statistical significance during Physo application in the presence of 4-DAMP and DH-β-E vs. Anta conditions (one-way repeated measures ANOVA followed by post hoc comparison analyses based on Tukey). Numbers of neurons (n) for every experiment are indicated in the text of Results section.