Sigh and Eupnea Rhythmogenesis Involve Distinct Interconnected Subpopulations: A Combined Computational and Experimental Study

Biphasic shape of sigh bursts requires inhibitory synaptic input. A−C, In vitro recordings of sigh and eupnea activities in control conditions and after blockade of glycinergic synapses. A, Integrated traces of preBötC recordings in control conditions (black, top) and in the presence of 1 µM strychnine (blue, bottom). Both recordings show two types of bursts corresponding to eupnea and sigh. B, Sequential slots of the amplitude (in arbitrary units) of inspiratory bursts versus time in control conditions (top) and under strychnine (bottom). Note that the amplitudes of both types of burst were significantly larger in the presence of strychnine. C, Averaged traces for eupneic bursts (n = 10; left) and sigh bursts (n = 8; right) in control (black) and strychnine (blue) conditions. D, In silico average voltage output of a two-compartment coupled model with (top trace) and without (bottom trace) synaptic inhibition ( ). E, Average profile of eupnea (left) and sigh bursts (right) obtained from traces in D. Orange stars indicate sigh events.

Calcium-dependent mechanisms are critically involved in sigh but not eupnea generation. A, In silico: Average model voltage with control value (g_Ca = 0.02 nS, top trace) and with 50% reduction (g_Ca = 0.01 nS, bottom trace) of calcium conductance. B, In vitro: Extracellular recordings of preBötC activity in control conditions (top trace) and after bath application of 4 µM cadmium to impair calcium conductances (bottom trace). A partial blockade of calcium conductances in silico and in vitro specifically prevents sigh bursts generation. C, In silico: Average model voltage with control value (top trace) and with 90% reduction (bottom trace) for SERCA activity. D, In vitro: Extracellular recordings of preBötC activity in control conditions (top trace) and after bath application of 100 µM CPA + 10 µM ryanodine to impair ER activity (bottom trace). A significant blockade of ER activity in silico and in vitro specifically prevents sigh bursts generation. Orange stars indicate sigh events.

A persistent sodium current is critically involved in sigh and eupnea generation. A, In silico experiment showing the effects of reducing g_NaP from 100% (2.5 nS for eupnea and 1 nS for sigh, top trace) to 80% (2 nS for eupnea and 0.8 nS for sigh, middle trace) and to 0% (bottom trace) on global network activity. B, In vitro: Extracellular recordings of preBötC activity in control conditions (top trace) and under increasing concentrations of riluzole (middle and bottom traces) to impair the persistent sodium current. C, Quantification of burst frequency changes under the partial and full blockade of g_NaP for fictive eupnea (white bars) and sighs (gray bars) in the model (left) and in vitro (right). Orange stars indicate sigh events.

Eupneic activity is more sensitive to [K⁺]_o than sigh activity. A, Voltage traces of in silico experiments where V_K, which is directly correlated to [K⁺]_o was progressively increased (from top to bottom). B, Bar plots representing the mean frequency for eupnea (left, unfilled bars) and sigh bursts (right, shaded bars) for different V_K. C, Extracellular recordings of spontaneous preBötC activity from slice preparation bathed in aCSF containing 4, 6, 8, and 10 mM of [K ⁺]_o (respectively, from top to bottom). D, Histograms of mean (+SEM) eupneic (unfilled bars) and sigh (shaded bars) burst frequencies for different [K⁺]_o . Sigh bursts were generated at a relatively constant frequency whereas the eupnea burst frequency increased with increasing [K⁺]_o both in silico and in vitro. *p < 0.05. Orange stars indicate sigh events.

PreBötC activity in low [K⁺]_o implicates neuronal activity from both subpopulations. A, Voltage traces (top: average; middle: eupnea compartment; bottom: sigh compartment) of an in silico experiment in control conditions (left) and with V_K set low (right). B, Schematic representation of the in vitro slice preparation from which an electrophysiological recording of preBötC activity was made simultaneously with calcium imaging performed on the contralateral respiratory network. Right, Image of a Ca²⁺-dye loaded slice obtained with a 40× objective. Yellow circles indicate rhythmically active cells. Bottom, Paired recordings of population electrical activity (top traces) and calcium transients (ΔF/F, bottom traces) in individual neurons (cells 1 to 19). All the cells displayed fluorescent changes synchronized to rhythmic electrical activity recorded on the contralateral side during both eupnea and sigh bursting in control conditions and during sigh-only activity in low [K⁺]_o . Orange stars indicate sigh events.

I_h activation is required for sigh but not for eupnea generation. A, Top, Simultaneous current-clamp recording of an inspiratory neuron (top trace) with integrated preBötC activity recording (bottom trace). Bottom left, Currents recorded in voltage clamp (bottom trace sets; i) evoked by hyperpolarizing voltage steps (top trace set; V) from a holding potential of −50 mV in control conditions (CTL) and in the presence of 50 µM ZD 7288 to block I_h . Bottom right, Amplitude of h current versus membrane potential under control conditions (black circles) and in the presence of ZD 7288 (gray circles). I_h was fully blocked by ZD 7288. B, In vitro extracellular recordings of preBötC activity in control (top trace) and in the presence of 50 µM ZD 7288. C, In silico experiments that included I_h in the eupnea compartment model only (top), the sigh compartment only (middle), and in both compartments (bottom). The sigh bursts exhibit a biphasic shape only when I_h is present in both compartments. D, Voltage traces of in silico experiment with changes in I_h from its control value g_h = 2 nS, (top) to g_h = 0 nS (bottom). Both in silico and in vitro blockade of I_h selectively prevents sigh-burst generation. Orange stars indicate sigh events.