Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network

Abstract

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons.

Keywords: mouse; neural network; neuroscience; pacemaker neuron; preBötzinger complex; prenatal development; respiratory rhythmogenesis.

Figure 1.. Functional isolation of embryonic inspiratory pacemaker neurons.

(A) left, Schematic representation of an in vitro embryonic slice preparation used for making electrophysiological macroelectrode recording of preBötC network activity on one side simultaneously with calcium imaging of the contralateral inspiratory network (red square). Right, Image of fluorescence expression by a Ca²⁺-dye loaded slice obtained with a 40X objective. Numbered yellow circles indicate rhythmically active neurons. (B) Simultaneous recordings of inspiratory population electrical activity (integrated trace in violet, Int preBötC) and calcium transients (ΔF/F, black and red traces) in 16 individual neurons (numbered 1 to 16). All the cells displayed fluorescent changes synchronized to rhythmic electrical activity recorded on the contralateral side in control conditions. (C) After blockade of chemical synapses by a bath- applied cocktail of CNQX (20 µM), AP5 (10 µM), strychnine (1 µM) and bicuculline (10 µM), neurons that remained rhythmically active (red traces) were considered to be pacemakers while those falling silent were considered as non-pacemaker cells (black traces). DOI: http://dx.doi.org/10.7554/eLife.16125.003

Figure 2.. Different discharge patterns expressed by embryonic inspiratory pacemaker neurons.

Patch-clamp recordings of individual pacemaker neurons from three different preparations expressing spontaneous plateau (A), oscillatory (B) or mixed plateau/oscillatory (C) burst firing patterns. (D) Histograms showing frequency distributions of the burst-generating dirve potential (DP) amplitude (D_1–3, left column) and duration (D_1–3, right column) for the three types of pacemaker neurons. D₄: Histograms representing mean DP amplitude ± SEM (left) and mean DP duration ± SEM (right) for pacemaker neurons expressing plateau-like (blue bars), burst-like (red bars) or mixed (green bars; light green bars correspond to short-lasting and long-lasting bursts when grouped separately) activity. Asterisks indicate significant differences (Mann-Whitney test; p<0.001), while numbers of neurons analyzed for each pacemaker phenotype are indicated in the corresponding bar. (E) Distribution plot for DP duration vs DP amplitude measured for neurons recorded for 8 to 10 min (blue, plateau bursting pacemakers (n = 8); red, oscillatory bursting pacemakers (n = 5); green, mixed pacemakers (n = 5). Note that the latter expressed two types of burst duration/amplitude relationship that overlapped with either the purely plateau or oscillatory bursters. DOI: http://dx.doi.org/10.7554/eLife.16125.004

Figure 3.. Voltage-dependence of pacemaker neuron properties.

(A) Current-voltage (I-V) relationships for plateau (black circles) and oscillatory bursting (open circles) pacemaker neurons. Individual circles indicate mean (± SEM) current response to an injected step-change voltage pulse; n = number of neurons analyzed for each pacemaker phenotype. Asterisks indicate statistically different values (t-test; p<0.001). (B,C) Patch-clamp recordings of plateau (B) and oscillatory bursting (C) pacemaker neurons displaying cycle frequencies that varied with membrane voltage when held at different indicated membrane potential levels with continuous depolarizing current injection. Right inset: A single burst (indicated by shading) in each pacemaker neuron subtype displayed at the same time scale for burst duration comparison. DOI: http://dx.doi.org/10.7554/eLife.16125.005

Figure 4.. Membrane conductances underlying the different discharge patterns.

Patch-clamp recordings of plateau (A), oscillatory (B) and mixed (C) bursting pacemaker neurons in control conditions (top traces), in the presence of either 10 µM Ril or 50 µM FFA (middle traces) or in the presence of 10 µM Ril + 50 µM FFA (bottom traces). The left and right panels in A illustrate two different plateau bursters under different indicated pharmacological treatments. Rhythmic burst activity of the plateau pacemaker was mostly affected (i.e., reduced) by blockade of I_CAN channels with FFA (A), whereas the oscillatory pacemaker activity was more sensitive to persistent sodium channel (I_NaP) blockade with Ril (B). Correspondingly, longer duration plateau driven discharge in the mixed pacemaker phenotype was blocked with FFA alone, whereas remaining short-duration oscillatory bursts were blocked by the addition of Ril (C). Shaded activity at right of each trace: recording excerpts on expanded time-scale to facilitate comparison. DOI: http://dx.doi.org/10.7554/eLife.16125.006

Figure 5.. Developmental changes in inspiratory network pacemaker composition.

(A) Bar histograms indicating the mean proportions (as% ) of individual pacemaker neuron subtypes recorded with patch-clamp at E16.5 (n = 27) and E18.5 (n = 35). Plateau bursting neurons, black shading; mixed plateau/oscillatory bursters, dark gray; oscillatory bursting neurons, light gray. (B) Calcium imaging protocol (see also Figure 1) to test the sensitivity of the preBötC pacemaker neuron subpopulation to I_NaP blockade at E16.5 and E18.5. Calcium transients in 12 monitored inspiratory neurons at E16.5 in control conditions (left column), after synapse blockade (middle column), then under additional exposure to 10 µM riluzole (right column). Black traces correspond to non-pacemaker neurons (i.e., inactive under cocktail); the blue trace denotes a pacemaker neuron whose spontaneous activity was subsequently blocked under riluzole (i.e., an I_NaP-dependent pacemaker); the red trace corresponds to a pacemaker neuron that remained rhythmically activity under both cocktail and riluzole (i.e., an I_CAN-dependent pacemaker). (C) Bar histograms representing the proportions of riluzole-sensitive (blue bar) vs riluzole-resistant (red bars) neurons detected at E16.5 and E18.5. Note the higher proportion of riluzole-sensitive (I_NaP-dependent) pacemaker neurons at the later embryonic stage. DOI: http://dx.doi.org/10.7554/eLife.16125.007

Figure 6.. The balance between simulated I_CAN and I_NaP in silico can determine pacemaker discharge phenotype.

(A) Computational model of an inspiratory pacemaker neuron with the main burst-generating currents I_CAN and I_NaP highlighted in yellow. For other abbreviations, see Material and methods and (Toporikova et al., 2015). (B–D) Model neuron voltage traces obtained with I_NaP conductance (g_NaP) held constant at 2.5 nS and g_CAN set to different steady state values (indicated in orange). Depending on g_CAN magnitude, the model neuron generated plateau bursting discharge (B, g_CAN = 2.5 nS), a mixed firing pattern (C, g_CAN = 1 nS) or a rhythmic oscillatory burst pattern (D, g_CAN = 0 nS). (E) Graph showing the type of model neuron discharge as a function of the proportions of g_NaP and g_CAN magnitudes. The gray dashed lines correspond to the values of constant g_NaP and g_CAN used, respectively, in the variable g_CAN simulations illustrated in (B-D) and in complementary simulations in which I_NaP was varied with g_CAN held constant (data not shown). Depending on the balance between g_NaP and g_CAN amplitudes, the model neuron could exhibit the three different patterns observed with patch-clamp recordings in vitro. DOI: http://dx.doi.org/10.7554/eLife.16125.008

Figure 7.. Mechanisms for inspiratory network rhythmogenesis change during embryonic development.

(A) Schematic representation of an in vitro slice preparation used for electrophysiological recording of spontaneous preBötC network activity (integrated traces, Int preBötC) in control conditions (top traces) or in the presence of either FFA (50 µM, middle trace, left) or Ril (20 µM, middle trace, right) alone or in combination (bottom traces) at E16.5. (B) Bar histograms representing the percentages of cycle frequency change (mean ± SEM) compared to control under the different pharmacological conditions illustrated in A. Asterisks indicate significant differences (t-test, p<0.05); n = number of experiments in each case. (C,D) same layout as (A,B) for experiments performed at E18.5. Note that unlike at E16.5 where both FFA and Ril significantly reduced rhythmogenesis, blockade of either I_CAN or I_NaP alone did not significantly perturb rhythm generation at E18.5. Also, blockade of both conductances at E16.5 was sufficient to completely suppress rhythm generation, but not at E18.5. DOI: http://dx.doi.org/10.7554/eLife.16125.009

Figure 8.. Developmental increase in burst-generating synaptic excitation amongst inspiratory network neurons.

Integrated preBötC network activity (top traces, Int preBötC) with simultaneous intracellular recordings of membrane current fluctuations (lower traces) in rhythmically active preBötC neurons under voltage clamp (holding potential set at −50 mV) obtained at E16.5 (A) and E18.5 (B). Expanded bottom traces in A and B show the excitatory synaptic currents (left side in each case, single burst cycle; right side in red, average of 15 cycles) recorded from individual inspiratory neurons during rhythmic network activity. Dashed lines indicate baseline and maximum amplitude levels of the synaptic currents. Note their larger amplitude at E18.5 for both single cycle and averaged events. (C) Bar histograms representing the mean maximal amplitude (± SEM) of excitatory synaptic current underlying burst discharge measured in 14 neurons at E16.5 (unshaded bar) and 17 neurons at E18.5 (gray bar). The asterisk indicates significant difference (p< 0.001). DOI: http://dx.doi.org/10.7554/eLife.16125.010