Phosphatidylinositol 4,5-bisphosphate regulates inspiratory burst activity in the neonatal mouse preBötzinger complex

Abstract

Neurons of the preBötzinger complex (preBötC) form local excitatory networks and synchronously discharge bursts of action potentials during the inspiratory phase of respiratory network activity. Synaptic input periodically evokes a Ca(2+)-activated non-specific cation current (I(CAN)) postsynaptically to generate 10-30 mV transient depolarizations, dubbed inspiratory drive potentials, which underlie inspiratory bursts. The molecular identity of I(CAN) and its regulation by intracellular signalling mechanisms during inspiratory drive potential generation remains unknown. Here we show that mRNAs coding for two members of the transient receptor potential (TRP) family of ion channels, namely TRPM4 and TRPM5, are expressed within the preBötC region of neonatal mice. Hypothesizing that the phosphoinositides maintaining TRPM4 and TRPM5 channel sensitivity to Ca(2+) may similarly influence I(CAN) and thus regulate inspiratory drive potentials, we manipulated intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)) and measured its effect on preBötC neurons in the context of ongoing respiratory-related rhythms in slice preparations. Consistent with the involvement of TRPM4 and TRPM5, excess PIP(2) augmented the inspiratory drive potential and diminution of PIP(2) reduced it; sensitivity to flufenamic acid (FFA) suggested that these effects of PIP(2) were I(CAN) mediated. Inositol 1,4,5-trisphosphate (IP(3)), the product of PIP(2) hydrolysis, ordinarily causes IP(3) receptor-mediated I(CAN) activation. Simultaneously increasing PIP(2) while blocking IP(3) receptors intracellularly counteracted the reduction in the inspiratory drive potential that normally resulted from IP(3) receptor blockade. We propose that PIP(2) protects I(CAN) from rundown by interacting directly with underlying ion channels and preventing desensitization, which may enhance the robustness of respiratory rhythm.

Figure 1. Model of I_CAN activation with hypothesized channel identity and PIP₂ regulation

Glutamate (triangle) activates group 1 metabotropic glutamate receptors (mGluRs), which then cause G_q-proteins to stimulate phospholipase C (PLC). PLC hydrolyses PIP₂ into diacylgylcerol (DAG) and IP₃, which activates IP₃ receptors (IP₃R) causing Ca²⁺ release from the endoplasmic reticulum (ER). Intracellular Ca²⁺ gates TRPM4 and TRPM5 channels, causing Na⁺ influx and K⁺ efflux that results in depolarization from baseline membrane potentials of –60 mV. PIP₂ also acts directly on TRPM4 and TRPM5 channels to maintain Ca²⁺ sensitivity (dotted arrow).

Figure 3. The effects of excess PIP₂ on inspiratory drive potentials

A, bar chart summarizing the effects of diC8-PIP₂ (diC8) on mean inspiratory drive potentials (±s.e.m.). Inspiratory drive potential amplitude and area are plotted as a per cent of control for the following conditions: 10 μm diC8 with Mg-ATP (dark grey bars) or diC8 without Mg-ATP (white bars). The same measure is shown for the effects of flufenamic acid (FFA, 100 μm) in the presence of diC8-PIP₂ (light grey bars). *Statistical significance at P < 0.05. B, representative traces showing the effects of Mg-ATP removal from the standard patch solution. Sequential traces represent perforated patch (pp), whole cell immediately after patch rupture (wc), and whole cell 30 min after patch rupture. Baseline membrane potential (V_m) was held at –60 mV. XII represents hypoglossal nerve motor output. Mg-ATP was excluded from all subsequent diC8 experiments in panels D–F. C, representative traces from a control experiment show drive potential stability and longevity under standard conditions in vitro. Standard patch solution was employed with Mg-ATP present. The drive potential did not change over 60 min. D, representative neuron showing diC8 enhancement of drive potential onset. Sequential traces show pp, wc and wc 5 min after diC8 dialysis. E, representative neuron showing diC8 prolongation of drive potentials and the effects of FFA. The sequence of traces are similar to D (above) with the additional FFA condition after 25 min of diC8 dialysis. Fa–c, the effects of diC8 and FFA on ectopic depolarizations in preBötC neurons. Fa, example of ectopic depolarizations observed immediately following patch rupture. Inset shows the superposition of ectopic depolarizations in control and diC8. Fb, the same cell 3 min after patch rupture. Fc, with diC8 still present, FFA was bath-applied and ectopic depolarizations disappeared entirely.

Figure 5. Excess PIP₂ augments drive potentials through an IP₃-independent mechanism

A, intracellular Xestospongin (Xes, 1 μm) attenuates inspiratory drive potentials by blocking IP₃ receptors. Perforated-patch (pp) and whole-cell (wc) control conditions are illustrated along with whole-cell Xes dialysis at steady state. B, typical data from an inspiratory neuron in response to co-application of Xes and diC8-PIP₂ (diC8). The inspiratory burst changed shape somewhat but neither amplitude nor area changed significantly. C, typical response to 10 μm intracellular PLC-resistant PIP₂. Again, neither the amplitude nor the area of the inspiratory burst changed significantly. D, bar chart plots the mean amplitude of inspiratory drive potentials (±s.e.m.) as per cent of control in the following conditions: diC8 alone (left white bar), Xes alone (right white bar), co-applied diC8 and Xes (dark grey bar), and PLC-resistant PIP₂ (light grey bar). Here ** denotes P < 0.01 and ns denotes ‘not significant’ at P ≫ 0.05.

Figure 2. The mRNA coding for TRPM4 and TRPM5 is expressed in the preBötC region

Total RNA was extracted from preBötC and positive control tissues and reverse transcribed. Amplified products of the expected sizes were obtained for TRPM4 (301 bp), TRPM5 (483 bp) and GADPH (452 bp). Negative control reactions were performed without reverse transcriptase and amplified nothing.

Figure 4. The effects of PIP₂ removal on inspiratory drive potentials

A, sequential traces show the response of a representative inspiratory neuron to intracellular 30 μg ml⁻¹ poly l-lysine (PLL) and FFA. Perforated patch (pp) and whole cell immediately following patch rupture (wc) represent controls. PLL reached steady state after 20 min of whole-cell dialysis. Then, FFA was applied; the illustrated trace was taken at 30 min. The bar graph plots the mean drive potential amplitude and area (±s.e.m.) as per cent of control in response to PLL applied alone (grey bars) and in combination with FFA (white bars). B, sequential traces show the effects of intracellular application of 50 μm wortmannin (WM) and FFA on a representative inspiratory neuron. The control trace is superimposed (in grey) on top of the WM trace for comparison. The bar graph plots the mean drive potential amplitude and area (±s.e.m.) as per cent of control after application of WM alone (grey bars) and co-application of WM and FFA (white bars). For both graphs, ** denote statistical significance at P < 0.01 compared with pp or wc control.