Role of parafacial nuclei in control of breathing in adult rats

Abstract

Contiguous brain regions associated with a given behavior are increasingly being divided into subregions associated with distinct aspects of that behavior. Using recently developed neuronal hyperpolarizing technologies, we functionally dissect the parafacial region in the medulla, which contains key elements of the central pattern generator for breathing that are important in central CO2-chemoreception and for gating active expiration. By transfecting different populations of neighboring neurons with allatostatin or HM4D Gi/o-coupled receptors, we analyzed the effect of their hyperpolarization on respiration in spontaneously breathing vagotomized urethane-anesthetized rats. We identify two functionally separate parafacial nuclei: ventral (pFV) and lateral (pFL). Disinhibition of the pFL with bicuculline and strychnine led to active expiration. Hyperpolarizing pFL neurons had no effect on breathing at rest, or changes in inspiratory activity induced by hypoxia and hypercapnia; however, hyperpolarizing pFL neurons attenuated active expiration when it was induced by hypercapnia, hypoxia, or disinhibition of the pFL. In contrast, hyperpolarizing pFV neurons affected breathing at rest by decreasing inspiratory-related activity, attenuating the hypoxia- and hypercapnia-induced increase in inspiratory activity, and when present, reducing expiratory-related abdominal activity. Together with previous observations, we conclude that the pFV provides a generic excitatory drive to breathe, even at rest, whereas the pFL is a conditional oscillator quiet at rest that, when activated, e.g., during exercise, drives active expiration.

Keywords: active expiration; control of breathing; expiratory oscillator; parafacial respiratory group; respiration; retrotrapezoid nucleus.

Figure 1.

Overall experimental design. Rats were transfected with: syn-HM₄DR-mCit (light yellow) in ventral parafacial region (pF_V:HM₄DR) or into lateral parafacial region (pF_L:HM₄DR), or synapsin-AlstR-GFP (dark green) into pF_V (pF_V:AlstR), or both pF_L:HM₄DR and pF_V:AlstR. All rats were subject to the same triad of experimental challenges, i.e., hypoxia, hypercapnia, and disinhibition of pF_L. For rats transfected with pF_L:HM₄DR, pF_V:AlstR, or pF_L:HM₄DR and pF_V:AlstR, these challenges were performed under control conditions (i–iii), following injections of Alst (light green) into pF_V (v–vii), and following application of CNO (dark yellow) to medullary surface (ix–xi). Rats transfected with pF_V:HM₄DR underwent the same triad of experimental challenges, i.e., hypoxia, hypercapnia, and disinhibition of pF_L. These challenges were performed under control conditions (iv, before CNO; xii, after CNO), and following application of CNO to medullary surface (viii). Data for different conditions were compared. Since Alst did not affect HM₄DR-transfected neurons and since CNO did not affect AlstR-transfected neurons, data for similar conditions were combined for analysis and comparisons. Thus we compared i + ii versus ix + x, iii versus xi, ii + iii versus vi + vii, i versus v, and iv + xii versus viii.

Figure 2.

Neuronal transfection in parafacial regions. A, Localization of injections into pF_V and pF_L. Ai, Transverse view of medulla at bregma −11.25 mm. Dashed squared blue boxes identify location of sections illustrating immunocytochemistry shown in B–D. Aii, Ventral view of medullary surface. Ai, Aii, Green circle shows location of AlstR injection sites for pF_V (Ci),yellow circle shows location of HM₄DR injection sites for pF_L (Bi) and pF_V (Di). Bi–Di, Micrographs of injection sites: neurons (blue) transfected with AlstR (in Ci) expressing GFP, or HM₄DR (in Bi, Di) expressing mCitrine, colocalized with NK1R (red). Bii–Dii, Expanded micrographs from merged figures in Bi–Di (dashed gray boxes): NeuN (blue), GFP, or mCitrine (green), and NK1R (red). Py, pyramidal tract; SP-5, spinal trigeminal tract; 7n, facial nucleus.

Figure 3.

Measurement of respiratory variables. Gray traces are raw data (not shown in Figs. 4–13). Black traces are integrated data, end-tidal CO₂, and frequency (shown in Figs. 4–13). Maximum and minimum values for each variable were measured from integrated traces (red dashed lines) and the differences, along with frequency (blue line + dots), were used to calculate f, T_I, T_E, V_T, GG_EMG, Dia_EMG, and Abd_EMG.

Figure 4.

Effect of CNO in rats with and without HM₄DRs. A, Integrated traces: gray arrows and vertical dashed lines indicate application of CNO in pF_L:HM₄DR rats (Ai) or in rats lacking HM₄DRs, i.e., pF_V:AlstR rats (Aii). B, Comparison of respiratory variables before and after CNO in pF_L:HM₄DR rats (Bi) and pF_V:AlstR rats (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or CNO group. C, Comparison of ratio changes between effects of CNO on pF_L:HM₄DR and pF_V:AlstR rats. Box-and-whisker plots show combined data, with data points from individual experiments. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in A. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 5.

Hyperpolarizing pF_L neurons significantly reduced effects of disinhibition of pF_L (B + S_pFL). A, Integrated traces from a single experiment: gray arrows and vertical dashed lines represent pipette placement for unilateral and bilateral B + S_pFL. Ai, Rest. Aii, During application of CNO to medullary surface (present for entire trace). B, Comparison of respiratory variables before and after B + S_pFL in pF_L:HM₄DR rats at rest (Bi) and in the presence of CNO (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or B + S_pFL group. C, Comparison between ratio changes induced by B + S_pFL in pF_L:HM₄DR rats at rest and in the presence of CNO. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 6.

Hyperpolarizing pF_L neurons reduced effects of hypercapnia (9% CO₂) on Abd_EMG only. A, Integrated traces from a single experiment: shaded area shows period of hypercapnia. Ai, Rest. Aii, During application of CNO to medullary surface (present for entire trace). B, Comparison of respiratory variables before and after hypercapnia in pF_L:HM₄DR rats at rest (Bi) and in the presence of CNO (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or 9% CO₂ group. C, Comparison between ratio changes induced by hypercapnia in pF_L:HM₄DR rats at rest and in the presence of CNO. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 7.

Hyperpolarizing pF_L neurons reduced effects of hypoxia (8% O₂) on Abd_EMG only. A, Integrated traces from a single experiment: shaded area shows period of hypoxia. Ai, Rest. Aii, During application of CNO to medullary surface (present for entire trace). B, Comparison of respiratory variables before and after hypoxia in pF_L:HM₄DR rats at rest (Bi) and in the presence of CNO (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or 8% O₂ group. C, Comparison between ratio changes induced by hypoxia in pF_L:HM₄DR rats at rest and in the presence of CNO. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 8.

pF_V provides facilitative drive to respiration at rest. A, Integrated traces from a single experiment: gray arrows and vertical dashed lines show the beginning of Alst injection in pF_V:AlstR rats (Ai), or in rats lacking AlstRs, i.e., pF_L:HM₄DR rats (Aii). B, Comparison of respiratory variables before and after Alst in pF_V:AlstR rats (Bi) and pF_L:HM₄DR rats (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or Alst group. C, Comparison of ratio changes between effects of Alst on pF_L:HM₄DR and pF_V:AlstR rats. Box-and-whisker plots show combined data, with data points from individual experiments. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in A. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 9.

Hyperpolarizing pF_V neurons reduced effects of disinhibition of pF_L (B + S_pFL) on Abd_EMG only. A, Integrated traces from a single experiment: gray arrows and vertical dashed lines represent pipette placement for unilateral and bilateral B + S_pFL. Ai, Rest. Aii, After Alst in pF_V (present for entire trace). B, Comparison of respiratory variables before and after B + S_pFL in pF_V:AlstR rats at rest (Bi) and in the presence of Alst (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or B + S_pFL group. C, Comparison between ratio changes induced by B + S_pFL in pF_V:AlstR rats at rest and in the presence of Alst. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 10.

Hyperpolarizing pF_V neurons reduced effects of hypercapnia (9% CO₂) on Abd_EMG and GG_EMG. A, Integrated traces from a single experiment: shaded area shows period of hypercapnia. Ai, Rest. Aii, After addition of Alst into pF_V (present for entire trace). B, Comparison of respiratory variables before and after hypercapnia in pF_V:AlstR rats at rest (Bi) and in the presence of Alst (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or 9% CO₂ group. C, Comparison between ratio changes induced by hypercapnia in pF_V:AlstR rats at rest and in the presence of Alst. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 11.

Hyperpolarizing pF_V neurons reduced effects of hypoxia (8% O₂) on Abd_EMG and GG_EMG. A, Integrated traces from a single experiment: shaded area shows period of hypoxia. Ai, Rest. Aii, After addition of Alst into pF_V (present for entire trace). B, Comparison of respiratory variables before and after hypoxia in pF_V:AlstR rats at rest (Bi) and in the presence of Alst (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or 8% O₂ group. C, Comparison between ratio changes induced by hypoxia in pF_V:AlstR rats at rest and in the presence of Alst. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 12.

Hyperpolarizing pF_V neurons during hypercapnia (9% CO₂) with HM₄DR only affects GG_EMG and Abd_EMG. A, Integrated traces from a single experiment: shaded area shows period of hypercapnia. Ai, Rest. Aii, During application of CNO to medullary surface (present for entire trace). B, Comparison of respiratory variables before and after hypercapnia in pF_V:HM₄DR rats at rest (Bi) and in the presence of CNO (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or 9% CO₂ group. C, Comparison between ratio changes induced by hypercapnia in pF_V:HM₄DR rats at rest and in presence of CNO. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005. ^#n = 7 for GG_EMG.

Figure 13.

Hyperpolarizing pF_V neurons during hypoxia (8%) with HM₄DR only affects GG_EMG and Abd_EMG. A, Integrated traces from a single experiment: shaded area shows period of hypoxia. Ai, Rest. Aii, During application of CNO to medullary surface (present for entire trace). B, Comparison of respiratory variables before and after hypoxia in pF_V:HM₄DR rats at rest (Bi) and in the presence of CNO (Bii). Lines connect data from individual experiments, and box-and-whisker plots show combined data. Data in Bi and Bii are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, GG_EMG, Dia_EMG, or Abd_EMG, regardless of whether it belonged to control or 8% O₂ group. C, Comparison between ratio changes induced by hypoxia in pF_V:HM₄DR rats at rest and in presence of CNO. Data in C are expressed as ratios of resting values, and red horizontal dashed line represents a ratio of 1. D, Table containing median, IQR, and p values, from data represented in B. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 14.

Schematic of minimal respiratory central pattern generator, which at its core consists of three essential components: (1) an inspiratory oscillator in preBötC that drives inspiration by exciting inspiratory premotor neuronal populations, e.g., rVRG and parahypoglossal region (pXII), and inhibits pF_L; (2) a (conditional) expiratory oscillator in pF_L that gates and drives expiration by exciting expiratory premotor neuronal populations, i.e., cVRG (Janczewski et al., 2002) and pXII, and assures alteration of phases by exciting neurons that inhibit preBötC, e.g., inhibitory neurons in either the preBötC or BötC; and (3) a source of tonic drive in pF_V that is responsive to CO₂/pH and integrates other sensory afferents affecting respiratory drive, via excitatory connections to preBötC, BötC, and respiratory premotor neurons, e.g., rVRG, cVRG, and pXII.