Fentanyl effects on respiratory neuron activity in the dorsolateral pons

Figures

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  Figure 1.. Respiratory motor output during fentanyl and naloxone (NLX) administration. Recordings from central vagus nerve (cVN), phrenic nerve (PN), and the dorsolateral pons were made using an in situ preparation of rat. A: continuous recording of (top to bottom) integrated instantaneous frequency of single dorsolateral pontine unit spiking activity (black, Unit Freq), raw signal from dorsolateral pontine extracellular recording (black, Extracell), integrated cVN activity (blue), and integrated PN activity (pink). Instantaneous respiratory rate [fictive breaths per minute (BPM)]) is shown below. Systemic application of fentanyl (300 nM) results in slowing of normal three-phase output, transition to low-amplitude, two-phase bursts, which then slowed resulting in sustained apnea. Systemic application of naloxone (1 μM) restores output in a similar but reversed manner to fentanyl-mediated decline. B: zoomed in view of epochs indicated in A. Avg10 is average of 10 breaths. Epochs are as follows: (1) baseline, (2) last three-phase breath in fentanyl (F3p), (3) first two-phase breath in fentanyl (F2p(f)), (4) last two-phase breath in fentanyl (F2p(l)), (5) Apnea, (6) last two-phase breath in…

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  Figure 2.. Discharge identity of dorsolateral pontine single units in the inspiratory class. Recordings from central vagus nerve (cVN, blue), phrenic nerve (PN, pink), and dorsolateral pontine units [raw data shown in gray (Extracell), sorted units shown in black (Unit)] were made using an in situ preparation of rat. Units were placed into subgroups depending on their pattern of activity over the respiratory cycle (A–E). Left column: example traces of a recorded unit with corresponding nerve output for each subgroup. Right column (histograms): average firing frequency per phase fraction bin for 10 consecutive breaths was plotted for individual units in each group. Each unit was assigned a number, which is indicated to the right of the histogram and is used consistently throughout the Figures. Inspiration (Insp) is indicated by gray shading. Expiration was duplicated to display phase transitions.

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  Figure 3.. Discharge identity of dorsolateral pontine single units in the expiratory class. Recordings from central vagus nerve (cVN, blue), phrenic nerve (PN, pink), and dorsolateral pontine units [raw data shown in gray (Extracell), sorted units shown in black (Unit)] were made using an in situ preparation of rat. Units were placed into subgroups depending on their pattern of activity over the respiratory cycle (A–E). Left column: example traces of a recorded unit with corresponding nerve output for each subgroup. Right column (histograms): average firing frequency per phase fraction bin for 10 consecutive breaths was plotted for individual units in each group. Each unit was assigned a number, which is indicated to the right of the histogram and is used consistently throughout the Figures. Inspiration (Insp) is indicated by gray shading. Expiration was duplicated to display phase transitions.

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  Figure 4.. Baseline firing frequency and location of dorsolateral pontine units. A: summary of firing frequency of dorsolateral pontine single units at baseline. Data are grouped by class (inspiratory or expiratory) (left) or inspiratory subgroup (right). Symbols represent the average peak firing frequency over 10 consecutive breaths for individual units. Line and error are means ± SE. **P < 0.01, ***P < 0.001 by unpaired t test (left) or one-way ANOVA and Tukey’s post hoc test (right). Symbols are keyed for subclass (see results). Symbol outline represents if the unit had activity during only respiration (respiratory, black) or had activity during respiration and “swallowing” (multifunctional, neon green). B: semi-schematic drawings of coronal slices through rat dorsolateral pons. Location of recorded units was identified post hoc in coronal slices (n = 42 rats). A7, noradrenergic neurons; E-dec, expiratory decrementing; E-plat, expiratory plateau; EIPS, expiratory/inspiratory phase-spanning; I-aug, inspiratory augmenting; I-dec, inspiratory decrementing; I-plat, inspiratory plateau; IC, inferior colliculus; IEPS, inspiratory/expiratory phase-spanning; KF, Kölliker–Fuse; LPB, lateral…

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  Figure 5.. Phasic activity of dorsolateral pontine single units during baseline, fentanyl-induced apnea, and naloxone recovery. A–C, left: phase fraction two-dimensional (2-D) histogram (heatmap) displays activity of individual recorded units (n = 47) during baseline (A, epoch “1avg10” from Fig. 1), fentanyl-induced apnea (B, epoch 5 from Fig. 1), and naloxone recovery (C, epoch “8avg10” from Fig. 1). Each row represents a single unit from an individual experiment (one unit/experiment). Rows were divided into the inspiratory (top; unit Nos. 1–23) and expiratory (bottom; unit Nos. 24–47) classes. Row number corresponds to unit No. in Figs. 2 and 3. Inspiration was divided into 20 consecutive fractional bins (1–20) and expiration was divided into 40 consecutive fractional bins (21–60). Only the expiratory phase was sampled during apnea (B). Z-axis scale (bottom left) indicates average firing frequency per bin for 10 consecutive breaths. A–C, right: average firing frequency per bin of all units in the inspiratory class (top) or expiratory class (bottom). Line and error are means ± SE. Expiration was duplicated to display phase transitions. D: baseline-subtracted difference plot for firing…

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  Figure 6.. Firing frequency of dorsolateral pontine units during baseline, fentanyl-induced apnea, and naloxone recovery. Summary of single unit peak firing frequency at baseline, during fentanyl-induced apnea, and after fentanyl recovery. Peak firing frequency at baseline and after recovery with naloxone was averaged from 10 consecutive breaths. Peak firing frequency at apnea was determined during the duration of apnea (≥30 s). Units were placed into subgroups based on discharge identity. preinspiratory/inspiratory (pre-I/I; n = 6, red square), inspiratory augmenting (I-aug; n = 9, red circle), inspiratory plateau (I-plat; n = 5, red triangle), inspiratory/expiratory phase-S (IEPS; n = 9, yellow circle), expiratory plateau (E-plat; n = 6, blue circle), and expiratory decrementing (E-dec; n = 5, blue star) were statistically compared. Inspiratory decrementing (I-dec; n = 1, red star), expiratory/inspiratory phase-spanning (EIPS) (n = 2 orange star), tonic respiratory modulated expressing decreased activity during postinspiration (TRM1; n = 2, blue green circle), and tonic respiratory modulated expressing an increase in activity at inspiratory/expiratory phase transition (TRM2; n = 2,…

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  Figure 7.. Activity of inspiratory units during fentanyl-mediated decline and naloxone-mediated recovery. A: phase fraction histograms of inspiratory class single unit activity from individual experiments (one unit/row, left column), or average activity of all inspiratory units (right column) at transitional epochs during fentanyl-mediated decline and naloxone-mediated recovery. Line and error are means ± SE. As depicted in Fig. 1. epochs included: (1) Baseline, (2, F3p) the last three-phase breath during fentanyl, [3, F2p(f)] the first two-phase breath during fentanyl, [4, F2p(l)] the last two-phase breath before apnea, (5) fentanyl-induced apnea, (6, NLX2p) the last two-phase breath during naloxone, [7, NLX3p(f)] the first three-phase breath during naloxone, and (8, NLX) a fully recovered breath post naloxone administration. Z-axis scale for left side of A is shown at top of right column. B: consecutive heatmaps were subtracted. Difference heatmaps display differences within individual experiments on each row (B, left) and difference histograms show differences in average activity (B, right). B, right: line and error are means ± SE. Bins with significant differences from zero are shown in…

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  Figure 8.. Activity of expiratory units during fentanyl-mediated decline and naloxone-mediated recovery. A: phase fraction two-dimensional (2-D) histogram (heatmap) of expiratory class single unit activity from individual experiments (one unit/row, left column) or average activity of all expiratory units (right column) at transitional epochs during fentanyl-mediated decline and naloxone-mediated recovery. Line and error are means ± SE. Z-axis scale for A (left) is at the top of right column. As depicted in Fig. 1. epochs included: (1) Baseline, (2, F3p) the last three-phase breath during fentanyl, [3, F2p(f)] the first two-phase breath during fentanyl, [4, F2p(l)] the last two-phase breath before apnea, (5) fentanyl-induced apnea, (6, NLX2p) the last two-phase breath during naloxone, [7, NLX3p(f)] the first three-phase breath during naloxone, and (8, NLX) a fully recovered breath post naloxone administration. B: consecutive heatmaps were subtracted. Difference heatmaps display differences with individual experiments on each row (B, left) and difference histograms show differences in average activity (B, right). B, right: line and error are means ± SE. Bins with significant differences from…

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  Figure 9.. Activity of expiratory class subgroups during fentanyl-mediated decline and naloxone-mediated recovery. The expiratory class was divided into two groups: inspiratory/expiratory phase-spanning (IEPS) (A, n = 7) and expiratory decrementing (E-dec)/ expiratory plateau (E-plat) (B, n = 7; 3 E-dec, 4 E-plat). As depicted in Fig. 1, epochs included: (1) Baseline, (2, F3p) the last three-phase breath during fentanyl, [3, F2p(f)] the first two-phase breath during fentanyl, [4, F2p(l)] the last two-phase breath before apnea, (5) fentanyl-induced apnea, (6, NLX2p) the last two-phase breath during naloxone, [7, NLX3p(f)] the first thre-phase breath during naloxone, and (8, NLX) a fully recovered breath post naloxone administration.

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  Figure 10.. Swallow-associated dorsolateral pontine neurons. Recordings from central vagus nerve (cVN), phrenic nerve (PN), and single unit activity in the dorsolateral pons were made using an in situ preparation of rat. Example traces for three experiments demonstrating dorsolateral pontine neurons involved with fictive breathing and swallowing are shown at baseline (left) and ∼30 s after perfusion of fentanyl (300 nM) (right). Dorsolateral pontine inspiratory (A), expiratory decrementing (B), and inspiratory/expiratory phase-spanning (IEPS, C) respiratory patterned neurons burst with spontaneous fictive swallows (orange highlight) at baseline. After fentanyl perfusion (300 nM), the IEPS neuron (C) ceases to fire. At the same time, the inspiratory neuron (A) and expiratory neuron (B) are still firing. Note in A, there is decreased firing of the inspiratory neuron during the swallow in fentanyl compared to the swallow at baseline.