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Research ArticleConfirmation, Integrative Systems

Developmental Nicotine Exposure Alters Synaptic Input to Hypoglossal Motoneurons and Is Associated with Altered Function of Upper Airway Muscles

Lila Buls Wollman, Jordan Clarke, Claire M. DeLucia, Richard B. Levine and Ralph F. Fregosi
eNeuro 11 November 2019, 6 (6) ENEURO.0299-19.2019; DOI: https://doi.org/10.1523/ENEURO.0299-19.2019
Lila Buls Wollman
Department of Physiology, The University of Arizona, Tucson, AZ 85724
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Jordan Clarke
Department of Physiology, The University of Arizona, Tucson, AZ 85724
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Claire M. DeLucia
Department of Physiology, The University of Arizona, Tucson, AZ 85724
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Richard B. Levine
Department of Physiology, The University of Arizona, Tucson, AZ 85724Department of Neuroscience, The University of Arizona, Tucson, AZ 85724
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Ralph F. Fregosi
Department of Physiology, The University of Arizona, Tucson, AZ 85724Department of Neuroscience, The University of Arizona, Tucson, AZ 85724
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  • Figure 1.
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    Figure 1.

    In vivo experimental model and EMG response to nasal occlusion. A, Schematic rendition of the in vivo experimental preparation, which combines head-out plethysmography and GG EMG recordings in lightly anesthetized neonatal rats, as described in Materials and Methods. B, Example trace showing diaphragm and GG EMG and integrated EMG along with volume and flow traces obtained from the plethysmograph. After 5 min of uninterrupted baseline recordings, a 10- to 15-s nasal occlusion was administered (rectangle). C, GG EMG amplitude during the nasal occlusion. The duration of the nasal occlusion was normalized by dividing the total duration of each occlusion into equal 20% time bins. EMG amplitude was normalized as a percentage of the largest burst recorded (see Materials and Methods). All animals in both groups showed increased GG burst amplitude as the nasal occlusion progressed. Post hoc analysis following two-way ANOVA revealed that DNE animals had a significantly blunted amplitude response compared to control at the 40%, 60%, and 80% time bins; *p < 0.05, **p < 0.01, ***p < 0.001.

  • Figure 2.
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    Figure 2.

    Elapsed time between the onset of nasal occlusion (dashed line) to first discernible GG muscle EMG burst, defined as onset latency. A, B Recordings from representative control (A) and (B) DNE pups, as indicated. The length of the solid line under the EMG tracing represents the latency, which is prolonged in the DNE animal following the onset of nasal occlusion. C, Onset latency in nine control and DNE pups. The horizontal lines represent the mean value. An unpaired t test revealed a significant difference between the groups (p = 0.0018).

  • Figure 3.
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    Figure 3.

    Failure to recover from nasal occlusion. A, An example tracing of one of the 12 animals that exhibited continuous breathing difficulties following nasal occlusion, as explained in Results. B, The number of animals in each treatment group that either recovered from nasal occlusion or failed to recover. Note that of the 12 animals that never recovered, nine were from the DNE group, which is a significant difference by χ2 analysis (p = 0.0290).

  • Figure 4.
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    Figure 4.

    Example traces of AMPA receptor-mediated sEPSCs recorded from XIIMNs. A, B, Representative traces of pharmacologically isolated sEPSCs from a control animal (A) and a DNE animal (B), both studied on P4. Each trace shows the entire 3-min recording period at baseline (upper trace in each panel) and during acute nicotine application (bottom trace in each panel). Inset panels are an expanded view, showing 10 s of the recording at the end of each trace. Note that the size and frequency of events at baseline is similar in the control and DNE animals. In contrast, with acute nicotine challenge (bottom trace in each panel) the DNE cell shows a decrease in sEPSC frequency, whereas there is a modest increase in the control cell.

  • Figure 5.
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    Figure 5.

    Individual and average IEI of sEPSCs and mEPSCs at baseline and during acute nicotine challenge. A, Individual and average IEI of AMPA receptor-mediated sEPSCs under baseline conditions and following acute nicotine challenge. Note that in control cells (circles, left of the vertical line) there was a slight although non-significant decline in IEI (i.e., an increase in frequency) on both P1–P2 (filled circles) and P3–P5 (open circles). The IEI in response to an acute nicotine challenge in cells from DNE animals was age-dependent (squares, right of the vertical line). Note that on P1–P2, nicotine challenge decreased the IEI, although as in controls this trend was not significant. Surprisingly, on P3–P5 acute nicotine challenge significantly increased the IEI (p = 0.0021). Moreover, the mean value for IEI in P3–P5 DNE cells during acute nicotine challenge is significantly different from corresponding data in P1–P2 DNE cells (p = 0.0001), and in P3–P5 cells from control animals. B, Individual and average IEI of AMPA receptor-mediated mEPSCs under baseline conditions and following acute nicotine challenge. Note that in the P1–P2 group, both control and DNE cells show a trend toward a decrease in IEI with acute nicotine challenge; however, this was not significant. Analysis of average IEI from control and DNE neurons at P3–P5 shows no differences at baseline and no change in frequency with acute nicotine challenge.

  • Figure 6.
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    Figure 6.

    DNE alters the modulation of glutamatergic sEPSCs in response to an acute nicotine challenge but only in cells from pups aged P3–P5. Cumulative probability distributions of glutamatergic sEPSC IEIs in control and DNE cells, at baseline and during acute nicotine challenge. At P1–P2 (A, B), acute nicotine challenge with 0.5 μM nicotine (black dashed lines) caused a left shift, toward shorter IEIs, of glutamatergic sEPSCs in both control and DNE cells [control, p = 0.049 (A) DNE, p < 0.0001 (B)]. In cells from P3–P5 animals (C, D), acute nicotine challenge caused a left shift, toward shorter IEIs in control cells (p < 0.0001; C), but the distribution shifted to the right, toward longer IEIs in the DNE cells (p = 0.012; D). Arrows indicate the direction of the shift with acute nicotine challenge and indicates significant differences with K–S test (see Materials and Methods). Dotted gray lines indicate the 95% confidence intervals of each curve.

  • Figure 7.
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    Figure 7.

    Influence of DNE on AMPA receptor-mediated mEPSCs recorded from XIIMNs. A, B, Representative traces of pharmacologically isolated mEPSCs from a control animal (A) and a DNE animal (B) studied on P4. Each trace shows the entire 3 min of recording at baseline (top trace) and during acute nicotine application (bottom trace). Inset panels show an expanded view of 10 s at the end of each trace, as in Figure 4. Note that the size and frequency of events is similar in the control and DNE cell at baseline. However, during acute nicotine challenge, mEPSC frequency increased in control cells but not in DNE cells.

  • Figure 8.
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    Figure 8.

    Cumulative probability distributions of glutamatergic mEPSC IEIs in control and DNE cells, at baseline and during acute nicotine challenge. In cells from animals aged P1–P2 (A, B), acute nicotine challenge with 0.5 μM nicotine (black dashed lines) caused a left shift, toward shorter IEIs, of glutamatergic mEPSCs in both control (p < 0.0001; A) and DNE cells (p < 0.0001; B). In cells from animals aged P3–P5 (C, D), acute nicotine challenge caused a significant left shift of glutamatergic mEPSCs in control cells (p = 0.004; C), but there was no change in the distribution of IEIs in DNE cells (p = 0.066; D). Arrows indicate the direction of the shift with acute nicotine challenge and indicates significant differences with K–S test (see Materials and Methods). Dotted gray lines indicate the 95% confidence intervals of each curve.

  • Figure 9.
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    Figure 9.

    Activation of postsynaptic AMPA receptors in control cells and DNE cells. A, Representative trace of the AMPA receptor-mediated inward current in XIIMNs from a control animal at P1. B, Individual values for the peak inward current in response to bath application of AMPA. Mean values within each treatment group are indicated by the horizontal lines. There were no differences in the magnitude of the postsynaptic inward current either within or between treatment groups.

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    Figure 10.

    Influence of DNE on diaphragm muscle fatigue and neuromuscular transmission failure (NTF) during repetitive muscle or phrenic nerve stimulation in diaphragm phrenic nerve-muscle strips. A, Representative force recording over a 5-min period of direct muscle stimulation. Fatigue was quantified as the % of the maximum force remaining at the end of the 5-min stimulation period (see Materials and Methods). B, Muscle twitch produced by a single stimulus pulse, and an expanded view of the muscle force produced by a 330-ms train of stimulation pulses, as described in Materials and Methods. An identical protocol was used when phrenic nerve stimulation was used to assess the magnitude of force decline. C, Representative force recording during 5 min of repeated phrenic nerve stimulation with superimposed direct muscle stimulation every 15 s. NTF was calculated as: the percentage force declines in each control and DNE preparation subjected to either muscle (D) or phrenic nerve (E) stimulation. F, Percentage neuromuscular transmission failure in control and DNE preparations. Horizontal lines in D–F indicate the mean. There were no significant differences in the percentage force loss between control and DNE preparations with either muscle or nerve stimulation, or the force loss due to neuromuscular transmission failure.

Tables

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    Table 1.

    Statistical tests and significance threshold used for each experiment in each experimental series

    ExperimentStatistical test, post hocSignificance threshold
    In vivo series
        1. Changes to EMG during nasal occlusionTwo-way mixed-model ANOVA with Tukey’s post hoc analysisp < 0.05
        2. Differences in EMG onset latencyUnpaired Student’s t testp < 0.05
        3. Autoresuscitationχ2 analysisp < 0.05
    In vitro series A
        1. Differences in baseline parametersTwo-way mixed model ANOVA with Tukey’s post hoc analysisp < 0.05
        2. Differences in IEI and amplitude of EPSCsK–S test of cumulative probability distributions, and three-way mixed model ANOVA with the Holm–Sidak post hoc analysisp < 0.05
        3. Differences in peak whole cell currentUnpaired Student’s t testp < 0.05
    In vitro series B
        4. Differences in nerve stimulation, muscle stimulation, and estimates of neuromuscular transmission failureUnpaired Student’s t testp < 0.05
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    Table 2.

    List of drug cocktails used to isolate AMPA receptor-mediated glutamatergic EPSCs and postsynaptic AMPA receptors, with the number of cells used in each condition

    Experiments and drugs usedAgeNumber of cells
    (control:DNE)
    Glutamate sEPSCs
    50 μM AP-5, 10 μM bicuculline, 0.4 μM strychnine
    + 0.5 μM nicotine
    P1–P2
    P3–P5
    6:6
    6:6
    Glutamate mEPSCs
    50 μM AP-5, 10 μM bicuculline, 0.4 μM strychnine, 1 μM TTX
    + 0.5 μM nicotine
    P1–P2
    P3–P5
    6:6
    6:6
    Postsynaptic receptors
    50 μM AP-5, 10 μM bicuculline, 0.4 μM strychnine, 1 μM TTX
    + 2.5 μM AMPA
    P1–P2
    P3–P5
    6:6
    6:6
    • The number of cells studied in each experiment are also shown.

    • View popup
    Table 3.

    Number of sEPSCs and mEPSCs recorded per neuron at baseline and during acute nicotine application

    ControlDNE
    sEPSC
    P1–P2BaselineAcute nicotine    65 ± 2374 ± 59    91 ± 38187 ± 105
    P3–P4BaselineAcute nicotine    145 ± 107211 ± 211    116 ± 8481 ± 60
    mESPCs
    P1–P2BaselineAcute nicotine    28 ± 1438 ± 20.8    28 ± 2261 ± 39
    P3–P4BaselineAcute nicotine    18 ± 1331 ± 15    41 ± 3833 ± 27
    • Values are mean ± SD.

    • View popup
    Table 4.

    Age, weight, resting membrane potential (mV), and input resistance (Rin) of XIIMNs from control and DNE animals

    ControlDNEp valuen (control:DNE)
    Age (d)
        P1–P2 P3–P51.7 ± 0.23.4 ± 0.11.4 ± 0.13.7 ± 0.2p = n/sp = n/s18:1818:18
    Weight (g)
        P1–P2 P3–P57.72 ± 0.3110.53 ± 0.44***7.4 ± 0.110.8 ± 0.3***p = n/sp = n/s18:1818:18
    Vm
        P1–P2 P3–P5–49 ± 3–48 ± 2–48 ± 2–47 ± 2p = n/sp = n/s18:1818:18
    Rin (MΩ)
        P1–P2 P3–P5221 ± 49185 ± 41184 ± 23189 ± 42p = n/sp = n/s4:44:4
    • View popup
    Table 5.

    Mean values for amplitude of glutamatergic sEPSCs, mEPSCs, and mEPSC rise time at baseline and during acute nicotine challenge

    BaselineAcute nicotine challenge
    sEPSC amplitude (pA)Control:
    P1–P2P3–P5DNE:P1–P2P3–P5–15.2 ± 2.7–15.5 ± 2.3 –18.5 ± 7.4–16.4 ± 2.8–16.0 ± 3.2–15.7 ± 3.9 –18.3 ± 8.9–14.8 ± 2.9
    mEPSC amplitude (pA)Control:
    P1–P2P3–P5DNE:P1–P2P3–P5–16.2 ± 2.6–14.9 ± 3.0 –16.0 ± 2.9–17.5 ± 1.9–14.6 ± 2.6–16.3 ± 3.7 –15.6 ± 3.8–17.4 ± 1.9
    mEPSC rise time (ms)Control:
    P1–P2P3–P5DNE:P1–P2P3–P5    2.1 ± 0.2 2.0 ± 0.1  2.1 ± 0.2 1.8 ± 0.1    2.1 ± 0.2 2.2 ± 0.1  2.0 ± 0.1 2.0 ± 0.1
    • There were no significant differences in any of these variables.

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Developmental Nicotine Exposure Alters Synaptic Input to Hypoglossal Motoneurons and Is Associated with Altered Function of Upper Airway Muscles
Lila Buls Wollman, Jordan Clarke, Claire M. DeLucia, Richard B. Levine, Ralph F. Fregosi
eNeuro 11 November 2019, 6 (6) ENEURO.0299-19.2019; DOI: 10.1523/ENEURO.0299-19.2019

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Developmental Nicotine Exposure Alters Synaptic Input to Hypoglossal Motoneurons and Is Associated with Altered Function of Upper Airway Muscles
Lila Buls Wollman, Jordan Clarke, Claire M. DeLucia, Richard B. Levine, Ralph F. Fregosi
eNeuro 11 November 2019, 6 (6) ENEURO.0299-19.2019; DOI: 10.1523/ENEURO.0299-19.2019
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Keywords

  • motor neuron
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