Inspiratory-phase short time scale synchrony in the brainstem slice is generated downstream of the pre-Bötzinger complex

Abstract

Respiratory neurons are synchronized on a long time scale to generate inspiratory and expiratory-phase activities that are critical for respiration. Long time scale synchrony within the respiratory network occurs on a time scale of more than hundreds of milliseconds to seconds. During inspiration, neurons are synchronized on a short time scale to produce synchronous oscillations, which shape the pattern of inspiratory motor output. This latter form of synchrony within the respiratory network spans a shorter time range of tens of milliseconds. In the neonatal mouse rhythmically active medullary slice preparation, we recorded bilateral inspiratory activity from hypoglossal (XII) rootlets to study where in the slice synchronous oscillations are generated. Based on previous work that proposed the origin of these oscillations, we tested the pre-Bötzinger complex (PreBötC) and the XII motor nucleus. Unilateral excitation of the PreBötC, via local application of a perfusate containing high K(+), increased mean inspiratory burst frequency bilaterally (296+/-66%; n=10, P<0.01), but had no effect on the relative power of oscillations. In contrast, unilateral excitation of the XII nucleus increased both mean peak integrated activity bilaterally (ipsilateral: 41+/-10%, P<0.01; contralateral: 17+/-7%; P<0.05, n=10) and oscillation power in the ipsilateral (50+/-17%, n=7, P<0.05), but not in the contralateral rootlet. Cross-correlation analysis of control inspiratory activity recorded from the left and right XII rootlets produced cross-correlation histograms with significant peaks centered around a time lag of zero and showed no subsidiary harmonic peaks. Coherence analysis of left and right XII rootlet recordings demonstrated that oscillations are only weakly coherent. Together, the findings from local application experiments and cross-correlation and coherence analyses indicate that short time scale synchronous oscillations recorded in the slice are likely generated in or immediately upstream of the XII motor nucleus.

Figure 1

Image of local unilateral perfusion. In this example, ACSF containing fast green was locally and unilaterally perfused onto the XII nucleus of a rhythmically active medullary slice preparation. Perfusion region is circumscribed to a zone approximately 0.5 mm in diameter as revealed by fast green labeled region over the left XII nucleus. Asterisks are over epoxy beads on a nylon thread that help to limit the labeled perfusion region. Arrows indicate the local perfusion pipette and the local uptake pipette.

Figure 2

Local unilateral perfusion of high K⁺ to the PBC bilaterally increases inspiratory burst frequency but not peak integrated activity or inspiratory burst duration. A. Representive raw (XII) and rectified and integrated (∫XII) hypoglossal rootlet recordings from the ipsilateral (Ipsi) and contralateral (Contra) hypoglossal rootlets in the control condition and following local unilateral perfusion of high K⁺ (80 mM). A and B. Local perfusion of high K⁺ to the unilateral PBC bilaterally increases inspiratory burst frequency. C and D. Unilateral excitation of PBC did not significantly affect peak integrated activity or inspiratory burst duration bilaterally. **P < 0.01; paired t-test v.s. control

Figure 3

Local unilateral perfusion of high K⁺ to the XII nucleus bilaterally increases peak integrated activity but not inspiratory burst frequency or inspiratory burst duration. A. Representive raw (XII) and rectified and integrated (∫XII) hypoglossal rootlet recordings from the Ipsi and Contra hypoglossal rootlets in the control condition and following local unilateral perfusion of high K⁺ (20 mM). A and B. Local perfusion of high K⁺ to the unilateral XII nucleus did not significantly affect inspiratory burst frequency measured bilaterally. C. Unilateral excitation of the XII nucleus increased peak integrated activity in the IPSI rootlet and, to a lesser extent, in the CONTRA rootlet. D. There was no significant effect on mean inspiratory burst duration measured bilaterally. *P < 0.05; **P < 0.01; paired t-test v.s. control

Figure 4

High K⁺ increases oscillation power when unilaterally applied to the XII nucleus. A and B. Representative raw filtered (1–200 Hz bandpass) inspiratory bursts recorded from the IPSI (A) and CONTRA (B) XII rootlet. The top traces are inspiratory bursts recorded in the control condition. The bottom traces are inspiratory bursts recorded following local perfusion of 20 mM K ⁺ to the IPSI XII nucleus. Same time scale applies to all inspiratory bursts. C. Ipsilateral average relative power spectra in the control condition and following local perfusion of 20 mM K ⁺ to the IPSI XII nucleus. Unilateral excitation of the XII nucleus increases oscillation power within the bin containing the dominant peak (36–46 Hz). D. Contralateral average relative power spectra in the control condition and following local perfusion of 20 mM K ⁺ to the CONTRA XII nucleus. Unilateral excitation of the XII nucleus increases oscillation power within the bin containing the dominant peak (29–39 Hz). When we averaged data for the overall population of experiments in which the XII nucleus was unilaterally excited, this increase in oscillation power was not statistically significant. The bins containing the dominant peak are marked by vertical dotted lines in the IPSI and CONTRA average relative power spectra.

Figure 5

Summary of the effects of unilateral excitation of the PBC or the XII nucleus on the relative power of oscillations. Local perfusion of ACSF containing high K⁺ (60 or 80 mM) to the unilateral PBC had no signifiicant effect on oscillation power bilaterally. Local perfusion of ACSF containing high K⁺ (20 mM) to the unilateral XII nucleus increased oscillation power in the IPSI, but had no significant effect on the oscillation power recorded from the CONTRA XII rootlet.

Figure 6

Crosscorrelation histograms (CCHs) of left and right hypoglossal rootlet activity produce three types of peaks. Those three types of peaks are (1) narrow and (2) broad central peaks and (3) a peak in the spike probability that gradually decreases and is not distinguishable from the baseline spike probability. The representative CCH in (A) displays a narrow peak that occurs at a time lag of 0 ms and has a half-width of 6 ms. The representative CCH in (B) displays a broad peak that occurs at a time lag of −2 ms and has a half-width of 22 ms. The representative CCH in (C) displays a peak that occurs at a time lag of 4 ms and is not distinguishable from the baseline. None of the CCHs exhibit harmonic peaks. For all CCHs, the bin width equals 2 ms. Dashed vertical line marks zero time lag.

Figure 7

Bilaterally recorded XII rootlet activity is weakly coherent. A and B. Representative raw filtered (1–200 Hz bandpass) inspiratory bursts recorded from the IPSI (A) and CONTRA (B) XII rootlet following unilateral excitation of the XII nucleus. C and D. IPSI and CONTRA average absolute power spectra each computed from 12 inspiratory bursts recorded following unilateral excitation of the XII nucleus. Dashed vertical lines mark the location of the dominant peaks in the IPSI and CONTRA power spectra at 46 and 38 Hz, respectively. E. Coherence plot computed from the IPSI and CONTRA average absolute power spectra in C and D. The dashed horizontal line marks the upper 95% confidence limit at 0.19. 46 and 38 Hz are marked by the dashed vertical lines. Although there are peaks in the IPSI (C) and CONTRA (D) average absolute power spectra at 46 and 38 Hz, respectively, the oscillations are not coherent at these frequencies. However, the small peaks that rise above the confidence limit between 22–100 Hz demonstrate that the left and right XII rootlet discharges are weakly coherent.