Perturbations of Respiratory Rhythm and Pattern by Disrupting Synaptic Inhibition within Pre-Bötzinger and Bötzinger Complexes

Ventral view of the adult rat medulla and histology illustrating targeted sites for pharmacology experiments in vivo. A, Photograph of adult rat brainstem ventral surface as exposed in the in vivo experimental preparations with an overview of targeted locations (caudal to facial motor nucleus, VII) of BötC and pre-BötC as routinely identified by electrophysiological mapping of neuronal activity profiles in the present experiments. B, Photomicrograph of ventral medullary surface and bilaterally arranged pipettes (blue dye-filled for visualization) as typically configured for near perpendicular penetrations of the ventral surface for simultaneous microinjections. C, D, Confocal microscopic images of parasagittal histologic sections (50 µm thick) showing, respectively, examples of targeted sites for microinjection of inhibitory antagonists in the pre-BötC ventral to the semicompact subdivision of nucleus ambiguous (NAsc), and in the BötC ventral to the compact subdivision of nucleus ambiguus (NAc). Targeted sites are marked by microinjected solution of fluorescent microspheres (green). NA and VII motoneurons are immunolabeled by ChAT antibody (red). Rostrocaudal spatial extent of the pre-BötC and BotC compartments are indicated. vs, ventral surface.

Confocal microscopic images of histologic sections illustrating post hoc validation of microinjection sites marked by fluorescent microspheres (green) in the pre-BötC or BötC regions in fixed sections from anesthetized adult rat in vivo preparations and juvenile rat in situ brainstem–spinal cord preparations. A, B, Coronal sections (30 µm thick) of fixed tissue at the level of pre-BötC (A) and BötC (B) from in vivo preparations. C, D, Coronal sections (30 µm) of fixed tissue at pre-BötC (C) and BötC (D) levels from juvenile rat in situ brainstem–spinal cord preparations. Subdivisions of nucleus ambiguus (NAc, compact subdivision; NAsc, semi-compact subdivision), labeled with ChAT antibody (red), provide regional landmarks for pre-BötC (ventral to NAsc) and BötC (ventral to NAc) levels of the medulla. Each image is taken from serial coronal histologic sections obtained from experiments targeting these regions. Labeling with microinjected solution of fluorescent microbeads, with varying extent of local spread, indicates the approximate center of the drug microinjection sites in these examples. vs, ventral surface.

Characteristic profiles of extracellularly recorded neuronal population activity and examples of perturbations of inspiratory motor output activity and blood pressure produced by pharmacological excitation of neurons within the pre-BötC or BötC regions in the adult rat in vivo. A, A typical example of pre-inspiratory/inspiratory (pre-I/I) population activity used for the identification of pre-BötC. B, An example of post-I and aug-E population activity (post-I/aug-E, simultaneously recorded in this example) used for the identification of BötC. In both A and B, the raw recording from the phrenic nerve (PN) and PN integrated activity (∫PN) are shown at the bottom. C, A 500 ms duration microinjection of L-Glu (Glutamate) in the pre-BötC produced an increase in the ∫PN burst frequency (see the trace for integrated PN activity, ∫PN), and a transient decrease in the ABP. The respiratory frequency (f_R; bottom trace, green) increased primarily due to reduction in expiratory phase duration (T_E trace, blue) at a relatively unchanged inspiratory duration (T_I trace, red). D, A microinjection of L-Glu in the BötC caused a rapid suppression of PN activity (see traces for ∫PN and inspiratory, T_I, and expiratory, T_E, durations) accompanied by an increase of ABP. In C and D, L-Glu (10 mm, 5 nl) was microinjected bilaterally during the brief (500 ms) pulse; the moments of injections are indicated by brown arrows. Traces for T_I, T_E, and f_R, represent corresponding running time intervals of these parameters before, during, and recovery from L-Glu microinjection.

Perturbations of respiratory activity by pharmacologically disrupting GABA_Aergic and glycinergic inhibition in the pre-BötC of anesthetized adult rat in vivo. A, Simultaneous bilateral microinjections of gabazine and strychnine (both 250 µm delivered by slow microinjections of 110 nl during the time interval indicated by the brown bar at the top and blue rectangle) caused an increase of respiratory frequency and a reduction in the integrated phrenic nerve activity amplitude (∫PN; black trace, top). The increase of respiratory frequency (f_R; green trace, bottom) was mainly due to reductions of expiratory phase duration (T_E, blue trace) accompanied by only a small increase of inspiratory phase duration (T_I, red trace). B–E, Group mean time series showing developing changes of normalized T_I (B, red curve), T_E (C, blue curve), f_R (D, green curve), and ∫PN amp (E, magenta curve) computed over the time window shown from the start (time = 0) of microinjection. Data were computed from ∫PN for this representative experimental group (n = 6). Solid colored curves are group mean time and mean normalized parameter values; gray shaded bands are ±1 SEM for the mean parameter values. Endpoints shown are mean time and normalized parameter values ±1 SEM for both at the maximal perturbation for the injection periods used.

Perturbations of respiratory activity by pharmacologically disrupting GABA_Aergic and glycinergic inhibition in the pre-BötC of juvenile rat perfused brainstem–spinal cord in situ. A, Example of experimental recordings illustrating perturbations of integrated PN (∫PN) and cVN (∫cVN) activities by simultaneous bilateral microinjections of gabazine and strychnine (both 30 µm; injection period indicated by the brown bar at the top and blue rectangle), which caused an increase of respiratory frequency (f_R) due to a reduction in T_E (blue) without significant changes in T_I, and a reduction in amplitude of ∫PN and ∫cVN. B, Example of perturbations where the ∫PN activity progressed to tonic activity, as indicated by the upward shift of the ∫PN signal baseline. Analysis of respiratory parameters in this case was performed up to the time point indicated by the vertical dot-dashed line. The progressive reduction in ∫cVN amplitude in both examples reflects in part a reduction and eventual loss of post-I activity (Fig. 10 shows a more detailed analysis).

Group data summarizing changes of respiratory activity parameters by pharmacologically disrupting GABA_Aergic and glycinergic inhibition in the pre-BötC of juvenile rat perfused brainstem–spinal cord in situ. A–D, Mean time series from the start of microinjection showing developing changes of normalized T_I (A, red curve), T_E (B, blue curve), respiratory frequency (f_R; C, green curve), and integrated PN discharge amplitude (D, amp, magenta curve), which were computed from integrated phrenic nerve activity for a representative experimental group (n = 6). Solid colored curves are group mean time and normalized parameter values; gray bands are ±1 SEM for the mean normalized parameter values as in Figure 4B–E .

Perturbations of respiratory activity by pharmacologically disrupting of GABA_Aergic and glycinergic inhibition in the BötC of adult rat in vivo. A, Gabazine and strychnine cocktail (110 nl, 250 µm) slowly injected during time period indicated (by the brown bar at the top and blue rectangle), progressively reduced respiratory frequency leading to transient apnea in this example (∫PN, black trace, top). Changes in the inspiratory (T_I, red) and expiratory (T_E, blue) phase durations and respiratory frequency (f_R) are shown at the bottom; f_R was reduced mainly due to prolongation of T_E. B–E, Mean time series from the start of microinjection showing developing changes of normalized T_I (B, red curve), T_E (C, blue curve), f_R (D, green curve), and integrated PN activity amplitude (E, magenta curve) for a representative experimental group (n = 6). Solid colored curves are group mean time and normalized parameter values; gray bands are ±1 SEM for the mean normalized parameter values.

Disruption of rhythmic respiratory activity by block/attenuation of GABA_Aergic and glycinergic inhibition in the BötC of in situ perfused brainstem–spinal cord preparations. A, B, Two examples from experiments using different preparations illustrating perturbations caused by microinjections of gabazine and strychnine (30 µm slowly injected during the period indicated by the brown bars at the top and blue rectangles). As in the vivo experiments, respiratory frequency was reduced by a prolonged T_E, integrated phrenic nerve discharge amplitude (∫PN) was also reduced, and ultimately apnea occurred. Simultaneously recorded integrated cVN (∫cVN) in A shows disruption of rhythmic activity and tonic discharge (shift of integrated activity baseline) during apneic period. B, Simultaneously recorded integrated pre-BötC pre-I/I population activity from another experiment also reflects the reduction of inspiratory frequency and termination of rhythmic activity during bilateral microinjections of the inhibitory receptor blockers in BötC.

Group data (n = 6) summarizing perturbations of respiratory rhythm and motor output pattern parameters by disrupting GABA_Aergic and glycinergic inhibition in the BötC of in situ perfused brainstem–spinal cord preparations. Solid colored curves in these time series are mean time and normalized parameter values and gray bands are ±1 SEM for the normalized values of T_I (A), T_E (B), respiratory frequency (f_R; C), and integrated inspiratory activity amplitude (D) computed from the start of microinjections from recordings of integrated PN activity.

Disturbances of three-phase respiratory pattern including disruption of post-I activity by bilaterally microinjected gabazine and strychnine (30 µm each) in the pre-BötC (A) or BötC (B) of in situ perfused juvenile rat brainstem–spinal cord preparations. Upper traces in A and B show seven consecutive overlaid and aligned integrated PN (red traces) and cVN (blue traces) activity signals in the pre-microinjection control period (light red and light blue larger amplitude traces, respectively) and also seven overlaid traces of recorded signals during microinjections (dark red and dark blue traces with reduced amplitudes). Simultaneously recorded pre-BötC pre-I/I population activity before and during microinjections (light and dark green traces, respectively) is also shown at the top in B to indicate activity perturbations in this region occurring with disruption of inhibition in BötC. Signals are aligned (cycle-triggered) at the onset of PN inspiratory activity indicated by the vertical dotted line in A and B. Raster plots below overlaid traces show consecutive series of inspiratory-onset aligned respiratory cycles with integrated PN inspiratory (red) and cVN including post-I (blue) activities before and during microinjections. Arrows on the raster plots indicate time interval over which the overlaid traces above the raster plots were obtained for the pre-microinjection period. During microinjections in the pre-BötC or BötC, the amplitude of inspiratory and post-I activity is progressively reduced, and post-I discharge is eliminated as seen on the aligned (darker) ∫cVN traces above the raster plots and in the raster plots with loss of post-I discharge indicated (white arrow).

Suppression of rhythmic inspiratory activity by bilateral microinjection of muscimol in the pre-BötC of adult rat in vivo and gabazine antagonism of GABA_A receptor activation. Muscimol (30 nl, 100 µm) microinjection (during yellow shaded area) rapidly reduced integrated PN activity and produced a long-lasting suppression of inspiratory activity that could be restored by bilateral microinjection of gabazine (110 nl, 250 µm) at the same site in the pre-BötC (blue shaded area). ∫PN, phrenic nerve integrated activity; f_R, respiratory frequency.

Illustration of prolonged suppression of rhythmic inspiratory activity by bilateral microinjection of muscimol (10 µm) in the pre-BötC (yellow shaded area) of juvenile rat perfused brainstem–spinal cord in situ. As in vivo, muscimol rapidly reduces phrenic nerve inspiratory discharge frequency and terminates inspiratory activity. ∫PN, phrenic nerve integrated activity; f_R, respiratory frequency.

Perturbation of inspiratory activity by bilateral microinjection of muscimol in the BötC of anesthetized adult rat in vivo. A, Muscimol (30 nl, 100 µm) slowly microinjected (brown bar at the top and yellow rectangle) in the BötC augments PN frequency due to a reduction of T_E. B–E, Mean time series for group data (n = 7) summarizing perturbations of respiratory rhythm and motor output pattern parameters. Solid colored curves are mean time and normalized parameter values and gray bands are ±1 SEM of T_I (B), T_E (C), respiratory frequency (f_R; D), and integrated inspiratory activity amplitude (E) computed from recordings of integrated phrenic nerve activity as in previous figures.