Models of Respiratory Rhythm Generation in the Pre-Bötzinger Complex. III. Experimental Tests of Model Predictions

Abstract

We used the testable predictions of mathematical models proposed by Butera et al. to evaluate cellular, synaptic, and population-level components of the hypothesis that respiratory rhythm in mammals is generated in vitro in the pre-Bötzinger complex (pre-BötC) by a heterogeneous population of pacemaker neurons coupled by fast excitatory synapses. We prepared thin brain stem slices from neonatal rats that capture the pre-BötC and maintain inspiratory-related motor activity in vitro. We recorded pacemaker neurons extracellularly and found: intrinsic bursting behavior that did not depend on Ca(2+) currents and persisted after blocking synaptic transmission; multistate behavior with transitions from quiescence to bursting and tonic spiking states as cellular excitability was increased via extracellular K(+) concentration ([K(+)](o)); a monotonic increase in burst frequency and decrease in burst duration with increasing [K(+)](o); heterogeneity among different cells sampled; and an increase in inspiratory burst duration and decrease in burst frequency by excitatory synaptic coupling in the respiratory network. These data affirm the basis for the network model, which is composed of heterogeneous pacemaker cells having a voltage-dependent burst-generating mechanism dominated by persistent Na(+) current (I(NaP)) and excitatory synaptic coupling that synchronizes cell activity. We investigated population-level activity in the pre-BötC using local "macropatch" recordings and confirmed these model predictions: pre-BötC activity preceded respiratory-related motor output by 100-400 ms, consistent with a heterogeneous pacemaker-cell population generating inspiratory rhythm in the pre-BötC; pre-BötC population burst amplitude decreased monotonically with increasing [K(+)](o) (while frequency increased), which can be attributed to pacemaker cell properties; and burst amplitude fluctuated from cycle to cycle after decreasing bilateral synaptic coupling surgically as predicted from stability analyses of the model. We conclude that the pacemaker cell and network models explain features of inspiratory rhythm generation in vitro.

Functional Imaging, Spatial Reconstruction, and Biophysical Analysis of a Respiratory Motor Circuit IsolatedIn Vitro

“…This excitatory waveform originates in the pre-BötC (Butera et al 1999b;Del Negro et al, 2001) and propagates, according to our data, with high fidelity. Despite marked differences in preMN and MN size/somatodendritic morphology, synaptic conductance/current densities are similar.…”

“…To accomplish this, we exploited neonatal rat medullary slice preparations, which are used for analysis of inspiratory rhythm and pattern generation mechanisms (Smith et al, 1991;Koshiya and Smith, 1999;Del Negro et al, 2001). These slices contain active rhythm-generating pre-BötC neurons and circuits transmitting rhythmic inspiratory drive to XII MNs.…”

Transient attenuation of CO2 sensitivity after neurotoxic lesions in the medullary raphe area of awake goats

“…Transection along the midline severs axonal connections between the bilateral pre-Bötzinger complex nuclei that lie dorsal to the midline raphe nuclei. Therefore, fibers of passage between pre-Bötzinger complex nuclei may play a role in coordinating rhythmic output (6). However, this alternative is not supported by our findings that transient, strong stimulation of raphe neurons by IA eliminated the altered breathing periods (Fig.…”

“…Alternatively, the microtubule may have destroyed fibers of passage between the bilaterally located pre-Bötzinger complex neurons. This alternative is suggested by findings that perturbations in respiratory rhythm occur after midline transection of medullary slices in solution (6). Transection along the midline severs axonal connections between the bilateral pre-Bötzinger complex nuclei that lie dorsal to the midline raphe nuclei.…”

Sodium Currents in Neurons From the Rostroventrolateral Medulla of the Rat

“…It has been suggested that I NaP plays an essential role in the activation of pacemaker neurons in the pre-Bötzinger Complex (pBC), a region within the rostroventrolateral medulla (RVLM), in vitro (Butera et al 1999;Del Negro et al 2001;Smith et al 2000). Del Negro et al (2002) experimentally characterized I NaP in pBC neurons.…”