No 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.

1. We have hypothesized that pacemaker neurons in the pre-Bötzinger complex (pre-BötC) form the kernel for respiratory rhythm generation. A prediction of this hypothesis is that oscillatory behavior in some respiratory neurons could persist in the absence of synaptic transmission. In this study we used extracellular recording of neuronal activity in slice preparations from neonatal rat medulla that generate respiratory rhythm in vitro to determine 1) whether pacemaker properties are present in pre-BötC and unique to respiratory neurons, 2) whether pacemaker properties are common to all respiratory neurons, and 3) the spatiotemporal patterns of pacemaker neuron activity. 2. Whole cell recordings from respiratory neurons verified that bathing the slices in a low-Ca2+/high-Mg2+ solution (low-Ca2+ solution) eliminated endogenous respiratory synaptic inputs and electrically evoked synaptic inputs. 3. Sixty-three neurons spontaneously generated rhythmic bursts of action potentials in low-Ca2+ solution. After we switched to control solution to reactivate the respiratory network, these neurons were classified on the basis of their spike discharge patterns relative to the respiratory cycle as: 1) inspiratory (I) neurons (n = 41), 2) tonic expiratory (tonic E) neurons (n = 4), and 3) tonic neurons (n = 18). 4. In other experiments we tested I and tonic E neurons identified first in control solution for bursting behavior in low-Ca2+ solution. Several I neurons (n = 5 of 33), but none of the tonic E neurons (n = 0 of 13), continued to burst rhythmically. 5. Bursting and nonbursting respiratory neurons were distributed throughout the ventrolateral reticular formation within the pre-BötC as well as in the ventral respiratory group (VRG) immediately caudal to the pre-BötC. 6. We conclude that subpopulations of VRG neurons in vitro have rhythmic bursting properties when synaptic transmission is abolished. Respiratory neurons, especially I neurons, were the most prevalent class of bursting cells. Only a small percentage of respiratory neurons, however, had pacemaker properties. These findings are consistent with the hypothesis that the respiratory oscillator includes specialized neurons with intrinsic oscillatory properties.

The pre-Bötzinger complex (pre-BötC), a bilaterally distributed network of rhythmogenic neurons within the ventrolateral medulla, has been proposed to be the critical locus for respiratory rhythm generation in mammals. To date, thin transverse medullary slice preparations that capture the pre-BötC have served as the optimal experimental model to study the region's inherent cellular and network properties. We have reduced the thin slices to isolated pre-BötC "islands" to further establish whether the pre-BötC has intrinsic rhythmicity and is the kernel for rhythmogenesis in the slice. We recorded neuron population activity locally in the pre-BötC with macroelectrodes and fluorescent imaging of Ca(2+) activities with Calcium Green-1AM dye before and after excising the island. The isolated island remained rhythmically active with a population burst profile similar to the inspiratory burst in the slice. Rhythmic population activity persisted in islands after block of GABA(A)ergic and glycinergic synaptic inhibition. The loci of pre-BötC Ca(2+) activity imaged in thin slices and islands were similar, and imaged pre-BötC neurons exhibited synchronized flashing after blocking synaptic inhibition. Population burst frequency increased monotonically as extracellular potassium concentration was elevated, consistent with mathematical models consisting entirely of an excitatory network of synaptically coupled pacemaker neurons with heterogeneous, voltage-dependent bursting properties. Our results provide further evidence for a rhythmogenic kernel in the pre-BötC in vitro and demonstrate that the islands are ideal preparations for studying the kernel's intrinsic properties.