Isolation of the kernel for respiratory rhythm generation in a novel preparation: The pre-Botzinger complex "island"

The pre-Botzinger complex (pre-BotC), 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 dat e, thin transverse medullary slice preparations that capture the pre-BotC h ave served as the optimal experimental model to study the region's inherent cellular and network properties. We have reduced the thin slices to isolat ed pre-BotC "islands" to further establish whether the pre-BotC has intrins ic rhythmicity and is the kernel for rhythmogenesis in the slice. We record ed neuron population activity locally in the pre-BotC with macroelectrodes and fluorescent imaging of Ca2+ activities with Calcium Green-1AM dye befor e 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-BotC Ca 2+ activity imaged in thin slices and islands were similar, and imaged pre- BotC neurons exhibited synchronized flashing after blocking synaptic inhibi tion. Population burst frequency increased monotonically as extracellular p otassium concentration was elevated, consistent with mathematical models co nsisting entirely of an excitatory network of synaptically coupled pacemake r neurons with heterogeneous, voltage-dependent bursting properties. Our re sults provide further evidence for a rhythmogenic kernel in the pre-BotC in vitro and demonstrate that the islands are ideal preparations for studying the kernel's intrinsic properties.