Physiological and Morphological Characterization of Genetically Defined Classes of Interneurons in Respiratory Rhythm and Pattern Generation in Neonatal Mice.

摘要

Breathing in mammals depends on an inspiratory-related rhythm that is generated by glutamatergic neurons in the preBotzinger complex (preBotC), a specialized site of the lower brainstem. Rhythm-generating preBotC neurons are derived from a single lineage that expresses the transcription factor (TF) Dbx1, but the cellular mechanisms of rhythmogenesis remain incompletely understood. To elucidate these mechanisms we comparatively analyzed Dbx1-expressing neurons (Dbx1 +) and Dbxl- neurons in the preBotC in knock-in transgenic mice. Whole-cell recordings in rhythmically active newborn mouse slice preparations showed that Dbx1 + neurons activate earlier in the respiratory cycle and discharge greater magnitude inspiratory bursts compared to Dbxl - neurons. Furthermore, Dbx1+ neurons required significantly less input current to discharge spikes (rheobase) in the context of network activity. The expression of intrinsic membrane properties indicative of A-current (IA) and hyperpolarization-activated current (Ih) was generally mutually exclusive in Dbx1 + neurons, which may indicate rhythmogenic function. In contrast, there was no such relationship in the expression of intrinsic currents I A and Ih in Dbxl- neurons. Confocal imaging and digital reconstruction of recorded neurons revealed dendritic spines on Dbxl- neurons, but Dbx1 + neurons were spineless. Dbx1 + neuron morphology was largely confined to the transverse plane whereas Dbxl- neurons projected dendrites to a greater extent in the parasagittal plane (rostrocaudally). A greater percentage of Dbx1 + neurons showed contralaterally projecting axons whereas Dbxl- neurons showed axons projecting in the rostral direction, which were severed by transverse cutting of the slice. Our data suggest that the rhythmogenic properties of Dbx1+ neurons include a higher level of intrinsic excitability that promotes burst generation in the context of network activity, which may be attributable to dendritic active properties that are recruited by excitatory synaptic transmission. Along with Dbxl, the TF Math1 has been shown to give rise to neurons that have important respiratory functions, including a potential role in coordinating the inspiratory and expiratory phases. To evaluate this role, we performed physiological and morphological characterizations of Math1+ neurons in transgenic mice and found that one out of six recorded Math1+ neurons showed expiratory activity. The expiratory Math1+ neuron appeared to be have a larger soma as well as a greater somatodendritic span in all axes (dorsal-ventral, medial-lateral and rostral-caudal) than the non-respiratory modulated Math1+ neurons. This suggests that respiratory modulated Math1+ neurons may be physiologically and morphologically specialized compared to non-rhythmic Mathl+ neurons. Their larger morphological span and rhythmic expiratory modulation could be indicative of a function in coordinating phasic activity between inspiratory and expiratory oscillators. Although our findings are still preliminary, the data thus far are consistent with a hypothesized respiratory network model wherein the Math1+ neurons function in coordinating the pattern of inspiration and expiration. Identifying and characterizing hindbrain interneurons according to developmental genetic origins as well as physiological properties provides complementary information to help elucidate the cellular mechanisms underlying the generation and coordination of the respiratory rhythm.