Distribution of calcium binding proteins, sodium channels and persistent sodium current in the rat ventral respiratory group

Abstract

The ventral respiratory group (VRG) is important in generating both the breathing rhythm and motor pattern. The VRG is generally subdivided into 4 regions, the Botzinger complex, preBotzinger Complex (pBc), rostral VRG, and caudal VRG based on neuron discharge patterns and axonal projections. Anatomical markers provide a potential bridge between the physiological analysis of the respiratory system and the neurochemical elements that are the ultimate building blocks of this system. An example of this approach is the coincidence of NK1 receptors with neurons in the pBc complex [1]. VRG labeling by antibodies to calcium binding proteins (parvalbumin, calbindin, and calretinin) in some respects complements the NK1 immunoreactivity. Parvalbumin has been suggested to label VRG neurons [2]. We have verified this by demonstrating that parvalbumin positive neurons project to the ipsilateral and contralateral VRG as well as to the phrenic nucleus. We have also found that a prominent gap in the column of VRG related parvalbumin cells [2] likely corresponds to the pBc since parvalbumin cells are rare in this zone and never co-localize with NK1 receptors. Calbindin and calretinin immunoreactive cells are scattered in the pBc and rostral VRG but rare in the Botzinger complex. Calbindin neurons are intermingled, but not colocalized with pBc NK1 cells. Calretinin is not colocalized with NK1, except for a small population of cells at the caudal ventral edge of the pBc, which likely corresponds to NK1 bulbospinal neurons [3]. Finally, preliminary evidence indicates glycine immunoreactivity in some parvalbumin neurons within the Botzinger complex region. In addition to this compartmentalized distribution of calcium binding proteins within the VRG, preliminary evidence is consistent with a differential distribution of Na channel alpha subunits. A slowly inactivating persistent Na current is postulated to underlie the pacemaker activity seen in a subset of pBc neurons [4]. Within the CNS at least 5 different Na channel alpha subunits have been identified, termed Nav1.1, 1.2, 1.3, 1.5 and 1.6. Of these Nav1.6 has been most strongly linked to persistent Na current. Immunohistochemical examination of Nav1.2, 1.3 and 1.6 demonstrated Nav1.2 is widely expressed in the VRG including some NK1 neurons. Nav1.3 was present in cranial motoneurons. In neurons acutely dissociated from the pBc of 1–15 day old rats, whole-cell voltage clamp recordings were used to analyze transient and persistent Na currents and single-cell RT-PCR was used to probe for Nav1.1, 1.2 and 1.6 mRNA. Whole-cell recordings were made using an external solution containing 50 mM NaCl. Slow ramp depolarization from -80 to +30 mV revealed a TTX-sensitive, persistent Na current. The current kinetics were dependent upon the ramp speed. A slow ramp (100 mV/s), elicited an inward non-inactivating current in 42% (10 of 24) of neurons sampled from the pBc. These results are consistent with a role for persistent Na current in regulation of the subthreshold behavior, including pacemaker activity. Moreover, single-cell RT-PCR revealed the presence of Nav1.6 in 40 of 72 cells (55%). The Nav1.1 subunit mRNA was present in 47 of 82 neurons (57%) and was co-expressed with Nav1.6 in 28% of cells. Preliminary findings on Nav1.2 mRNA are consistent with the immunohistochemistry with it present in 7 of 18 (38%) pBc neurons.