Activation state of the hyperpolarization-activated current modulates temperature-sensitivity of firing in locus coeruleus neurons from bullfrogs

Abstract

Locus coeruleus neurons of anuran amphibians contribute to breathing control and have spontaneous firing frequencies that, paradoxically, increase with cooling. We previously showed that cooling inhibits a depolarizing membrane current, the hyperpolarization-activated current ( Ih) in locus coeruleus neurons from bullfrogs, Lithobates catesbeianus (Santin JM, Watters KC, Putnam RW, Hartzler LK. Am J Physiol Regul Integr Comp Physiol 305: R1451–R1464, 2013). This suggests an unlikely role for Ih in generating cold activation, but led us to hypothesize that inhibition of Ih by cooling functions as a physiological brake to limit the cold-activated response. Using whole cell electrophysiology in brain slices, we employed 2 mM Cs+ (an Ih antagonist) to isolate the role of Ih in spontaneous firing and cold activation in neurons recorded with either control or Ih agonist (cyclic AMP)-containing artificial intracellular fluid. Ih did not contribute to the membrane potential ( Vm) and spontaneous firing at 20°C. Although voltage-clamp analysis confirmed that cooling inhibits Ih, its lack of involvement in setting baseline firing and Vm precluded its ability to regulate cold activation as hypothesized. In contrast, neurons dialyzed with cAMP exhibited greater baseline firing frequencies at 20°C due to Ih activation. Our hypothesis was supported when the starting level of Ih was enhanced by elevating cAMP because cold activation was converted to more ordinary cold inhibition. These findings indicate that situations leading to enhancement of Ih facilitate firing at 20°C, yet the hyperpolarization associated with inhibiting a depolarizing cation current by cooling blunts the net Vm response to cooling to oppose normal cold-depolarizing factors. This suggests that the influence of Ih activation state on neuronal firing varies in the poikilothermic neuronal environment.