Hyperpolarization-activated currents in the growth cone and soma of neonatal rat dorsal root ganglion neurons in culture.
Dissociated dorsal root ganglion neuron growth cones and somata from neonatal rats were voltage and current clamped with the use of the perforated-patch whole cell configuration to study the occurrence and properties of slow hyperpolarization-activated currents (Ih) at both regions. Under voltage-clamp conditions Ih, blockable by 2 mM extracellular CsCl, was present in 33% of the growth cones tested. Its steady-state activation as a function of voltage could be fitted with a single Boltzmann function with a midpoint potential of -97 mV. The time course of current activation could be best described by a double-exponential function. The magnitude of the fully activated conductance was 3.5 nS and the reversal potential amounted to -29 mV. At the soma, Ih was found in 80% of the somata tested, which is much higher than occurrence at the growth cone. The steady-state activation curve of Ih at the soma, fitted with a single Boltzmann function, had a midpoint potential of -92 mV, which was more positive than that in the growth cone. The double-exponential activation of the current was faster than in the growth cone. The fully activated conductance of 5.1 nS and the reversal potential of -27 mV were not significantly different from the values obtained at the growth cone. Membrane hyperpolarization by current-clamp pulses elicited depolarizing sags in 30% and 78% of the tested growth cones and somata, respectively, which is in agreement with our voltage-clamp findings. Termination of the hyperpolarizing current pulse evoked a transient membrane depolarization or an action potential at both sites. Application of 2 mM extracellular CsCl hyperpolarized the membrane potential reversibly by approximately 5 mV and blocked the depolarizing sags and action potentials following the current injections at these regions. Thus Ih contributes to the resting membrane potential and modulates the excitability of both the growth cone and the soma. Intracellular perfusion with the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) was only possible at the soma by the use of the conventional whole cell configuration. Addition of 100 microM cAMP to the pipette solution shifted the midpoint potential of the Ih activation curve from -108 to -78 mV. The current activation time course was also accelerated. The reversal potential and the fully activated conductance underlying Ih were not changed by cAMP. These results imply that cAMP primarily affects the gating kinetics of Ih. Our results show for the first time quantitative differences in Ih properties and occurrence at the growth cone and soma membrane. These differences may reflect differences in intracellular cAMP concentration and in the expression of Ih.
Wang
Z
Z
Department of Physiology, Leiden University, The Netherlands.
Van Den Berg
R J
RJ
Ypey
D L
DL
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar