Isolation of Somatic Na⁺Currents by Selective Inactivation of Axonal Channels with a Voltage Prepulse

Abstract

We present a simple and effective method for isolating the somatic Na ϩ current recorded under voltage clamp from neurons in brain slices. The principle is to convert the axon from an active compartment capable of generating uncontrolled axonal spikes into a passive structure by selectively inactivating axonal Na ϩ channels. Typically, whole-cell currents from intact neurons under somatic voltage clamp contain a mixture of Na ϩ current and axial current caused by escaped axonal spikes. We found that a brief prepulse to voltages near spike threshold evokes the axonal spike, which inactivates axonal but not somatic channels. A subsequent voltage step then evokes only somatic Na ϩ current from electrotonically proximal sodium channels under good voltage-clamp control. Simulations using a neuron compartmental model support the idea that the prepulse effectively inactivates currents from the axon and isolates well controlled somatic currents. Na ϩ currents recorded from corticalpyramidalneuronsinslices,usingtheprepulse,werefoundtohavevoltagedependencenearlyidenticaltothatofcurrentsrecordedfrom acutely dissociated pyramidal neurons. In addition, studies in dissociated neurons show that the prepulse has no visible effect on the voltage dependence and kinetics of Na ϩ currents elicited by the subsequent voltage step, only decreasing the amplitude of the currents by 10 -20%. The technique was effective in several neuronal types in brain slices from male and female neonatal rats and mice, including raphé neurons, cortical pyramidal neurons, inferior olivary neurons, and hypoglossal motoneurons.

Cell adhesion molecule L1 contributes to neuronal excitability regulating the function of voltage-gated Na+ channels

“…3A). Indeed, I Na recorded from cortical pyramidal neurons in slices using such pre-pulse showed a voltage dependence nearly identical to that of I Na recorded from pyramidal neurons lacking axonal and dendritic processes (Milescu et al, 2010). Consistently with the impaired firing activity described above, the J Na evoked at increasing membrane potentials was reduced in the absence of L1 (Fig.…”

“…Space-clamp problems affecting I Na recordings in cultured neurons were circumvented by a voltage-step protocol preceded by a brief pre-pulse at −40 mV that inactivates axonal I Na currents, thus allowing isolation of the I Na somatic component (Milescu et al, 2010) (Fig. 3A).…”

Development of Electrophysiological Properties of Nucleus Gigantocellularis Neurons Correlated with Increased CNS Arousal

“…In slice recording, where imperfect space clamp sometimes occurs, the inward currents may also contain “axial currents” (indicated by the out-of-place inward current in Fig. 2Aa) induced by an axonal action potential escaping the imperfect space clamp [24]. The axial current may contain other currents than the main Na + current.…”

“…The single voltage step is too brief to allow for axial current to contaminate the inward current the voltage step evoked. When neurons are held at −40 mV, near the AP threshold, the axonal AP and, hence, the axial current are inactivated [24]. Therefore, the inward currents measured with holding potential (Vh) at −40 mV are devoid of the “axial current” (note that the “axial current” is no longer detectable in Fig.…”

Polarized localization of voltage-gated Na + channels is regulated by concerted FGF13 and FGF14 action

“…Current density-to-voltage relationships were plotted (I-V relationship). The prepulse protocol to separate axonal and somatic currents was performed as described previously (39). A prepulse from the holding potential of −90 mV to −45 mV for 4 ms was followed by a brief recovery phase at −55 mV for 0.5 ms.…”