Commentary: Synaptic Excitation in Spinal Motoneurons Alternates with Synaptic Inhibition and Is Balanced by Outward Rectification during Rhythmic Motor Network Activity

摘要

In a recent study, Guzulaitis and Hounsgaard ( 2017 ) (GH2017) used whole cell voltage clamp (VC) on the reversal potential for inhibition or excitation to assess their synaptic currents (Johnston and Wu, 1995 ; Brette and Destexhe, 2012 ). GH2017 concluded that inhibition and excitation alternated during rhythmic scratching, and a voltage-dependent intrinsic conductance was masking this input such that it appeared as balanced excitation and inhibition in previous published work (Berg et al., 2007 ; Petersen et al., 2014 ). Nevertheless, this reasoning relies entirely on the validity of the clamp and, as we will see below, there is a clamp error, which complicates the interpretation of their data. Errors associated with voltage-clamp is a common problem as noted in previous reports (Spruston et al., 1993 ; Williams and Mitchell, 2008 ; Petersen, 2017 ). The membrane current (I) is composed of intrinsic, leak, excitatory and inhibitory currents with individual conductances and reversal potentials, which collectively form a membrane resistance ( R _( m )) and an equilibrium potential ( E _( m )). When recording these using a pipette electrode, its resistance ( R _( s )), sometimes called access or series resistance, is in series with R _( m )(Figure 1A ). When there is no electrode current the membrane potential V _( m )= E _( m ). However, during VC, a non-zero current introduces a drop in potential over R _( s ), which can only be partially compensated with the amplifier electronics (Brette and Destexhe, 2012 ). R _( s )therefore has an uncompensated part (blue, R _( us ), Figures 1A,B ), which generates an unaccounted drop in potential from the clamp potential ( V _( c )) proportional to the pipette current: (1) V m = V c ? I · R u s GH2017 report: “Voltage clamp (VC) experiments were performed on motoneurons when access resistance was low ( R _( a )< 20 MΩ) and possible to compensate by 60-80%.” This means that R _( us )= 20 ? 40% · 20 MΩ = 4–8 MΩ. When clamping at 0 mV the applied current is likely large. The authors do not report I for their clamp experiments (Figures 8–9), but their IV-plots suggest up to 10 nA (Figures 5E, 6). Hence, when trying to clamp at 0 mV, V _( m )is really ?10 nA ·4 MΩ = ?40 mV with 80% R _( s )-compensation. Figure 1 Caveats using voltage clamp to resolve excitation and inhibition. (A) Whole-cell VC can be decomposed into electrical components including the pipette series resistance ( R _( s )). (B) Partial compensation for R _( s )introduces a disparity between clamped potential ( V _( c )) and V _( m )due to uncompensated resistance (red). (C) Reciprocal model for rhythmic V _( m )has alternating E/I. ( R _( m )= 20 MΩ , E _( leak )= ?70 mV ). (D) Balanced model has concurrent E/I and also rhythmic V _( m ). (E) Outward currents measured using VC is assumed to be inhibition when clamping 0mV (black). The actual clamp is at ?30 mV (red). (F) Balanced E/I spuriously appears as reciprocal when the actual clamp is below synaptic reversal potential (“same phase” cf. red in F and black in E ). (G) VC-recording of a putative motoneuron with blocked spikes (with intracellular QX314) at different holding potentials (gray: current, red: mean, blue: nerve). Reversal of phase (arrow) is consistent with the balanced scheme (F) although with a smaller out-of-phase inhibition (indicated). (H) Blocking inhibition (strychnine) increases firing rate also consistent with the balanced scheme. (G) provided by A. Alaburda (current levels indicated, right) and (H) adapted with permission (Vestergaard and Berg, 2015 ). To better understand the issue, we consider steady-state where all current passes through the resistors. From Ohm's law the voltage drop over R _( us )is V _( m )? V _( c )= I · R _( us ). Similarly, the voltage drop over the membrane is E _( m )? V _( m )= I · R _( m ). Combining these we can eliminate I and isolate V _( m ): (2) V m = V c R m + E m R u s R m + R u s Hence, for a good clamp ( V _( m )≈ V _( c )) it is required that R _( m )? R _( us ). GH2017 report a membrane conductance of 49.2 nS (Figure 5B), which gives R _( m )= 20 MΩ . With these values ( E _( m )= ?70 mV ) clamping at 0 mV gives (3) V m = 0 ? 70 m V · 8 M Ω 28 M Ω = ? 20 m V Whereas R _( us )is assumed constant, R _( m )may change dramatically due to synaptic and intrinsic conductance. GH2017 nicely document a nonlinearity starting at ?30 mV (Figures 5, 6), and a conductance of 314 nS ( R _( m )= 3.2 MΩ ). Here, the low R _( m )even becomes smaller than R _( us )and therefore the clamp deteriorates further: (4) V m = 0 ? 70 m V · 8 M Ω 11.2 M Ω = ? 50 m V The clamp is unlikely to be this bad, since the reduction in R _( m )occurs above ?50mV. Also, E _( m ), which we assume constant, may depolarize due to change in the weighted average (Figure 1B ), which mitigates the effect. The exact level of clamping of V _( m )with ( V _( c )= 0mV) is difficult to estimate and may change in time. A reasonable guess is