Emergence of a spermine-sensitive, non-inactivating conductance in mature hippocampal CA1 pyramidal neurons upon reduction of extracellular Ca2+: dependence on intracellular Mg2+ and ATP

Abstract

Large and protracted elevations of intracellular [Ca(2+)] and [Na(+)] play a crucial role in neuronal injury in ischemic conditions. In addition to excessive glutamate receptor activation, other ion channels may contribute to disruption of intracellular ionic homeostasis. During episodes of ischemia, extracellular [Ca(2+)] falls significantly. Here we report the emergence of an inward current in hippocampal CA1 pyramidal neurons in acute brain slices from adult mice upon reduction/removal of [Ca(2+)](e). The magnitude of the current was 100-300pA at -65mV holding potential, depending on intracellular constituents. The current was accompanied by intense neuronal discharge, observed in both whole-cell and cell-attached patch configurations. Sustained currents and increased neuronal firing rates were both reversed by restoration of physiological levels of [Ca(2+)](e), or by application of spermine (1mM). The amplitudes of the sustained currents were strongly reduced by raising intracellular [Mg(2+)], but not by extracellular [Mg(2+)] increases. Elevated intracellular ATP also reduced the current. This conductance is similar in several respects to the "calcium-sensing, non-selective cation current" (csNSC), previously described in cultured mouse hippocampal neurons of embryonic origin. The dependence on intracellular [ATP] and [Mg(2+)] shown here, suggests a possible role for this current in disruption of ionic homeostasis during metabolic stress that accompanies excessive neuronal stimulation.

Figure 1. Removal of extracellular divalent cations produces a non-inactivating inward current accompanied by neuronal firing

A: Whole cell recording, with K⁺-containing intracellular solution, in normal ACSF (2mM Ca²⁺, 1mM Mg²⁺). After onset of a sustained inward current triggered by Ca²⁺/Mg²⁺ removal (indicated by black bar), data acquisition was interrupted (at grey dotted bar) for application of voltage ramp protocols (see figure 6). Following resumption of continuous data acquisition, the current and spiking were terminated by restoration of Ca²⁺ and Mg²⁺. Dashed line indicates zero current in this and all figures. B: Conditions of this experiment are identical to those in A, except that the intracellular solution contained Cs⁺ as the major cation plus 2 mM TEA (“Int 6”), in lieu of K⁺. A similar inward current and spiking activity was recorded. C: The conditions of this experiment are identical as for “B”; however, no neuronal firing is observed in this neuron. The inset is an expanded region of the area indicated by the black box, to show small excitatory currents generated by Ca²⁺/Mg²⁺ -free solution in this neuron.

Figure 2. Increased activity produced by removal of divalent cations, recorded in cell-attached mode

A: Recording of a CA1 pyramidal neuron perfused in the absence of extracellular divalent cations, as indicated by the top black bar. B: Expanded regions corresponding to those depicted in the recording shown in A, showing a tonic firing pattern (seen in ~75 % of neurons tested). C&D: Identical experimental conditions as in A, but in this example, a burst firing pattern was seen following removal of extracellular divalent cations (seen in ~15% of neurons tested).

Figure 3. Effects of reduction of extracellular divalent cations on seal resistance

A: Current-voltage relationships from cell-attached recordings in the presence (black circles) or absence (grey triangles) of extracellular divalent cations (mean ±SEM of 5 cells each). Seal resistance (R_s) was adjusted by mouth suction to ~ 2 GOhm and divalent cations removal had a minor effect on seal resistance. B: Recordings performed as for A, except that R_s was adjusted by mouth suction to ~0.5 GOhm, and resulted in significantly greater decreases in seal resistance following divalent cations removal.

Figure 4. Inward currents produced by removal of Ca²⁺ alone

Whole cell recording (holding potential =−65 mV), showing inward current produced by Ca²⁺ removal, retaining extracellular Mg²⁺ constant at 1 mM. Current spikes are truncated.

Figure 5. Effects of moderate decreases in extracellular Ca²⁺

A: Mean differences of peak minus the base leak amplitudes of sustained inward currents, generated by nominally Ca²⁺-free solution (grey bar) or 0.1mM Ca²⁺_e (white bar). [Mg²⁺]_e was constant at 1 mM. B: Representative recording of a neuron perfused with ACSF containing 100 μM [Ca²⁺]_e. The intracellular solution contains K⁺ as the major cation (Int “2”). Neuronal discharge also accompanies the sustained inward current.

Figure 6. Inhibition of currents evoked by Ca²⁺ removal by intracellular [Mg²⁺] and [ATP]

Currents were evoked by voltage ramps (−40 to +40 mV, see inset for waveform) from a holding potential of 0 mV. The intracellular solution contained Cs²⁺ as the major cation. Thin black curves indicate superfusion with normal ACSF, and grey curves indicate superfusion in nominally Ca²⁺-free ACSF (1mM Mg²⁺). Panels A–C show the effects of increasing Mg²⁺_i concentrations. D shows the effects of addition of ATP to the intracellular solution. The calculated free [Mg²⁺]_i and [ATP] are shown for each panel (calculated with Winmaxc, see Methods). The dotted curve on panel “A” indicates reintroduction of [Ca²⁺]_e to the perfusate (curve “c”). The thick black curve on panel A is the result of subtracting curve “b” from curve “a”.

Figure 7. Estimates of currents induced by removal of extracellular Ca²⁺ at −65 mV holding potential in whole cell recordings, and effect of spermine

A: The intracellular solution contains K⁺ as the major cation (Int “1–4”). B: The intracellular solution contains Cs⁺ as the major cation (Int “5–8”). Hatched bars signify reintroduction of Ca²⁺ (and Mg²⁺ if it was removed previously) to the perfusate. *, significant, (p<0.05), Mann-Whitney rank sum test. Each bar represents the mean value of 5–9 cells. Non-responding cells were not included in the calculations of the mean currents. Free [Mg²⁺]_i and [ATP] concentration are shown on the corresponding group of bars. The total [Mg²⁺]_i and [ATP] are identical to that shown for figure 6. The currents measured with 0.08 mM free [Mg²⁺]_i is statistically significantly higher than in the presence of 0.07 mM free [Mg²⁺]_i plus 2.2 mM ATP, irrespective if the major intracellular cation is K⁺ or Cs⁺ (p<0.005). C: Whole cell recordings of neurons perfused with either nominally Ca²⁺-free ACSF (grey bar) as compared to the presence of 0.1 mM spermine in the perfusate (grey-white hatched bar), or 1 mM spermine (white-black hatched bar). The intracellular solution for these experiments is “Int 6”. In panel D, a representative recording of a voltage clamped neuron in the whole cell mode is shown, where 1 mM spermine ameliorates against the –low-[Ca²⁺]_e–induced current; current spikes are truncated.

Figure 8. Characterization of neuronal firing accompanying the sustained inward current

A: Left graph: Current clamp recording of a patched neuron in the whole cell mode, during superfusion with nominally [Ca²⁺]_e-free ACSF; right graph: Interspike voltage (mV) of the recording depicted on the left graph. B: Left graph: Quantification of current spike frequency as a function of interspike Vm, for the same neuron as shown in A. B, Right graph: Quantification of current spike frequency over gradual injection of current on the patched neuron through the pipette. Top x-axis depicts the measured Vm during injection of current. C: Voltage clamp recording of a patched neuron in the whole cell mode, during superfusion with nominally [Ca²⁺]_e-free ACSF, but in the presence of intracellular Mg²⁺ and ATP (“Int 4”). Current spikes are truncated.