Ionic Mechanism Underlying Rebound Depolarization in Medial Prefrontal Cortex Pyramidal Neurons

Abstract

Rebound depolarization (RD) occurs after membrane hyperpolarization and converts an arriving inhibitory signal into cell excitation. The purpose of our study was to clarify the ionic mechanism of RD in synaptically isolated layer V medial prefrontal cortex (mPFC) pyramidal neurons in slices obtained from 58- to 62-day-old male rats. The RD was evoked after a step hyperpolarization below -80 mV, longer than 150 ms in 192 of 211 (91%) tested neurons. The amplitude of RD was 30.6 ± 1.2 mV above the resting membrane potential (-67.9 ± 0.95 mV), and it lasted a few 100 ms (n = 192). RD could be observed only after preventing BK channel activation, which was attained either by using paxilline, by removal of Ca⁺⁺ from the extra- or intracellular solution, by blockade of Ca⁺⁺ channels or during protein kinase C (PKC) activation. RD was resistant to tetrodotoxin (TTX) and was abolished after the removal of Na⁺ from the extracellular solution or application of an anti-Nav1.9 antibody to the cell interior. We conclude that two membrane currents are concomitantly activated after the step hyperpolarization in the tested neurons: a. a low-threshold, TTX-resistant, Na⁺ current that evokes RD; and b. an outward K⁺ current through BK channels that opposes Na⁺-dependent depolarization. The obtained results also suggest that a. low-level Ca⁺⁺ in the external medium attained upon intense neuronal activity may facilitate the formation of RD and seizures; and b. RD can be evoked during the activation of PKC, which is an effector of a number of transduction pathways.

Keywords: BK channels; Ca++ channels; Nav1.9 channels; amyloid β-peptide (1–42); prefrontal cortex; pyramidal neurons; rats; rebound depolarization.

Figure 1

Measurements of the amplitude and duration of rebound depolarization (RD) in medial prefrontal cortex (mPFC) pyramidal neurons. (Aa) Standard protocol applied to study RD. A 600-pA hyperpolarizing current prepulse lasting 1000 ms preceded 200-ms current pulses applied in 20-pA increments from −100 pA to +180 pA. The 200-ms current pulses were followed by a hyperpolarizing current postpulse at −200 pA that lasted 1300 ms. (b) Membrane potential changes evoked by current pulses −20 pA (*) and 0 pA (RD) in the bath in the absence of Ca⁺⁺. (c) An RD taken from (b). Double-headed arrow 1 indicates maximum depolarization above the resting membrane potential attained after repetitive tetrodotoxin (TTX)-resistant spikelets. Arrow 2 indicates the beginning of RD when the depolarization phase of the RD attained 10% of its maximum level. Arrow 3 indicates the RD end, which was defined as the time point at which the RD repolarization phase intersected with the resting membrane potential level. Double-headed arrow 4 indicates the RD duration, defined as the time interval between the beginning and the end of the RD. (B) Voltage changes (b1,b2) evoked by a current pulse to 0 pA (a) followed by postpulses to −200 pA (a1) and postpulse 0 pA (a2). The dotted horizontal line in panel (Aa) indicates 0 pA and the dotted horizontal lines in panel (Ab,c,Bb) and other figures indicate the resting membrane potential level.

Figure 2

Effects of Ca⁺⁺ and TTX in the extracellular solution on RD. (A) Portion of the current protocol applied to evoke RD shown in (B–D). Current pulses were preceded by a −300-pA hyperpolarizing current prepulse lasting 1000 ms, followed by a −300-pA current postpulse lasting 1300 ms. Membrane potential changes evoked in the presence of 1 mM of Ca⁺⁺ and the absence of TTX (B), in the absence of Ca⁺⁺ and the absence of TTX (C), and in the absence of Ca⁺⁺ and the presence of TTX (0.5 μM) (D) in the extracellular solution.

Figure 3

Effects of the amplitude and duration of the prepulse on RD in the absence of Ca⁺⁺ in the extracellular solution. (Aa) Current protocol applied to study the effect of the prepulse amplitude on RD. Hyperpolarizing 1000-ms current prepulses from −700 pA to −250 pA were applied in 50-pA increments preceding the threshold current pulse to 0 pA, lasting 200 ms. Current pulses that evoked RD were followed by hyperpolarization to −300 pA, lasting 1300 ms. (b) RDs evoked by the current protocol shown in (a). RDs were evoked only at prepulse potentials more negative than −79 mV. (Ba) Current protocol applied to study the effect of the prepulse duration on the RD duration. The prepulse amplitude was −600 pA. The prepulse duration was changed from 1000 to 0 ms. Only 2 prepulses with durations of 200 and 600 ms are shown. The 200-ms threshold current pulse that evoked RD was 0 pA. The postpulse amplitude was −200 pA and lasted 1300 ms. (b) RD was evoked at a prepulse duration of 600 ms, but not at a prepulse duration of 200 ms (protocol of current pulses is shown in a). (c) Relationship between prepulse duration (horizontal axis) and RD duration (vertical axis).

Figure 4

Effects of the extracellular concentration of Na⁺ on RD in the absence of Ca⁺⁺ in the extracellular solution. (A) Portion of the current protocol applied to evoke the membrane potential changes shown in (B–D) (compare to Figure 1Aa). (B) RD evoked in the presence of 125 mM of NaCl in the extracellular solution. (C) Membrane potential changes recorded after replacing 125 mM of NaCl with 125 mM of choline-Cl in the extracellular solution. (D) RD evoked after choline chloride washout (replacing 125 mM of choline chloride with 125 mM of NaCl).

Figure 5

Effects of HCN channel blockers on RD in the absence of Ca⁺⁺ in the extracellular solution. (Aa) Current protocol applied to evoke the membrane potential changes shown in (b,c) (compare to Figure 1Aa). RD evoked before (b) and during (c) bath application of ZD 7288 (50 μM). Insets to (b,c) show amplified voltage responses to the beginning of the rectangular hyperpolarizing current steps. Voltage sag was present before (inset to b) and was absent during bath application of ZD 7288 (50 μM, inset to c). (B) Examples of RDs evoked in two pyramidal neurons, in which voltage sag was present (a) and absent (b). Insets show amplified voltage responses to the beginning of the rectangular hyperpolarizing current steps. The current protocol applied to evoke RD is shown in Figure 1Aa. (C) Duration of RD before (control) and after 15-min bath application of either ZD 7288 (a, ZD 7288, 50 μM) or Cs⁺ (b, Cs⁺, 3 mM); n.s., non-significant.

Figure 6

Effects of an anti-Nav1.9 channel antibody, IgG and F⁻ on RD in the absence of Ca⁺⁺ in the extracellular solution. (Aa) Current protocol applied to evoke RD (compare Figure 1Aa). (b) Voltage responses to subthreshold −20-pA and threshold 0-pA current pulses lasting 200 ms are shown. Voltage responses were recorded after the 15-min pipette presence of anti-Nav1.9 channel antibody (4 μg/ml, anti-Nav1.9, 15 min). (c) Voltage responses to currents steps shown in (a) after loading the cell for 60 min with anti-Nav1.9 channel antibody (4 μg/ml, anti-Nav1.9, 60 min). In (b,c) are shown recordings obtained from the same neuron. (B) Current protocol applied to evoke RD shown in (b,c). (a) Voltage response to −20 pA subthreshold and 0 pA threshold current pulses lasting 200 ms recorded after the 15 (b) and 60 min (c) pipette presence of IgG (4 μg/ml; IgG, 15 min and IgG, 60 min, respectively). (C) Mean RD durations after 15-min (a) and 60-min (b) intracellular applications of anti-Nav1.9 antibody (anti-Nav1.9) or IgG (IgG). (D) RD current thresholds recorded during 15, 25 and 35 min in the presence of either only Cl⁻ (control) or Cl⁻ and F⁻ (F⁻) in the pipette solution; n.s., non-significant.

Figure 7

Effects of BK and SK channel blockers on RD in the presence of Ca⁺⁺ (1 mM) in the extracellular solution. (Aa) The current protocol (compare to Figure 1Aa) applied to evoke membrane potential changes shown in (b–d). Membrane potential changes evoked before (b), after a 15-min application of paxilline (10 μM) (c), and after paxilline washout (d). (c) Membrane potential changes to 200-ms pulses to −40 pA (subthreshold step) and to −20 pA (threshold step). (B) Absence of RD in the presence of only Ca⁺⁺ (1 mM) in the extracellular solution (Ba,b, Ca⁺⁺). Duration of RD and the presence of Ca⁺⁺ (1 mM) together with paxilline (a, 10 μM, Ca⁺⁺ + paxilline). Absence of RD in the bath presence of Ca⁺⁺ (1 mM) together with apamin (b, 50 nM, Ca⁺⁺ + apamin).

Figure 8

Effects of intracellular application of EGTA or BAPTA on RD. (Aa) Current protocol applied to evoke RDs shown in (Ab,c,Ba,b) (compare Figure 1Aa). (b) Voltage responses evoked by pulse amplitudes of 0 pA (subthreshold) and +20 pA (threshold required to evoke RD). RD was evoked at the beginning of cell “dialysis” with EGTA (10 mM) in the absence of Ca⁺⁺ in the extracellular solution (control). (c) RD was not evoked after 60 min of cell “dialysis” with EGTA in the bath presence of Ca⁺⁺ (1 mM, Ca⁺⁺ + EGTA) (b and c show the results obtained from the same neuron). (Ba) RD was evoked at the beginning of cell “dialysis” with BAPTA (100 μM) in the absence of Ca⁺⁺ (control). (b) RD was evoked in the same neuron after 60 min of cell “dialysis” with BAPTA despite the presence of Ca⁺⁺ in the extracellular solution (1 mM, Ca⁺⁺ + BAPTA). Voltage responses were evoked by 200-ms current steps to 0 pA (subthreshold) and to +20 pA (threshold current step required to evoke RD). (C) The RD duration measured at the beginning of cell “dialysis” with EGTA (a, control) or BAPTA (b, control) in the absence of Ca⁺⁺ in the extracellular solution. RD could not be evoked after 60 min of cell dialysis with EGTA (a, Ca⁺⁺ + EGTA) in the bath presence of Ca⁺⁺ (1 mM). RD was evoked after 60 min of cell dialysis with BAPTA (b, Ca⁺⁺ + BAPTA), despite the presence of Ca⁺⁺ (1 mM) in the extracellular solution; n.s., non-significant.

Figure 9

Effects of Ba⁺⁺, Li⁺ and ouabain on RD in the absence of Ca⁺⁺ in the extracellular solution. (A) Duration of RD before (control) and after a 15-min application of Ba⁺⁺ (Ba⁺⁺, 200 μM) to the extracellular solution (a) and before (control) and after replacing 125 mM NaCl with 125 mM LiCl (Li⁺) in the extracellular solution (b). (B) Duration of RD before (control) and after a 15-min application of 100 μM of ouabain (ouabain). The threshold RDs were evoked by application of a standard protocol shown in Figure 1Aa.

Figure 10

Effects of Cd⁺⁺, NNC 55-0396, isradipine and NPS 2143 on RD. (Aa) Current protocol applied to evoke voltage changes shown in (b,c) (compare Figure 1Aa). (b) RD was not evoked in the presence of Ca⁺⁺ (1 mM) and the absence of Cd⁺⁺ in the bath in response to 200-ms current pulses from −20 pA to +160 pA. (c) RD evoked by a threshold 200-ms current pulse to 0 pA in the bath presence of Ca⁺⁺ (1 mM) and Cd⁺⁺ (50 μM). (d) Absence of the RD in the bath in the presence of only Ca⁺⁺ (Ca⁺⁺, 1 mM). RD could be evoked in the bath in the presence of Ca⁺⁺ (1 mM) together with Cd⁺⁺ (50 μM, Ca⁺⁺ + Cd⁺⁺). (Ba) Current protocol applied to evoke voltage changes shown in (b,c) (compare Figure 1Aa). (b) RD was not evoked in the presence of Ca⁺⁺ (0.1 mM) in the bath in response to 200-ms current pulses from −20 pA to +160 pA. (c) RD evoked by a threshold 200-ms current pulse to 0 pA in the presence of Ca⁺⁺ (0.1 mM) together with the T-type Ca⁺⁺ channel blocker NNC 55-0396 (50 μM) in the bath. (d) The RD duration in the presence of only 0.1 mM of Ca⁺⁺ (Ca⁺⁺) or Ca⁺⁺ (0.1 mM) together with the T-type channel blocker NNC 55-0396 (50 μM) (d, Ca⁺⁺ + NNC 55-0396) in the extracellular solution. (e) The RD duration in the presence of only Ca⁺⁺ (Ca⁺⁺, 0.1 mM) or Ca⁺⁺ (0.1 mM) together with the L-type channel blocker isradipine (10 μM; Ca⁺⁺ + isradipine) in the extracellular solution. (Ca) Current protocol applied to evoke voltage changes shown in (b,c) (compare Figure 1Aa). (b) RD was not evoked in the presence of only Ca⁺⁺ (0.1 mM) in the bath in response to 200-ms current pulses from −20 pA to +160 pA. (c) RD was evoked in response to a 200-ms threshold current pulse to 0 pA in the presence of Ca⁺⁺ (0.1 mM) together with calcium sensing receptor (CaSR) blocker (NPS 2143, 3 μM) in the bath. (d) The RD was not evoked in the presence of only 0.1 mM of Ca⁺⁺ (Ca⁺⁺). RD duration in the bath presence Ca⁺⁺ (0.1 mM) together with NPS 2143 (3 μM) (Ca⁺⁺ + NPS 2143).

Figure 11

Effects of phorbol 12-myristate 13-acetate (PMA), 4αPMA, Aβ_1–42 and IgG on RD. (Aa) Current protocol applied to evoke voltage changes shown in (b–d) (compare Figure 1Aa). (b) RD was not evoked in response to 200-ms current pulses from −20 pA to +160 pA in the presence of Ca⁺⁺ (1 mM) in the bath. (c) RD was evoked by the 200-ms threshold current pulse to 0 pA in the bath presence of PMA (1 μM) and Ca⁺⁺ (1 mM). (d) The absence of RD in the presence of an inactive analog of PMA (4α-phorbol 12-myristate 13-acetate, 4α-PMA, 2 μM) together with Ca⁺⁺ (1 mM) in the bath. (e) Absence of RD in the bath presence of only 1 mM of Ca⁺⁺ (Ca⁺⁺) or Ca⁺⁺ (1 mM), together with an inactive analog of the protein kinase C (PKC) activator 4α-PMA (2 μM, Ca⁺⁺ + 4α-PMA). Duration of RD in the bath presence of Ca⁺⁺ (1 mM) together with the PKC activator PMA (1 μM, Ca⁺⁺ + PMA). The voltage traces shown in (b,c) were obtained from the same neuron. (Ba) Current protocol applied to evoke voltage changes shown in (b–d) (compare Figure 1Aa). (b) RD was not evoked by current pulses from −20 pA to +160 pA in the presence of Ca⁺⁺ (0.3 mM) in the bath. (c) RD was evoked by a threshold 0 pA 200-ms pulse when the pipette solution contained Aβ_1–42 (10 μM) and Ca⁺⁺ (0.3 mM) was present in the bath. (d) Absence of RD when the pipette solution contained IgG (4 μg/ml) and the extracellular solution contained Ca⁺⁺ (0.3 mM). The voltage traces shown in (b,c) were obtained from the same neuron. (e) The absence of RD in the bath presence of Ca⁺⁺ (0.3 mM) only or in the bath presence of Ca⁺⁺ (0.3 mM) together with IgG (4 μg/ml, Ca⁺⁺ + IgG) in the pipette solution. The RD was evoked in 7 from 9 tested neurons in the bath presence Ca⁺⁺ (0.3 mM), together with the presence of Aβ_1–42 (10 μM) in the pipette solution (Ca⁺⁺ + Aβ_1–42).