Cav 1.3 channels play a crucial role in the formation of paroxysmal depolarization shifts in cultured hippocampal neurons

Abstract

Objective: An increase of neuronal Ca_v 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca²⁺ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Ca_v 1.2 and Ca_v 1.3, in PDSs remained unknown. Moreover, Ca²⁺ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking.

Methods: Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Ca_v 1.2 mutant (Ca_v 1.2DHP^-/- mice) or from Ca_v 1.3^-/- knockout mice. To investigate the role of Ca_v 1.2 and Ca_v 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist).

Results: Distinct PDS could be elicited upon LTCC potentiation in Ca_v 1.2DHP^-/- neurons but not in Ca_v 1.3^-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Ca_v 1.3^-/- neurons but not in Ca_v 1.2DHP^-/- neurons. Because only the Ca_v 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity.

Significance: Our data suggest that the LTCC requirement of PDS relates primarily to Ca_v 1.3 channels rather than to Ca_v 1.2 channels and CAN channels in hippocampal neurons. Hence, Ca_v 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

Keywords: CAN channels; Epileptogenesis; L-type voltage-gated calcium channels; Primary cultured hippocampal neurons.

Figure 1. Proposed mechanisms of PDS generation.

A Recording of an original type of PDS that illustrates activation by summing up of excitatory inputs (indicated by the blue arrows) to a giant post-synaptic potential that triggers action potential firing (1), which turns after spike-decline into a plateau phase (2). After the plateau, the membrane potential may return back to baseline (dashed line) or transiently to an even more hyperpolarized potential (afterhyperpolarization) as in the example shown (3). B The two schemes illustrate the conductances that have been proposed to underlie the formation of original PDS. Phase 1 in A is mediated by excitatory synaptic input, presumably via AMPA type glutamate receptors. The decline in action potential amplitude is thought to be due to progressive inactivation of voltage-gated sodium channels. The plateau phase (2) may be largely due to L-type mediated Ca²⁺ inward current (I_Ca ²⁺), allegedly together with cationic inward current via CAN channels (I_CAN). Afterhyperpolarizations (3) are considered to be due to K⁺-efflux and/or Cl^--influx via Ca^2+-activated K⁺ and Cl^- channels. Note that this figure is based on a textbook figure published by Speckmann and Walden (1993) to illustrate the proposed role of CAN channels. It has been modified with respect to the prevailing view of an initiation of the PDS by excitatory postsynaptic potentials^{, ,} , whereas in the original depiction by Speckmann and colleagues the onset was described to be caused directly by inward calcium current.

Figure 2. Bicuculline-induced PDS require Ca_v1.3 channels.

A, B Suprathreshold electrical events recorded in Ca_v1.2DHP^-/- neurons (A) or Ca_v1.3^-/- neurons (B) after co-administration of bicuculline (10 μM) and BayK (3 μM). C-F Analysis of the area (C, D) and duration (E, F) of all suprathreshold electrical events recorded in the presence of bicuculline in Ca_v1.2DHP^-/- neurons (C, E) or Ca_v1.3^-/- neurons (D, F) under three conditions of LTCC activity: endogenous activity (DMSO control), potentiated (BayK, 3 μM) and inhibited (isradipine, 3 μM). The figures in the bars indicate the total number of events evaluated. All statistically significant differences are indicated by horizontal brackets. G, H Overlay of typical suprathreshold electric events recorded from Ca_v1.2DHP^-/- neurons (G) or Ca_v1.3^-/- neurons (H) from the experiments analysed in C to F.

Figure 3. Caffeine-induced PDS require Ca_v1.3 channels.

A, B Suprathreshold electrical events recorded in Ca_v1.2DHP^-/- neurons (A) or Ca_v1.3^-/- neurons (B) after co-administration of caffeine (1 mM) and BayK (3 μM). C-F Analysis of the area (C, D) and duration (E, F) of suprathreshold electrical events recorded in the presence of caffeine in Ca_v1.2DHP^-/- neurons (C, E) or Ca_v1.3^-/- neurons (D, F) under three conditions of LTCC activity: endogenous activity (DMSO control), potentiated (BayK, 3 μM) and inhibited (isradipine, 3 μM). The figures in the bars indicate the total number of events evaluated. All statistically significant differences are indicated by horizontal brackets. G, H Overlay of typical suprathreshold electric events recorded from Ca_v1.2DHP^-/- neurons (G) or Ca_v1.3^-/- neurons (H) from the experiments analysed in C to F.

Figure 4. Potentiation of Ca_v1.2 but not Ca_v1.3 channels gives rise to pronounced afterdepolarizations.

A, B Voltage response to brief current injections (50 to 1050 ms duration, increased in 4 steps of 250 ms) that cause depolarizations up to -20 mV in 0.5 μM tetrodotoxin-silenced neurons are shown together with their afterpotentials upon LTCC channel potentiation with 3 μM BayK. Distinct afterdepolarizations (ADP) can be seen in Ca_v1.3^-/- neurons (A) but not in Ca_v1.2DHP^-/- neurons (B). C Summary graph of experiments such as the one illustrated by original traces in A and B for ADP areas of 14 Ca_v1.3^-/- neurons and of 10 Ca_v1.2DHP^-/- neurons. Data (shown as mean + standard deviation) were normalized to the ADP area at 1050 ms current-injection duration, which is set to 1 for Ca_v1.3^-/- neurons and 0.077 for Ca_v1.2DHP^-/- neurons, which corresponds to the mean ADP area at this current-injection duration in relation to the mean ADP area of Ca_v1.3^-/- neurons. n.s., not significant. (***) indicates that the statistical test had to be been done on the raw data rather than the normalized one for this data point. D, E Overlay of voltage responses to similar current injections in the presence of BayK and isradipine of Ca_v1.3^-/- neurons and of Ca_v1.2DHP^-/- neurons. Afterpotentials are blocked by 3 μM isradipine. Note that the y-axis shown in B also applies for the traces in D and E. The graphs in the inserts compare the maximum ADP areas (shown as mean + SEM) that were elicited in the presence of BayK or isradipine (isra) of Ca_v1.3^-/- neurons (D, n= 10) and of Ca_v1.2DHP^-/- neurons (E, n=15). n.s., not significant.

Figure 5. Implication of Ca_v1.2 and Ca_v1.3 channels in seizure-like activity.

A, B Examples of seizure-like activity (SLA) induced by co-application of bicuculline (10 μM) and BayK (3 μM) in Ca_v1.2DHP^-/- neurons (A) or Ca_v1.3^-/- neurons (B). C-F Analysis of the area (C, D) and duration (E, F) of SLA induced by 10 μM bicuculline in Ca_v1.2DHP^-/- neurons (C, E) or Ca_v1.3-/- neurons (D, F) under three conditions of LTCC activity: endogenous activity (DMSO control), potentiated (BayK, 3 μM) and inhibited (isradipine, 3 μM). The figures in the bars indicate the total number of events evaluated. All statistically significant differences are indicated by horizontal brackets. G, H Overlay of typical SLA recorded from Ca_v1.2DHP^-/- neurons (G) or Ca_v1.3^-/- neurons (H) from the experiments analysed in C to F.