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. 2016 Jul 22;17(7):1191.
doi: 10.3390/ijms17071191.

Revisiting the Lamotrigine-Mediated Effect on Hippocampal GABAergic Transmission

Yu-Yin Huang  1   2 , Yu-Chao Liu  3 , Cheng-Ta Lee  4 , Yen-Chu Lin  5 , Mong-Lien Wang  6 , Yi-Ping Yang  7   8   9 , Kaung-Yi Chang  10 , Shih-Hwa Chiou  11   12   13
Affiliations

Revisiting the Lamotrigine-Mediated Effect on Hippocampal GABAergic Transmission

Yu-Yin Huang et al. Int J Mol Sci. .

Abstract

Lamotrigine (LTG) is generally considered as a voltage-gated sodium (Nav) channel blocker. However, recent studies suggest that LTG can also serve as a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel enhancer and can increase the excitability of GABAergic interneurons (INs). Perisomatic inhibitory INs, predominantly fast-spiking basket cells (BCs), powerfully inhibit granule cells (GCs) in the hippocampal dentate gyrus. Notably, BCs express abundant Nav channels and HCN channels, both of which are able to support sustained action potential generation. Using whole-cell recording in rat hippocampal slices, we investigated the net LTG effect on BC output. We showed that bath application of LTG significantly decreased the amplitude of evoked compound inhibitory postsynaptic currents (IPSCs) in GCs. In contrast, simultaneous paired recordings from BCs to GCs showed that LTG had no effect on both the amplitude and the paired-pulse ratio of the unitary IPSCs, suggesting that LTG did not affect GABA release, though it suppressed cell excitability. In line with this, LTG decreased spontaneous IPSC (sIPSC) frequency, but not miniature IPSC frequency. When re-examining the LTG effect on GABAergic transmission in the cornus ammonis region 1 (CA1) area, we found that LTG markedly inhibits both the excitability of dendrite-targeting INs in the stratum oriens and the concurrent sIPSCs recorded on their targeting pyramidal cells (PCs) without significant hyperpolarization-activated current (Ih) enhancement. In summary, LTG has no effect on augmenting Ih in GABAergic INs and does not promote GABAergic inhibitory output. The antiepileptic effect of LTG is likely through Nav channel inhibition and the suppression of global neuronal network activity.

Keywords: GABAergic interneuron; Lamotrigine; hyperpolarization-activated current; inhibitory postsynaptic current; voltage-gated sodium channel.

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Figures

Figure 1
Figure 1
Suppression of somatic GABAergic transmission onto GCs by LTG. (A) Schematic of experiment configuration: A stimulating electrode placed at a distance of 100–200 µm from a recorded GC within the GCL. ML, molecular layer; GCL, granule cell layer; (B) (Top) Exemplar average compound IPSCs (cIPSCs) (15–20 sweeps) recorded in the control, in LTG (100 µM), after LTG washout and after the addition of Gabazine (SR95531); (Bottom) Plot of the peak amplitudes of cIPSC against time; (C) Plot of the mean peak amplitude of cIPSC (n = 10) against time. Data were normalized to the baseline before LTG application. Symbols indicate the mean; error bars indicate SEM; (D) Dose-response relationship of cIPSC inhibition by LTG (1, 10, 30, 100 and 1000 µM). Data fitted to a single Hill equation with IC50 = 121.2 µM and Hill coefficient = 1.51. Each point represents the average from 5–14 experiments, as given in parentheses; error bars indicate SEM.
Figure 2
Figure 2
LTG had little effect on GABA release at BC-GC synapses. (A) Schematic diagram showing the BC-GC paired recording configuration. OML, outer molecular layer; IML, inner moleucular layer; (B) The 25-Hz bursts of five presynaptic APs (red) and postsynaptic unitary IPSC (uIPSC) traces (black, average of 25–30 sweeps) in the control (Ctrl) and after bath perfusion of LTG (100 µM); (C) Summary of the normalized uIPSC1 mean peak amplitude from five BC-GC pairs against time. Symbols indicate the mean; error bars indicate SEM; (D) Mean ratio of uIPSCn/uIPSC1 plotted against the number within the train (n).
Figure 3
Figure 3
LTG decreased the frequency of spontaneous IPSCs (sIPSCs), but not miniature IPSCs (mIPSCs) in GCs. (A1) (Top) Recording configuration: Whole-cell voltage-clamp recordings from a GC at −70 mV in the DG. CA1, cornus ammonis region 1; (Middle and bottom) Traces of sIPSCs recorded before (black) and after (gray) LTG (100 µM) application; (A2,A3) Cumulative distributions of sIPSC inter-event intervals (A2) and amplitudes (A3) from the control (black; n = 10) and LTG (gray; n = 10). Insets show the bar graph summaries of the averages. ** p < 0.01; (B1) (Top) Traces of mIPSCs recorded at −70 mV in the control (black); (Bottom) Events in the presence of LTG (100 µM) application (gray); (B2,B3) Cumulative distributions of mIPSC inter-event intervals (B2) and amplitude (B3) in the control (black, n = 10) and LTG (gray, n = 10). Insets show the bar graph summaries of the averages.
Figure 4
Figure 4
LTG suppressed BC excitability. (A) Reconstruction of a biocytin-filled BC whose axon arborized in the GCL. Soma and dendrites are shown in red, and axons are in black; (B) Exemplar traces of APs evoked by 1-s depolarizing current pulses (400, 800, 1200 pA) in the presence of synaptic blockers. Black traces, control; gray traces, LTG. Cells were held at −70 mV by holding the current adjustment throughout the experiment; (C) Mean AP frequency plotted against the injected current (n = 10). ** p < 0.01; (D) Exemplar voltage traces recorded during 1-s hyperpolarizing current pulses (−100 pA and −300 pA, respectively) under whole-cell current-clamp before (black traces) and after 100 µM LTG (gray traces) application; (E,F) Summary bar graphs of the effect of 100 µM LTG on (E) input resistance and (F) the sag ratio (voltage change at the end of the 1-s pulse/maximal voltage change or −300 pA current injection) with no significant difference under control and LTG conditions (n = 9). ns, no significance; (G) Summary of the plot of the spontaneous spike frequency against time illustrating the effect of 100 µM LTG (n = 10). BCs were slightly depolarized to fire persistent APs by sustained somatic current injection (100–200 pA); (H) (Top) A stimulating electrode (monopolar glass pipette) placed in the GCL at a distance of 100–300 µm from the recorded BC; (Bottom) 10 consecutive spikes of APs in the control (stimulus intensity 25 µA) (left), in the presence of 100 µM LTG at the same stimulus intensity (middle) and in the presence of 100 µM LTG after the increase in stimulation intensity (right, 29 µA); (I) Spike probability plotted against stimulus intensity in the control (black) and after LTG (gray). The dashed line indicates that LTG increases the threshold for spike initiation (current leading to 50% successes); (J) Summary of the LTG effect on the AP threshold. Data were normalized to the control from five BCs. * p < 0.05.
Figure 5
Figure 5
LTG decreased sIPSC frequency in CA1 PCs without affecting mIPSC frequency. (A1) (Top) Whole-cell voltage-clamp recordings from a CA1 PC; (Middle and bottom) Traces of sIPSCs recorded at −70 mV in the control (black) and LTG (100 µM) (gray); (A2,A3) Cumulative distributions of sIPSC inter-event intervals (A2) and amplitudes (A3) from the control (black) and LTG (gray). Insets show the bar graph summaries of the cell averages (n = 8) for the frequency (A2) and amplitude (A3). ** p < 0.01; (B1) (Top) Traces of mIPSCs recorded at −70 mV in control conditions (black); (Bottom) Events in the presence of LTG (100 µM) application (gray); (B2,B3) Cumulative distributions of mIPSC inter-event intervals (B2) and amplitudes (B3) from the control (black) and LTG (gray). Insets show the bar graph summaries of the ell averages (n = 6) for the frequency (B2) and amplitude (B3), respectively. * p < 0.05.
Figure 6
Figure 6
LTG suppressed oriens-lacunosum moleculare (O-LM) IN excitability without affecting Ih. (A) Reconstruction of a biocytin-filled O-LM IN. Soma and dendrite are shown in light blue, and axons are in black. SLM, stratum lacunosum moleculare; SR, stratum radiatum; SP, stratum pyramidal; SO, stratum oriens; (B) Exemplar AP traces from whole-cell current-clamp recordings of control (black) and LTG (gray) in an O-LM IN evoked by 1-s depolarizing current steps in the presence of synaptic blockers; (C) The AP frequency was plotted against the stepwise stimulus intensity (100~600 pA) and revealed significant inhibition by LTG (n = 8). ** p < 0.01; (D) Voltage responses to 1-s hyperpolarizing (−100 pA and −300 pA) current pulses from the same cell were recorded; (E) Summary of the input resistance. ns, no significance; (F) Summary of the sag ratio (voltage change at the end of the 1-s pulse/maximal voltage change or −300 pA current injection) with no significant difference under the control and LTG condition; (G) (Top) In a resting membrane potential around −60 mV, an O-LM IN generated spontaneous firing and was abolished by LTG (100 µM); (Bottom) Plot of normalized induced spontaneous spike frequency against time illustrating the effect of 100 µM LTG (n = 8); (H) Currents activated by hyperpolarizing pulse from a holding potential of −50 mV−120 mV with an increment of 10 mV under voltage clamp mode from a representative O-LM IN in the control condition, with LTG (100 µM) and 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidiniumchloride (ZD7288) (50 µM); (I) Plotting the ZD7288-sensitive current (current of the control or LTG with digital subtraction of the ZD current) against hyperpolarizing voltage steps before and after LTG application demonstrated that LTG had no significant effect on Ih (n = 6); (J) The same protocol as (H) was made in CA1 PC; (K) Plotting the ZD7288-sensitive current against hyperpolarizing voltage steps before and after LTG application demonstrated that LTG had no significant effect on Ih (n = 6).
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