Phenobarbital but Not Diazepam Reduces AMPA/kainate Receptor Mediated Currents and Exerts Opposite Actions on Initial Seizures in the Neonatal Rat Hippocampus

Abstract

Diazepam (DZP) and phenobarbital (PB) are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We have now compared the effects of DZP and PB on ictal-like events (ILEs) in an in vitro model of mirror focus (MF). Using the three-compartment chamber with the two immature hippocampi and their commissural fibers placed in three different compartments, kainate was applied to one hippocampus and PB or DZP to the contralateral one, either after one ILE, or after many recurrent ILEs that produce an epileptogenic MF. We report that in contrast to PB, DZP aggravated propagating ILEs from the start, and did not prevent the formation of MF. PB reduced and DZP increased the network driven giant depolarizing potentials suggesting that PB may exert additional actions that are not mediated by GABA signaling. In keeping with this, PB but not DZP reduced field potentials recorded in the presence of GABA and NMDA receptor antagonists. These effects are mediated by a direct action on AMPA/kainate receptors since PB: (i) reduced AMPA/kainate receptor mediated currents induced by focal applications of glutamate; (ii) reduced the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature excitatory postsynaptic currents (EPSCs); (iii) augmented the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in MF. Therefore, PB exerts its anticonvulsive actions partly by reducing AMPA/kainate receptors mediated EPSCs in addition to the pro-GABA effects. We suggest that PB may have advantage over DZP in the treatment of initial neonatal seizures since the additional reduction of glutamate receptors mediated signals may reduce the severity of neonatal seizures.

Keywords: AMPA; diazepam; immature hippocampus; phenobarbital; seizures.

Figure 1

Diazepam (DZP) aggravates the propagating ictal-like events (ILEs) and does not prevent the formation of a mirror focus by repeated ILEs. (A,B) The scheme of the triple chamber preparation with the two intact hippocampi and their connecting inter-hemispheric commissure in independent chambers (left). Field recordings were made in the two hippocampi. (A) Kainate (KA) applied to one hippocampus (ipsilateral: ipsi-) generated seizure activity referred to as ILE that propagated to the contralateral hippocampus (contra-). The ILEs included high frequency gamma oscillations (GOs, 40–120 Hz) as shown in the time–frequency (TF) analysis (A₁). (B) Application of DZP to contralateral hippocampus (2 μM, 15 min) before a second application of KA increased the ILE and GOs. TF analysis of ILE recorded in contra- is shown in (B₁). (C) Superimposed ILEs [from (A,B)] recorded in contra- before (black) and after DZP application (red). DZP increased the amplitude and duration of ILE. (C₁) Power spectra representation of ILEs recorded before (black) and after (red) DZP treatment [from (C)] after band-pass (40–150 Hz) filtering. (D) Example of power spectra of ILEs before and after DZP treatment [from (A,B)] used for calculation of power of ILEs. (D₁) Normalized average power histogram of contralateral ILEs before and after DZP. Note that DZP application significantly increased average power of ILEs (by 19.2 ± 3.9%, n = 7 experiments, p < 0.05). (E) After 15 transient applications of KA in ipsi- with continuous application of DZP in contra-, disconnection of the two hippocampi by applications of TTX to the commissural chamber revealed that contralateral hippocampus generated spontaneous ILEs.

Figure 2

Diazepam aggravates the severity of epileptiform activities in the isolated mirror focus. (Aa) Superimposed ILEs generated spontaneously in the contralateral hippocampus after formation of a MF before (black) and after (gray) DZP application. (Ab) Power spectra of spontaneous ILEs before and after DZP treatment [from (Aa)]. (Ac) Average power histogram of spontaneous ILEs before and after DZP. DZP application significantly increased power of ILEs (by 80 ± 19.8%, n = 4, p < 0.001). (Ba) Extracellular recording in slices obtained from contralateral hippocampus after the formation of a MF. Slices generated spontaneous IILEs and multi-unit activities (MUAs) continuously that were reversibly aggravated by DZP (2 μM). (Bb) Quantification of IILEs from a single experiment [from (Ba)]. (Bc) Average quantification histogram of IILEs. (Bd) Quantification of spikes from single experiment [from (Ba)] and (Be) the average quantification histogram. Note that DZP increased the frequency of IILEs (by 128.7 ± 12.2%, n = 6, p < 0.001) and spikes (by 88.6 ± 16.2%, n = 6, p < 0.001).

Figure 3

Diazepam increases ongoing neuronal activity and network driven GDPs. (A) Extracellular recording of the Giant Depolarizing Potentials (GDPs) and multi-unit activities (MUAs) in control slices to record network driven events and individual neuronal spikes, respectively. Non-filtered faster display traces are depicted below to show spikes. Note the increase of frequency of GDPs (B) and spikes (D) by DZP: (B–D) quantification of recorded GDPs and spikes from (A), Bin = 20 s. (C–E) Quantification of mean frequency of GDPs and spikes. DZP enhanced the frequency of GDPs (C) by 215.1 ± 13.6 (p < 0.001, n = 14) and spikes (E) by 170 ± 14.8% (p < 0.001, n = 14).

Figure 4

DZP enhances GABA excitation in naive neurons. (A) Experimental design. Simultaneous whole cell patch clamp recordings of a CA3a pyramidal neuron and field recordings in the CA3c pyramidal layer were made in control slices and GABA (100 μM) was focally applied (100 ms) by puff in the CA3c part of the pyramidal layer. (B) GABA puff generated a field and whole cell recorded GDPs (Ba) that was strongly augmented by DZP (Bb). The responses returned to control values after wash out of DZP (Bc). (C–E) Quantification of the effects of DZP on whole cell recorded postsynaptic currents: the amplitudes of the currents, the decay time and the area of whole cell recorded responses were significantly augmented by DZP. (F) Quantification histogram of mean number of spikes associated with puff GABA generated GDPs (a time window of 1 s from the GDP onset from GABA puff was analyzed).

Figure 5

Phenobarbital but not diazepam blocks AMPA/kainate mediated inter ictal-like events (IILEs). (Aa,Ba) Extracellular recordings of spontaneous and evoked (electrical stimulation every 30 s) IILEs generated in control slices in the presence of GABA receptors antagonists – bicuculline (10 μM), CGP (2 μM), – and NMDA receptors antagonist – APV (40 μM). Non-filtered faster display traces of evoked (Ab,Bb,Cb) and spontaneous (Ac,Bc,Cc) IILEs. Generation of IILEs in these conditions is mediated by AMPA/kainate receptors as they were reversibly blocked by CNQX (C). Note that PB but not DZP completely blocked IILEs and reduced the frequency of spikes (Ad,Bd).

Figure 6

Phenobarbital but not diazepam reduces AMPA/kainate mediated currents. (A–C) Whole cell patch clamp recordings of currents induced by focal application of glutamate (100 μM, 100 ms) in the presence APV (40 μM). (Ae–Ce) Histograms of normalized averaged amplitudes of glutamate induced currents showing the effects of PB (Ae), DZP (Be), and CNQX (Ce). Note that phenobarbital but not diazepam significantly reduced the amplitude AMPA/kainate receptor mediated postsynaptic currents (67.2 ± 8.1%, p < 0.001, n = 4). NS, not statistically significant.

Figure 7

Phenobarbital reduces AMPA receptor mediated currents in nucleated patches. (A) Currents evoked in nucleated patches from CA3 pyramidal neurons from control slices. L-Glutamate (Glu, 1 mM) was applied in 2 ms pulses at a holding potential of −60 mV. Each trace is the average of 4–8 sweeps. Note that PB [(A) red trace] reduced the amplitude of AMPA/kainate mediated currents. (B) PB significantly reduced (by 25.5 ± 0.5%, p < 0.001, n = 8) the averaged amplitude of AMPA/kainate receptor mediated currents recorded in nucleated patches.

Figure 8

Phenobarbital does not reduce the NMDA receptor mediated currents. (A,Da,b) Whole cell patch clamp recordings (V_clamp = +40 mV) of currents induced by focal application of glutamate (100 μM, 100 ms, arrows) in the presence of bicuculline (10 μM), CGP (2 μM), and CNQX (20 μM; antagonist of AMPA/kainate receptors). Averaged NMDA receptor mediated currents before (Aa), in the presence (Ab), and after wash out of PB (Ac) recorded from the same pyramidal cell. (Ba) Histogram of averaged (n = 11) amplitudes of NMDA receptor mediated currents. (Bb) Histogram of averaged amplitudes normalized to control. Averaged NMDA receptor mediated currents before (Ca) and in the presence of DZP (Cb). (Cc) Histogram of averaged (n = 5) current amplitudes normalized to control. NS, not statistically significant.

Figure 9

Pre- and postsynaptic actions of PB on AMPA/kainate receptor mediated excitatory postsynaptic synaptic currents (EPSCc). (A) In the presence of APV (40 μM) and bicuculline (10 μM), single fibers were electrically stimulated to evoke a mono synaptic AMPA receptor mediated EPSCs. The stimulation was adjusted to induce ∼20–50% failures [shown as red trace in (Aa2)]. (Aa–c) Single experiment obtained with repeated stimuli (30 stimuli every 10 s). (Aa) Control, (Ab) PB strongly enhanced the number of failures and reduced the average amplitude of EPSCs [see also (Ae)], (Ac) wash out of PB. (Ad) Superimposed averaged responses (the failures were discarded) from (Aa–c) to illustrate the effects of PB. Note the reduction of the averaged amplitude of EPSCs in the presence of PB (red trace). (Ae) Quantification of the mean number of normalized failures. Failure rate presented as “percentage of failure” (number of failure/total number of stimulation). Note the highly significant increase of the number of failures in the presence of PB (p < 0.001, n = 4 experiments). (B) PB reduces the amplitude of miniature AMPA receptor mediated EPSCs (mEPSCs). APV (40 μM) and bicuculline (10 μM) were applied throughout the recordings as well as TTX (1 μM) to generate mEPSCs. (Ba–c) Single traces to illustrate the actions of PB on AMPA receptor mediated mEPSCs. Note that PB (middle trace) reduced the amplitude of the minis. (Bd) Superimposed averaged traces of single mEPSCs to illustrate the effects of PB [from (Ba–c)]. Note the reduction of the mEPSCs amplitude (red trace). (Be) Quantification of the effects of PB. Note that PB reduced significantly (by 28.9%; from 25.9 ± 1.4 to 20.1 ± 0.9 pA, p < 0.01, n = 5 cells from different slices) the averaged amplitude of mEPSCs. PB also reduced the frequency of mini EPSCs (see text). (C) Cumulative probability distributions of mEPSCs amplitude in the presence of PB (red curve) demonstrate a leftward shift as compared with control and wash out. (D) Cumulative probability distribution of mEPSCs inter-event intervals in the presence of PB demonstrates a rightward shift as compared with control and wash out.

Figure 10

Phenobarbital reduces AMPA/kainate receptors field potentials in mirror focus neurons. (A) Extracellular field recording of spontaneous epileptiform activities in slices obtained from contralateral hippocampus after mirror focus formation. Recordings were performed in the presence of GABA(A) and GABA(C) receptors antagonist PTX (100 μM), GABA(B) receptor antagonist CGP (2 μM), and NMDA receptor antagonist APV (40 μM). (Ab) Example of epileptiform activities recorded in an “epileptic” slice from mirror focus before application of antagonists. (Ac–e) Examples of ILEs in the presence of GABA and NMDA receptors antagonists: before application (Ac), in the presence (Ad), and after PB wash out (Ae). Note the significant decrease of amplitude and frequency of ictal-like events. Mean interval between ictal-like events (double arrows) significantly increased (by 318.1 ± 66.5%, not shown). (B) Power spectrum [from (Ac–d)] showing the decrease of amplitude and frequency by PB. (C) Average power histogram of spontaneous ictal-like events before and after application of PB. PB significantly decreased the power of epileptiform events (by 26.9 ± 7.2%, n = 5, p < 0.001).