Depression of Neuronal Excitability and Epileptic Activities by Group II Metabotropic Glutamate Receptors in the Medial Entorhinal Cortex

Abstract

Whereas the ionotropic glutamate receptors are the major mediator in glutamatergic transmission, the metabotropic glutamate receptors (mGluRs) usually play a modulatory role. Whereas the entorhinal cortex (EC) is an essential structure involved in the generation and propagation of epilepsy, the roles and mechanisms of mGluRs in epilepsy in the EC have not been determined. Here, we studied the effects of activation of group II metabotropic glutamate receptors (mGluRs II) on epileptiform activity induced by picrotoxin or deprivation of extracellular Mg²⁺ and neuronal excitability in the medial EC. We found that activation of mGluRs II by application of the selective agonist, LY354740, exerted robust inhibition on epileptiform activity. LY354740 hyperpolarized entorhinal neurons via activation of a K⁺ conductance and inhibition of a Na⁺-permeable channel. LY354740-induced hyperpolarization was G protein-dependent, but independent of adenylyl cyclase and protein kinase A. However, the function of Gβγ was involved in mGluRs II-mediated depression of both neuronal excitability and epileptiform activity. Our results provide a novel cellular mechanism to explain the antiepileptic effects of mGluRs II in the treatment of epilepsy.

Introduction

Glutamate is the principal excitatory neurotransmitter in the brain where it interacts with ionotropic (NMDA, AMPA and kainate) and metabotropic glutamate receptors (mGluRs) (Dingledine et al., 1999; Riedel et al., 2003). Whereas the ionotropic glutamate receptors mediate the fast excitatory synaptic transmission, the mGluRs usually play a modulatory role in the brain. mGluRs are G protein-coupled receptors that are linked to various intracellular second messenger cascades (Conn and Pin, 1997; Pin and Acher, 2002). Group I mGluRs (mGluR₁ and mGluR₅) are coupled to G_q/11 proteins. Activation of this receptor group results in the activation of phospholipase C (PLC) leading to an increase in intracellular Ca²⁺ release and the activation of protein kinase C (PKC) (Conn and Pin, 1997; Pin and Acher, 2002). Group II (mGluR₂ and mGluR₃) and III (mGluR₄ and mGluR_6–8) mGluRs are coupled to G_i/o proteins. These two groups of receptors are negatively linked to adenylyl cyclase (AC) leading to a reduction in the intracellular level of cyclic AMP and an inhibition of protein kinase A (PKA) (Conn and Pin, 1997; Pin and Acher, 2002). There is compelling evidence demonstrating that these three groups of mGluRs modulate epilepsy in distinct manners; activation of group I mGluRs facilitates whereas activation of group II and III mGluRs inhibits epilepsy (Moldrich et al., 2003). However, the cellular and molecular mechanisms whereby mGluRs modulate epilepsy have not been fully determined.

The entorhinal cortex (EC) is part of a network in the limbic system closely involved in the consolidation and recall of memories (Dolcos et al., 2005; Haist et al., 2001; Squire et al., 2004; Steffenach et al., 2005), Alzheimer’s disease (Hyman et al., 1984; Kotzbauer et al., 2001), schizophrenia (Arnold et al., 1991; Falkai et al., 1988; Joyal et al., 2002; Prasad et al., 2004) and especially the temporal lobe epilepsy (Avoli et al., 2002; Spencer and Spencer, 1994). Anatomically, the EC mediates the majority of the connections between the hippocampus and other cortical areas (Witter et al., 1989; Witter et al., 2000a). Sensory inputs converge onto the superficial layers (layers II–III) of the EC (Burwell, 2000) which give rise to dense projections to the hippocampus; the axons of the stellate neurons in layer II of the EC form the major component of the perforant path that innervates the dentate gyrus and CA3 (Steward and Scoville, 1976) whereas those of the pyramidal neurons in layer III form the temporoammonic pathway that synapses onto the distal dendrites of pyramidal neurons in CA1 and the subiculum (Steward and Scoville, 1976; Witter et al., 2000a; Witter et al., 2000b). Moreover, neurons in the deep layers of the EC (layers V–VI) relay a large portion of hippocampal output projections back to the superficial layers of the EC (Dolorfo and Amaral, 1998a; Dolorfo and Amaral, 1998b; Kohler, 1986; van Haeften et al., 2003) and to other cortical areas (Witter et al., 1989). The EC expresses mGluRs (Fotuhi et al., 1994; Ohishi et al., 1993a; Ohishi et al., 1993b; Shigemoto et al., 1992) and functional changes induced by mGluRs have been observed in the EC. For instance, activation of group I mGluRs in entorhinal layer III neurons facilitates persistent firing (Yoshida et al., 2008); activation of group III mGluRs enhances glutamate release (Evans et al., 2000) and depresses spontaneous inhibition (Woodhall et al., 2001), whereas activation of group II mGluRs (defined as mGluRs II thereafter) inhibits glutamate release (Wang et al., 2012) in the EC. However, the potential effects of mGluRs II on neuronal excitability and epilepsy in the EC have not been determined. In the present study, we examined the effects of mGluRs II on neuronal excitability and the epileptiform activity induced by application of the GABA_A receptor blocker, picrotoxin (PTX), or by deprivation of extracellular Mg²⁺ in entorhinal slices. Our results demonstrate that activation of mGluRs II remarkably inhibits the epileptiform activities induced by PTX or by deprivation of extracellular Mg²⁺ and hyperpolarizes the medial entorhinal neurons. The hyperpolarization induced by mGluRs II is mediated by activation of a K⁺ conductance and inhibition of a Na⁺-permeable conductance. Gβγ is involved in mGluRs II-induced hyperpolarization and depression of epileptiform activity. Our results provide a novel cellular and molecular mechanism to explain the antiepileptic effects of mGluRs II in the EC.

Methods

Slice preparation

Horizontal brain slices (400 μm) including the EC, subiculum and hippocampus were cut using a vibrating blade microtome (VT1000S; Leica, Wetzlar, Germany) from 13- to 20-day-old Sprague Dawley rats as described previously (Cilz et al., 2014; Ramanathan et al., 2012; Wang et al., 2013; Xiao et al., 2009b) with slight modification (Xiao et al., 2014). Briefly, after being deeply anesthetized with isoflurane, rats were decapitated and their brains were dissected out in ice-cold saline solution that contained (in mM) 130 N-methyl-D-glucamine (NMDG)-Cl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 5.0 MgCl₂, and 10 glucose, saturated with 95% O₂ and 5% CO₂ (pH 7.4, adjusted with HCl). Slices were then incubated in the above solution except NMDG-Cl was replaced with NaCl at 35°C for 1 h for recovery and then kept at room temperature (~24°C) until use. All animal procedures conformed to the guidelines approved by the University of North Dakota Animal Care and Use Committee.

Recordings of the spontaneous epileptiform activity

Spontaneous epileptiform activity was induced by including PTX (100 μM) (Deng et al., 2006; Kurada et al., 2014; Wang et al., 2013) or no Mg²⁺ (Deng and Lei, 2008; Zhang et al., 2013) in the extracellular solution as described previously. An electrode containing the extracellular solution was placed in layer III of the medial EC to record epileptiform activity. After stable spontaneous epileptiform activity occurred, the selective agonist for mGluRs II, LY354740 (3 μM), was applied in the bath. The epileptiform events were initially recorded by Clampex 9 and subsequently analyzed by Mini Analysis 6.0.1.

Whole-cell recordings from entorhinal neurons

Whole-cell patch-clamp recordings using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) in current- or voltage-clamp mode were made from pyramidal neurons in layer III of the medial EC visually identified with infrared video microscopy (Olympus BX51WI) and differential interference contrast optics unless stated otherwise. The recording electrodes were filled with (in mM) 100 K⁺-gluconate, 0.6 EGTA, 2 MgCl₂, 8 NaCl, 33 HEPES, 2 ATPNa₂, 0.4 GTPNa and 7 phosphocreatine (pH 7.4). The extracellular solution comprised (in mM) 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 2.5 CaCl₂, 1.5 MgCl₂ and 10 glucose, saturated with 95% O₂ and 5% CO₂ (pH 7.4). Data were filtered at 2 kHz, digitized at 10 kHz, acquired on-line and analyzed after-line using pCLAMP 9.2 software (Molecular Devices, Sunnyvale, CA). The whole-cell recording configuration was used to record action potential (AP) firing from layer II stellate neurons and layer III pyramidal neurons. Because these neurons usually do not exhibit spontaneous AP firing, a positive current was injected via the recording pipettes to bring the resting membrane potential (RMP) to ~ −40 mV to induce AP firing. The preceding extracellular solution was supplemented with bicuculline (10 μM) and CGP55845 (1 μM) to block GABA_A and GABA_B responses, respectively, and DNQX (10 μM) and dl-APV (50 μM) to block glutamatergic transmission. Under these circumstances, any observed effects in response to the activation of mGluRs II should be a direct action on the recorded neurons. Agonist of mGluRs II (LY354740) was applied after the AP firing had been stable for 5~10 min. To avoid potential desensitization induced by repeated applications of the agonist, one slice was limited to only one application of LY354740. Frequency of APs was calculated by Mini Analysis 6.0.1 (Synaptosoft Inc., Decatur, GA). RMPs and holding currents (HCs) at −60 mV were recorded from layer III pyramidal neurons in the extracellular solution supplemented with TTX (0.5 μM) to block synaptic transmission. I-V curves were obtained by using a ramp protocol from −120 mV to 60 mV at a speed of 0.045 mV/ms. We compared the I-V curves recorded before and during the application of LY354740 for ~5 min when its effect was maximal. Pharmacological inhibitors were applied in one of the following ways. First, slices were pretreated with the inhibitors for >2h and the same concentration of drugs were continuously applied in the bath, unless stated otherwise. Second, drugs were included in the recording pipettes and waited for >20 min after the formation of whole-cell configuration.

Recordings of AP firing by perforated patches from layer V pyramidal neurons

Because we observed significant rundown of the AP firing frequency in layer V pyramidal neurons using whole-cell recording configuration, we utilized perforated patch-clamp recording configuration to record APs from layer V pyramidal neurons as described previously (Deng and Lei, 2007; Deng et al., 2010). Recording pipettes were tip-filled with the above-mentioned K⁺-gluconate-containing intracellular solution and then back-filled with the intracellular solution containing freshly prepared amphotericin B (200 μg/ml, Calbiochem, San Diego, CA). Patch pipettes had resistance of 6–8 MΩ when filled with the preceding solution. A 5-mV hyperpolarizing test pulse was applied every 3 s to monitor the changes of the series resistance and the process of perforation. Stable series resistances (50–70 MΩ) were usually obtained ~30 min after the formation of gigaohm seals. For those cells showing abrupt reduction in series resistance during membrane perforation suggesting the simultaneous formation of whole-cell configuration, experiments were terminated immediately. Perforated-patch configuration was verified by examining the series resistance again at the end of the experiments. Data were included for analysis only from those cells showing <15% alteration of series resistance.

Immunocytochemistry

Detailed experimental procedures for immunocytochemistry were described previously (Deng et al., 2009; Ramanathan et al., 2012; Xiao et al., 2014; Xiao et al., 2009a). Briefly, rats (18-day-old) were anaesthetized with pentobarbital sodium (50 mg/kg) and then perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Brains were rapidly removed and postfixed in the same fixative for an additional 2 h. After postfixation, brains were cryoprotected with 30% sucrose in PBS for 12 h and then cut into 20 μm slices in thickness horizontally in a Leica cryostat (CM 3050 S) at −21°C. Slices were washed in 0.1 M PBS and then treated with 0.3% H₂O₂ to quench endogenous peroxidase activity. After being rinsed in 0.1 M PBS containing 1% Triton X-100 and 1.5% normal donkey serum for 30 min, slices were incubated with the primary antibodies (rabbit anti-mGluR₂, AB9209, Millipore; mouse anti-mGluR₃, ab78366, Abcam) at a dilution of 1:200 at 4°C for 12 h. Slices were incubated at room temperature initially with biotinylated donkey anti-rabbit or mouse IgG (ABC Staining System, Santa Cruz Biotechnology Inc.) for 1 h, and then with avidin-biocytin complex (ABC Staining System) for 30 min. After each incubation, slices were washed three times for a total of 30 min. Diaminobenzidine (ABC Staining System) was used for a color reaction to detect the positive signals. Finally, slices were mounted on slides, dehydrated through an alcohol range, cleared in xylene and covered with cover-slips. Slides were visualized and photographed with a Leica microscope (DM 4000B). We stained 5–6 nonadjacent sections and each staining was repeated by using 3 rats.

Western blot

Brain tissues for western blot experiments were taken from 7 rats (18 days old). For each rat, horizontal brain slices were cut initially and the medial EC region was punched out from the slices under a microscope. The isolated brain region was lysed in tissue protein extraction buffer containing protease inhibitors (Pierce, Rockford, IL). The lysates were centrifuged at 10,000 g for 10 min to remove the insoluble materials and protein concentrations in the supernatant were determined (Bradford, 1976). An equivalent of 40 μg total protein was loaded to each lane. Proteins were separated by 12 % SDS–PAGE and transferred to the polyvinylidene difluoride (PVDF, Immobilon-P, Millipore, Billerica, MA) membranes using an electrophoretic transfer system (BioRad, Hercules, CA). Blots were blocked with 5% powdered milk, and then incubated with either mGluR₂ (1:500) or mGluR₃ (1:500) primary antibody overnight at 4°C followed by incubation with the secondary antibody (donkey anti-rabbit or mouse IgG-HRP, 1:2000) for 1 h at room temperature. Tris-buffered saline with 1% Tween −20 was used to wash the blots 3 times (10 min each) after incubation with both primary and secondary antibodies. Immunoreactive bands were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and detected by a Biospectrum Imagining System (UVP, Upland, CA).

Data analysis

Data are presented as the means ± S.E.M. LY354740 concentration-response curve was fit by using the Hill equation: I = I_max × {1/[1+(EC₅₀/[ligand])ⁿ]}, where I_max is the maximum response, EC₅₀ is the concentration of ligand producing a half-maximal response, and n is the Hill coefficient. Student’s paired or unpaired t test or analysis of variance (ANOVA) was used for statistical analysis as appropriate; P values are reported throughout the text and significance was set as P<0.05. N number in the text represents the cells or slices examined.

Results

Expression of mGluRs II in the EC

We initially detected the expression of mGluRs II which include mGluR₂ and mGluR₃ in the EC using immunocytochemistry and western blot. The EC can be divided into 6 layers (layer I-VI) (Mulders et al., 1997). Layer I is the molecular layer which has a scarcity of cells, whereas layer IV is the cell-sparse, fiber-rich narrow layer which constitutes the lamina dissecans. Thus the EC is actually classified as the superficial layers (layer II-III) that provide innervations to the hippocampus and the deep layers (layer V-VI) that receive hippocampal outputs. Immunoreactivities for both mGluR₂ and mGluR₃ were detected in each layer of the EC (Fig. 1, upper panel). Western blot showed that a protein band of 95 kDa corresponding to the molecular mass of mGluR₂ (Petralia et al., 1996; Phillips et al., 1998; Phillips et al., 2000; Tanabe et al., 1992) and a band of 100 kDa close to the molecular mass of mGluR₃ (Corti et al., 2007; Petralia et al., 1996; Tanabe et al., 1992) were identified in the lysates of the EC (Fig. 1, low panel). These data demonstrate that the EC expresses both mGluR₂ and mGluR₃.

Fig. 1.

The EC expresses both mGluR₂ and mGluR₃. A, Immunostaining (upper) and western blot (lower) of mGluR₂ in the EC. The right upper panel is the enlargement of the selected region in the left panel. Lower panel shows that a band of ~95 kDa protein was detected in the lysates of the EC. Pre-absorption of the mGluR₂ antibody with its corresponding blocking peptide blocked the detection of the band demonstrating the specificity of the antibody. B, Immunostaining (upper) and western blot (lower) of mGluR₃ in the EC. The right upper panel is the enlargement of the selected region in the left panel. Lower panel shows that a band of ~100 kDa protein was detected in the lysates of the EC. Likewise, pre-absorption of mGluR₃ antibody with the corresponding blocking peptide blocked the detection of the band demonstrating the specificity of the antibody.

Activation of mGluRs II depresses epileptiform activities in the EC

Because the EC is closely associated with epilepsy, we next examined the roles of mGluRs II in epilepsy by recording the epileptiform activities induced by bath application of PTX or deprivation of Mg²⁺ in the extracellular solution in entorhinal slices. Bath application of LY354740 (3 μM), a highly selective and potent agonist of mGluRs II, almost completely inhibited PTX-induced epileptiform activity (0.9±0.7% of control n=13 slices, P<0.0001, Fig. 2A, 2B). The EC₅₀ of LY354740 was measured to be 24 nM (Fig. 2C). We used 3 μM (a near saturating concentration) for the rest of the experiments for better comparison unless stated otherwise. The effect of LY354740 was mediated by activation of mGluRs II because prior application of the selective mGluRs II antagonist, LY341495 (1 μM), blocked LY354740-induced depression of epileptiform activity (109±13% of control, n=7 slices, P=0.78, Fig. 2D). Furthermore, bath application of LY354740 also almost completely blocked the epileptiform activity induced by deprivation of Mg²⁺ from the extracellular solution (3.5±2.4% of control, n=10 slices, P<0.001, Fig. 2E, 2F) and bath application LY341495 (1 μM) blocked LY354740-induced depression of epileptiform activity (97±6% of control, n=9 slices, P=0.63, Fig. 2G, 2H). These results together indicate that activation of mGluRs II exerts powerful antiepileptic activity.

Fig. 2.

Activation of mGluRs II inhibits epileptiform activity induced by PTX or deprivation of extracellular Mg²⁺. A, Epileptiform activity induced by PTX before, during and after the application of LY354740. The arrows indicate the expansion of the duration of the recording represented by the horizontal bar. B, Pooled time course of the frequency of PTX-induced epileptiform activity in response to LY354740. C, Concentration-response curve of LY354740. Numbers in the parentheses were the numbers of slices recorded. D, Bath application of the selective mGluRs II antagonist, LY341495 (1 μM), blocked the depression of epileptiform activity induced by LY354740. E, Epileptiform activity induced by deprivation of extracellular Mg²⁺ before, during and after the application of LY354740. Note that application of LY354740 completely blocked the epileptiform events induced by deprivation of extracellular Mg²⁺. The arrows indicate the expansion of the duration of the recording represented by the horizontal bar. F, Pooled time course showing the depressant effect of LY354740 on the frequency of epileptiform activity induced by deprivation of Mg²⁺ in the extracellular solution. G, Epileptiform activity induced by Mg²⁺-free extracellular solution before, in the presence of the mGluRs II antagonist, LY341495 (1 μM) alone, and during the application of both LY341495 and LY354740. The arrows indicate the expansion of the duration of the recording represented by the horizontal bar. H, Pooled time course of the frequency of epileptiform activity induced by Mg²⁺-free extracellular solution before, in the presence of LY341495 alone and during the application of both LY341495 and LY354740.

Activation of mGluRs II decreases neuronal excitabilities in the EC

One potential mechanism whereby mGluRs II depress epileptiform activity is due to their inhibition of glutamate release in the EC as described previously (Wang et al., 2012). Because a high density of mGluRs II has been detected in the entorhinal neurons (Fig. 1), we examined the effects of mGluRs II on the excitabilities of the principal neurons in each layer of the EC. To circumvent the potential influences of mGluRs II-induced changes in GABA and glutamate release on neuronal excitability, we included, in the extracellular solution, DNQX (10 μM) and dl-APV (50 μM) to block glutamatergic and bicuculline (10 μM) and CGP55845 (1 μM) to block GABAergic transmission. Because the stellate neurons are the principal cell type in layer II (Alonso and Klink, 1993), we studied the actions of mGluRs II on the excitability of the stellate neurons. Bath application of the selective mGluRs II agonist, LY354740, transiently and slightly inhibited the AP firing frequency of the stellate neurons (Control frequency: 2.18±0.28 Hz, LY354740: 1.64±0.30 Hz, 73±4% of control, n=7, P<0.001, Fig. 3A₁-A₂). The inhibitory effect of LY354740 could be completely reversed after wash for ~15 min (2.42±0.43 Hz, 108±10% of control, n=7, P= 0.3 vs. control baseline, Fig. 3A₁-A₂). We further examined the effects of LY354740 on the excitability of layer III pyramidal neurons. Bath application of LY354740 completely depressed the AP firing frequency of layer III pyramidal neurons (Control frequency: 1.95±0.23 Hz, LY354740: 0±0 Hz, n=6, P<0.001, Fig. 3B₁-B₂). The depressant effect of LY354740 was only partially reversible even after wash for 1h (0.81±0.25 Hz, 43±12% of control, n=6, P=0.005 vs. control baseline, Fig. 3B₁-B₂). We also tried to use whole-cell recordings to probe the effects of mGluRs II on the excitability of layer V pyramidal neurons. However, these cells displayed significant rundown of the basal AP firing frequency in whole-cell recording configuration. We therefore used perforated patch recordings to record AP firing from layer V pyramidal neurons. Under these circumstances, application of LY354740 significantly inhibited the AP firing frequency of layer V pyramidal neurons (Control: 1.80±0.27 Hz, LY354740: 1.36±0.20 Hz, 76±3% of control, n=7, P=0.003, Fig. 3C₁-C₂). However, the LY354740-induced depression of AP firing frequency was irreversible after wash for 25 min (1.26±0.18 Hz, 72±7% of control, n=7, P=0.02 vs. control baseline, Fig. 3C₁-C₂). For the remaining experiments, we focused on layer III pyramidal neurons to determine the underlying mechanisms because of the following reasons. First, layer III pyramidal neurons are selectively lost in epileptic animals and patients (Du and Schwarcz, 1992; Du et al., 1993) highlighting their importance in epilepsy. Second, as demonstrated above, activation of mGluRs II exerts the most powerful inhibition on layer III pyramidal neurons.

Fig. 3.

Activation of mGluRs II depresses the AP firing frequency in entorhinal neurons. A₁-A₂, Application of LY354740 transiently inhibited the AP firing frequency of layer II stellate neurons. A₁, APs recorded for a layer II stellate neuron before, during and after the application of LY354740. A₂, Pooled time course of AP firing frequency from 7 stellate neurons in response to LY354740. B₁-B₂, Application of LY354740 persistently inhibited the AP firing frequency of layer III pyramidal neurons. B₁, APs recorded for a layer III pyramidal neuron before, during and after the application of LY354740. B₂, Pooled time course of AP firing frequency from 6 layer III pyramidal neurons before, during and after the application of LY354740. C₁-C₂, Application of LY354740 significantly inhibited the AP firing frequency of layer V pyramidal neurons recorded by perforated patches. C₁, APs recorded for a layer V pyramidal neuron before, during and after the application of LY354740. C₂, Pooled time course of AP firing frequency from 7 layer V pyramidal neurons in response to LY354740.

Activation of mGluRs II hyperpolarizes layer III pyramidal neurons via activation of a K⁺ conductance and inhibition of a Na⁺-permeable channel

We then tested the hypothesis that LY354740 depresses AP firing by generating membrane hyperpolarization in layer III pyramidal neurons of the medial EC by recording the changes of the RMPs in the extracellular solution supplemented with TTX (0.5 μM) to block AP firing. A negative current (−50 pA for 500 ms) was injected every 5 s to assess the changes of the input resistance. Under these circumstances, application of LY354740 generated membrane hyperpolarization (Control: −61.2±1.3 mV, LY354740: −72.6±2.6 mV, n=5, P=0.002, Fig. 4A-B) and reduced the input resistance (Control: 290±44 MΩ, LY354740: 142±21 MΩ, n=5, P=0.025, Fig. 4A-B) suggesting that application of LY35470 increases membrane conductance. We further used voltage-clamp mode and recorded the HCs at −60 mV, a potential close to the RMPs of these neurons. Under these conditions, application of LY354740 generated an outward HC (67.1±10.5 pA, n=5, P=0.003, Fig. 4C). In current clamp, application of LY354740 hyperpolarized layer III pyramidal neurons by −5.7±0.4 mV (n=9, P<0.0001, Fig. 4D). These results demonstrate that activation of mGluRs II decreases neuronal excitability by hyperpolarizing layer III pyramidal neurons.

Fig. 4.

Activation of mGluRs II induces hyperpolarization in layer III pyramidal neurons. A, Activation of mGluRs II generated hyperpolarization and reduced the input resistance. A negative current (−50 pA for 500 ms) was injected every 5 s to assess the changes of input resistance. Insets are the voltage traces taken before (a) and during (b) the application of LY354740. Note that LY354740 induced membrane hyperpolarization and reduced the voltage responses induced by the negative current injections suggesting a reduction in input resistance. To exclude the influence of LY354740-induced membrane hyperpolarization on input resistance, a constant positive current (+58 pA indicated by the horizontal bar) was injected briefly to elevate the membrane potential to the initial level. Under these circumstances, the voltage responses induced by the negative current injections (−50 pA) were still smaller compared with control suggesting that LY354740-induced reduction in input resistance is not secondary to its effect on membrane hyperpolarization. B, Summarized data for LY354740-induced changes in RMPs and input resistance. Filled circles denote the averaged values. C, Bath application of LY354740 induced an outward holding current. D, Application of LY354740 hyperpolarized a layer III pyramidal neuron. E, The first application of NE did not block the hyperpolarization induced by the second application of LY354740 in a layer III pyramidal neuron. A 15 pA positive current was continuously injected to elevate the membrane potential to the initial level to prevent potential influences of membrane potential alteration in response to NE on the hyperpolarization induced by subsequent application of LY354740. F, The first application of LY354740 failed to block the hyperpolarization induced by the second application of NE in a layer III pyramidal neuron. A 30 pA positive current was consistently injected to elevate the membrane potential to the initial level to prevent potential influences of membrane potential alteration in response to LY354740 on the hyperpolarization induced by the second application of NE. G, Summarized data for the occlusion experiments by application of NE and LY354740. Note that application of NE does not occlude the hyperpolarization in response to the following application of LY354740 and vice versa.

We then tried to determine the cellular and molecular mechanisms underlying mGluRs II-mediated hyperpolarization. Because activation of α₂ adrenergic receptors by norepinephrine (NE) in layer III pyramidal neurons of the EC also induces hyperpolarization via a PKA-dependent activation of TREK-2 channels (Xiao et al., 2009a), we wondered whether the same mechanism is involved in mGluRs II-mediated hyperpolarization. If so, the hyperpolarization induced by mGluRs II should occlude that mediated by α₂ adrenergic receptors and vice versa. We therefore performed the following occlusion experiments. First application of NE (1st NE, 100 μM) induced significant hyperpolarization (−4.0±0.8 mV, n=7, P=0.002, Fig. 4E and 4G). We then injected positive current to elevate the membrane potential to the initial level to prevent potential effect of membrane potential alteration induced by NE on the hyperpolarization in response to second application of LY354740 (2nd LY354740). In the presence of NE, application of LY354740 still significantly hyperpolarized layer III pyramidal neurons (2nd LY354740: −5.3±1.0 mV, n=7, P=0.002, Fig. 4E and 4G). In a reverse way, first application of LY354740 (1st LY354740) induced significant hyperpolarization (−7.1±0.8 mV, n=5, P=0.001, Fig, 4F and 4G) but failed to block the hyperpolarization induced by the second application of NE (2nd NE: −2.2±0.3 mV, n=5, P=0.003, Fig. 4F and 4G). The hyperpolarization induced by the first application of NE or LY354740 was not significantly different from that induced by the second application of NE or LY354740 (Fig. 4G), suggesting that different mechanisms are involved in the hyperpolarization induced by mGluRs II and α₂ receptors.

We then tested whether the hyperpolarization in response to mGluRs II activation was Ca²⁺-dependent or not. We replaced extracellular Ca²⁺ with the same concentration of Mg²⁺ and adding EGTA (1 mM) to chelate potential tracing Ca²⁺. Under these circumstances, bath application of LY354740 still induced a comparable hyperpolarization (−5.3±1.3 mV, n=6, P=0.75 vs. control, Fig. 5A, 5K) suggesting that extracellular Ca²⁺ is not required for LY354740-induced hyperpolarization. We further determined the potential roles of intracellular Ca²⁺ in LY354740-mediated hyperpolarization by including BAPTA (20 mM) in the recording pipettes. We waited for ~20 min after the formation of whole-cell recording configuration. In this situation, application of LY354740 still induced a comparable hyperpolarization (−4.4±0.7 mV, n=8, P=0.13, Fig. 5B and 5K). These results demonstrate that mGluRs II-induced hyperpolarization is independent of intracellular and extracellular Ca²⁺. We next measured the reversal potential of the current generated by activation of mGluRs II to further determine the involved ionic mechanisms. The extracellular solution contained TTX (1 μM) and 0 Ca²⁺ to block voltage-gated Na⁺ and Ca²⁺ channels and the above K⁺-gluconate-containing intracellular solution was used. We used a ramp protocol to measure the I-V curve before and during the application of LY354740. Under these circumstances, the LY354740-generated currents showed double phases. From −120 mV to −50 mV (phase I), the I-V curve was linear with a reversal potential of −92.4±1.2 mV (n=4, Fig. 5C-D), close to the calculated K⁺ reversal potential (−85.5 mV), demonstrating that activation of mGluRs II opens a K⁺ conductance to generate hyperpolarization. From −50 mV to 60 mV (phase II), the LY354740-generated currents reversed at −1.3±8.2 mV (n=4, Fig. 5C-D) suggesting that activation of mGluRs II could inhibit a cation channel to generate hyperpolarization. The I-V curve observed in layer III pyramidal neurons resembled that generated by the activation of mGluRs II in baroreceptor neurons (Sekizawa et al., 2009). We linearly fit phase II and the time constant was −4.4±0.7 pA/mV (n=4, Fig. 5D). Because we have already demonstrated that extracellular Ca²⁺ is unnecessary for LY354740-induced hyperpolarization, we tested the roles of extracellular Na⁺ by replacing the above extracellular NaCl with the same concentration of NMDG-Cl or choline-Cl. In this situation, application of LY354740 induced a significantly lower level of hyperpolarization (NMDG: −0.86±0.15 mV, n=10. P<0.001 vs. baseline, P<0.001 vs. control condition, Fig. 5E, and 5K; choline: −1.19±0.25 mV, n=4, P=0.017 vs. baseline, P<0.001 vs. control condition, Fig. 5F and 5K). These results suggest that the hyperpolarization generated by activation of mGluRs II is dependent on extracellular Na⁺ in some extent. We further recorded the I-V curve in the extracellular solution containing NMDG-Cl instead of NaCl. In this situation, application of LY354740 shifted the reversal potential for phase I to −82.6±0.3 mV (n=6, P<0.0001 vs. control, Fig. 5G and 5H) and slowed phase II (time constant: −1.4±0.3 pA/mV, n=6, P=0.002 vs. the counterpart in control condition, Fig. 5H). These results suggest that both K⁺ channels and Na⁺-permeable channels are involved in mGluRs II-induced hyperpolarization. Because the extracellular solution contained TTX, the Na⁺-permeable channels which were sensitive to mGluRs II should be TTX-resistant. If so, the layer III neurons should exhibit a tonic TTX-resistance, Na⁺-permeable conductance. We therefore tested this possibility by recording the RMPs in the presence of TTX (1 μM) first and then switching to the extracellular solution in which NaCl was replaced by NMDG-Cl or choline-Cl. Replacement of NaCl with NMDG-Cl induced a significant hyperpolarization (−7.2±1.3 mV, n=5, P=0.005, Fig. 5I and 5K). Similarly, substitution of extracellular NaCl with the same concentration of choline-Cl induced a comparable hyperpolarization (−6.5±1.5 mV, n=6, P=0.009, Fig. 5J and 5K). These data together demonstrate that layer III pyramidal neurons in the EC express a tonic Na⁺-permeable conductance.

Fig. 5.

The hyperpolarization induced by mGluRs II is related to the opening of a K⁺ conductance and the inhibition of a TTX-resistant, persistent Na⁺-permeable channel. A, Depletion of extracellular Ca²⁺ by replacing extracellular Ca²⁺ with Mg²⁺ and including EGTA (1 mM) in the extracellular solution failed to alter LY354740-induced hyperpolarization. B, Inclusion of BAPTA (20 mM) in the recording pipettes did not change LY354740-induced hyperpolarization. C, I-V curves recorded by a ramp protocol before (red) and during (black) the application of LY354740. D, Net current generated by subtraction of the I-V curve during the application of LY354740 from that in control condition showed double phases. E, Replacement of extracellular NaCl with the same concentration of NMDG-Cl significantly reduced LY354740-induced hyperpolarization. F, Substitution of extracellular NaCl with the same concentration of choline-Cl significantly decreased LY354740-induced hyperpolarization. G, I-V curves recorded by a ramp protocol before (red) and during (black) the application of LY354740 in the extracellular solution in which NaCl was replaced by NMDG-Cl. H, Net current generated by subtraction of the I-V curve during the application of LY354740 from that in control condition in the extracellular solution in which NaCl was replaced by NMDG-Cl. I, In the presence of TTX (1 μM) to block voltage-gated Na⁺ channels, switching to the extracellular solution in which NaCl was replaced with the same concentration of NMDG-Cl generated membrane hyperpolarization in a layer III pyramidal neuron. J, In the presence of TTX (1 μM), switching to the extracellular solution in which NaCl was replaced with the same concentration of choline-Cl generated membrane hyperpolarization in a layer III pyramidal neuron. K, Summary bar graph. ** P<0.01 vs. control (LY354740 alone); ## P<0.01 vs. baseline.

We further verified the roles of K⁺ channels. LY354740-induced hyperpolarization was significantly reduced when intracellular Cs⁺-gluconate solution was used (−2.1±0.9 mV, n=13, P=0.036, Fig. 6A and 6K). Elevation of extracellular K⁺ concentration to 10 mM significantly reduced LY354740-induced hyperpolarization (−2.7±0.7 mV, n=7, P=0.001 vs. baseline, P=0.001 vs. control, Fig. 6B and 6K) whereas replacing extracellular K⁺ with the same concentration of Na⁺ significantly enhanced LY354740-induced hyperpolarization (−27.7±4.5 mV, n=6, P=0.002 vs. baseline, P<0.001 vs. control, Fig. 6C and 6K). These results further confirmed the requirement of K⁺ channels. However, LY354740-induced hyperpolarization was not significantly altered in the extracellular solution containing tetraethylammonium (TEA, 10 mM, −6.7±0.5 mV, n=5, P<0.001 vs. baseline, P=0.11 vs. control, Fig. 6D and 6K), Cs⁺ (3 mM, −6.3±0.9 mV, n=5, P=0.002 vs. baseline, P=0.48 vs. control, Fig. 6E and 6K), 4-aminopyridine (4-AP, 2 mM, −4.3±1.5 mV, n=7, P=0.03 vs. baseline, P=0.33 vs. control, Fig. 6F and 6K) or Ba²⁺ (2 mM, −8.9±1.3 mV, n=12, P<0.0001 vs. baseline, P=0.052 vs. control, Fig. 6G, 6K) although some of these K⁺ channel blockers alone induced significant depolarization. Because mGluRs II have been shown to activate the inwardly rectifier K⁺ channels in a variety of neurons (Cox and Sherman, 1999; Holmes et al., 1996; Knoflach and Kemp, 1998; Lee and Sherman, 2009; Muly et al., 2007; Watanabe and Nakanishi, 2003), we paid specific attention to this type of channels. LY354740-induced hyperpolarization was insignificantly altered when two inwardly rectifier K⁺ channel blockers, tertiapin-Q (250 nM, −5.5±1.2 mV, n=6, P=0.001 vs. baseline, P=0.86 vs. control, Fig. 6H and 6K) or SCH23390 (20 μM, −5.1±0.4 mV, n=13, P<0.0001 vs. baseline, P=0.331 vs. control, Fig. 6I, 6K), were included in the extracellular solution, suggesting that the involved K⁺ channels are unlikely to be the inwardly rectifier K⁺ channels. Furthermore, the LY354740-induced hyperpolarization was insensitive to 100 μM Zn²⁺ (−5.6±0.5 mV, n=6, P<0.001 vs. baseline, P=0.87 vs. control, Fig. 6J, 6K).

Fig. 6.

Role of K⁺ channels in the hyperpolarization induced by activation of mGluRs II in the EC. A, Replacement of intracellular K⁺ with the same concentration of Cs⁺ significantly reduced LY354740-induced hyperpolarization. B, Elevation of extracellular K⁺ concentration to 10 mM by itself generated depolarization and significantly reduced LY354740-induced hyperpolarization. C, Depletion of extracellular K⁺ by replacing extracellular K⁺ with the same concentration of Na⁺ significantly enhanced LY354740-induced hyperpolarization. D, Inclusion of TEA (10 mM) in the extracellular solution failed to alter LY354740-induced hyperpolarization. E, Inclusion of Cs⁺ (3 mM) in the extracellular solution failed to alter LY354740-induced hyperpolarization. F, Inclusion of 4-AP (2 mM) in the extracellular solution failed to alter LY354740-induced hyperpolarization. G, Bath application of Ba²⁺ (2 mM) induced depolarization but failed to change LY354740-induced hyperpolarization. H, Bath application of the inwardly rectifier K⁺ channel blocker, tertiapin-Q (250 nM), failed to change LY354740-mediated hyperpolarization. I, Bath application of another inwardly rectifier K⁺ channel blocker, SCH23390 (20 μM), failed to alter LY354740-mediated hyperpolarization. J, Bath application of Zn²⁺ (100 μM) did not alter LY354740-induced hyperpolarization. K, Summary bar graph. ** P<0.01 vs. control.

Signal transduction mechanisms underlying mGluRs II-mediated hyperpolarization

mGluRs II are negatively linked to AC leading to a reduction in the intracellular level of cyclic AMP and an inhibition of PKA (Conn and Pin, 1997; Pin and Acher, 2002). We next examined the signaling mechanisms underlying mGluRs II-induced hyperpolarization. Application of the selective mGluRs II antagonist, LY341495 (1 μM), blocked LY354740-induced hyperpolarization (0.3±0.1 mV, n=5, P=0.09, Fig. 7A and 7K), further confirming the requirement of mGluRs II. We determine the roles of G proteins in mGluRs II-mediated hyperpolarization. We included the G-protein inactivator, GDP-β-S (3 mM), in the recording pipettes and waited for >20 min after the formation of whole-cell configuration. Under these circumstances, bath application of LY354740 failed to induce hyperpolarization but induced a delayed depolarization instead (3.7±0.7 mV, n=7, P=0.001, Fig. 7B and 7K). Furthermore, inclusion of GTP-γ-S (0.5 mM) in the recording pipettes induced significant hyperpolarization (RMP difference between the time just after the formation of whole-cell configuration and 10 min after: −7.6±1.1 mV, n=5, P=0.002, Fig. 7C) and occluded LY354740-induced hyperpolarization (0.7±0.3 mV, n=5, P=0.06 vs. baseline, P<0.001 vs. LY354740 alone, Fig. 7C and 7K). These data together suggest that mGluRs II-induced hyperpolarization is mediated by activation of G proteins. We then tested the involvement of AC and PKA. Pretreatment of slices with and continuous bath application of the selective AC inhibitor, MDL 12330A (50 μM), failed to alter LY354740-induced hyperpolarization (−6.9±1.0 mV, n=6, P=0.19 vs. LY354740 alone, Fig. 7D and 7K). Moreover, application of SQ22536 (500 μM), another selective AC inhibitor, in the same fashion did not significantly change LY354740-induced hyperpolarization (−5.0±0.9 mV, n=7, P=0.47 vs. LY354740 alone, Fig. 7E and 7K), demonstrating that AC is not required for the hyperpolarization induced by activation of mGluRs II. We then tested the roles of PKA by including the selective PKA inhibitor, Rp-cAMPS (200 μM), in the recording pipettes. In the presence of Rp-cAMPS, application of LY354740 still induced comparable hyperpolarization (−5.9±1.1 mV, n=7, P=0.84 vs. LY354740 alone, Fig. 7F and 7K). Similarly, inclusion of PKI_14–22 (5 μM), another selective PKA inhibitor, in the recording pipettes, failed to alter significantly LY354740-induced hyperpolarization (−6.7±1.1 mV, n=5, P=0.32 vs. LY354740 alone, Fig. 7G and 7K), demonstrating that PKA is not required for LY354740-induced hyperpolarization. Moreover, LY354740-indued hyperpolarization was insensitive to the PKC inhibitor, calphostin C (5 μM, −4.7±0.6 mV, n=5, P=0.17 vs. LY354740 alone, Fig. 7H and 7K) and the broad-spectrum kinase inhibitor, staurosporine (5 μM, −6.8±0.7 mV, n=5, P=0.143, Fig. 7I, 7K), suggesting that mGluRs II-mediated hyperpolarization may be phosphorylation-independent. We finally tested whether activation of mGluRs II hyperpolarizes entorhinal neurons via Gβγ. Pretreatment of slices with and continuous bath application of gallein (30 μM), a Gβγ inhibitor (Irannejad and Wedegaertner, 2010; Lehmann et al., 2008; Ukhanov et al., 2011), significantly reduced LY354740-induced hyperpolarization (−1.1±0.6 mV, n=7, P<0.001 vs. LY354740 alone, Fig. 7J-K), demonstrating the requirement of Gβγ.

Fig. 7.

Activation of mGluRs II hyperpolarizes layer III pyramidal neurons via Gβγ. A, Bath application of the selective mGluRs II antagonist, LY341495 (1 μM), blocked LY354740-induced hyperpolarization. B, Application of LY354740 induced depolarization instead of hyperpolarization when GDP-β-S ( 3 mM) was dialyzed into the cells via the recording pipettes. C, Perfusion of GTP-γ-S (0.5 mM) into the cells via the recording pipettes induced hyperpolarization and subsequent application of LY354740 induced depolarization instead of hyperpolarization. D, Pretreatment of slices with and continuous bath application of the AC inhibitor, MDL 12330A (40 μM), had no effect on LY354740-induced hyperpolarization. E, Pretreatment of slices with and continuous bath application of the AC inhibitor, SQ22536 (500 μM), had no effect on LY354740-induced hyperpolarization. F, Intracellular application of the PKA inhibitor, Rp-cAMPS (200 μM), via the recording pipettes did not block LY354740-induced hyperpolarization. G, Intracellular application of the PKA inhibitor, PKI_14–22 (5 μM), via the recording pipettes did not block LY354740-induced hyperpolarization. H, Pretreatment of slices with and continuous bath application of the PKC inhibitor, calphostin C (5 μM), failed to affect LY354740-induced hyperpolarization. I, Intracellular application of the broad-spectrum kinase inhibitor, staurosporine (5 μM), did not block LY354740-induced hyperpolarization. J, Pretreatment of slices with and continuous bath application of the Gβγ inhibitor, gallein (30 μM), reduced LY354740-induced hyperpolarization. K, Summary bar graph. ** P<0.01 vs. LY354740 alone.

Inhibition of Gβγ significantly reduced mGluRs II-mediated depression of epileptiform activity

We then tested the role of Gβγ in mGluRs II-induced depression of epileptiform activity. Pretreatment of slices with and continuous bath application of gallein (30 μM) significantly attenuated the reduction of the frequency of epileptiform activity induced by LY354740 in PTX-induced epileptic model (66±8% of control, n=12 slices, P=0.001, Fig. 8A₁-A₂) or zero Mg²⁺-induced epileptic model (63±5% of control, n=13 slices, P<0.001, Fig. 8B₁-B₂), suggesting that Gβγ is required for mGluRs II-mediated depression of epileptiform activity.

Fig. 8.

Inhibition of Gβγ significantly attenuates mGluRs II-induced depression of epileptiform activity. A₁-A₂, Pretreatment of slices with and continuous bath application of the Gβγ inhibitor, gallein (30 μM), reduced the extent of inhibition of epileptiform activity induced by PTX. A₁, Epileptiform events recorded in the presence of gallein alone, gallein with LY354740 and wash in the presence of gallein. A₂, Time course of the epileptiform events pooled from 12 slices. B₁-B₂, Application of gallein (30 μM) in the same fashion, attenuated the extent of inhibition of epileptiform activity induced by zero Mg²⁺. B₁, Epileptiform events recorded in the presence of gallein alone, gallein with LY354740 and wash in the presence of gallein. B₂, Time course of the epileptiform events pooled from 13 slices.

Discussion

Our results demonstrate that activation of mGluRs II remarkably inhibits the epileptic activity induced by PTX or deprivation of extracellular Mg²⁺ and hyperpolarizes entorhinal neurons. The hyperpolarization induced by mGluRs II is mediated by activation of a K⁺ conductance. Inhibition of a Na⁺-permeable conductance also contributes to mGluRs II-induced depression of neuronal excitability in the EC. mGluRs II-mediated hyperpolarization requires the function of Gβγ but is independent of AC and PKA. Inhibition of Gβγ significantly reduced mGluRs II-mediated depression of epileptiform activity.

Ionic mechanisms

Our results demonstrate that activation of mGluRs II hyperpolarizes layer III pyramidal neurons in the EC by activating a K⁺ conductance and inhibiting a persistent, TTX-resistant Na⁺-permeable channel based on the following lines of evidence. First, the I-V curve in response to the activation of mGluRs II exhibits double phases with a reversal potential close to the K⁺ reversal potential for phase I and a reversal potential close to a cation channel for phase II. Second, alterations of the driving force for K⁺ by elevation or depletion of extracellular K⁺ concentration significantly reduced or increased, respectively, LY354740-induced hyperpolarization. Third, replacement of extracellular NaCl with NMDG-Cl significantly shifted the reversal potentials of phase I and phase II to the right suggesting that both K⁺ and Na⁺-permeable channels are involved in mGluRs II-induced hyperpolarization. Fourth, replacement of extracellular NaCl with NMDG-Cl or choline-Cl induced a hyperpolarization by itself and significantly reduced LY354740-induced hyperpolarization suggesting that activation of mGluRs II inhibits a tonic Na⁺-permeable channel contributing to hyperpolarization. Consistent with our results, both K⁺ channels and cation channels are involved in mGluRs II-induced depression of baroreceptor neurons (Sekizawa et al., 2009) and activation of mGluRs II inhibits TTX-resistant Na⁺ channels (Yang and Gereau, 2004). Furthermore, both activation of a K⁺ conductance and inhibition of a cation channel are shown to be involved in serotonin-induced hyperpolarization in the EC (Ma et al., 2007)

Whereas phase II of the I-V curve generated by the activation of mGluRs II might be due to an inhibition of a cation channel, an alternative explanation could be that Na⁺ just plays a gating role for the K⁺ channels facilitated by mGluRs II if the involved K⁺ channels are Na⁺-activated K⁺ channels. Because Na⁺-activated K⁺ channels can be coupled to TRPC-like channels (Kolaj et al., 2014), a possibility is that substitution of extracellular NaCl with NMDG-Cl depressed Na⁺-activated K⁺ channels and therefore decreased the hyperpolarization induced by the activation of mGluRs II. However, the threshold voltage for the activation of Na⁺-activated K⁺ channels seems to be more positive, whereas activation of mGluRs II can hyperpolarize layer III pyramidal neurons at potentials close to the RMPs.

Whereas activation of mGluRs II has been shown to induce hyperpolarization by activating the inwardly rectifier K⁺ channels in the neocortex (Lee and Sherman, 2009), bed nucleus of the stria terminalis and basolateral amygdala (Holmes et al., 1996; Muly et al., 2007), thalamus (Cox and Sherman, 1999) and cerebellum (Knoflach and Kemp, 1998; Watanabe and Nakanishi, 2003), other types of K⁺ channels are also involved in the hyperpolarization mediated by the activation of mGluRs II (Hermes and Renaud, 2011; Kolaj et al., 2014; Sekizawa et al., 2009). Our results demonstrate that the hyperpolarization induced by mGluRs II in layer III pyramidal neurons is unlikely to be mediated by activation of the inwardly rectifier K⁺ channels based on the following pieces of evidence. First, phase I of the I-V curve showed no inward rectification. Second, the inwardly rectifier K⁺ channels are sensitive to Ba²⁺, SCH23390 and tertiapine-Q. However, mGluRs II-induced hyperpolarization was insensitive to these inwardly rectifier K⁺ channel blockers. Third, inwardly rectifier K⁺ channels are sensitive to TEA, whereas LY354740-induced hyperpolarization was insensitive to TEA. The results that mGluRs II-mediated hyperpolarization is insensitive to TEA, Cs⁺ and 4-AP, the classical K⁺ channel blockers, suggest that activation of mGluRs II hyperpolarizes layer III pyramidal neurons by activation of two-pore domain K⁺ (K_2P) channels. Among the K_2P channels, TASK-1 (Han et al., 2002), TASK-3 (Han et al., 2002; Kim et al., 2000), TREK-1 (Fink et al., 1996; Ma et al., 2011), TREK-2 (Bang et al., 2000; Han et al., 2002), TWIK-1 (Lesage et al., 1996) and TRESK (Kang et al., 2004; Sano et al., 2003) are sensitive to Ba²⁺. Our results that mGluRs II-mediated hyperpolarization is insensitive to Ba²⁺ indicate that the K_2P channels activated by mGluRs II are not these 6 channel types. Furthermore, Zn²⁺ has been shown to preferentially inhibit TRESK (Czirjak and Enyedi, 2006) and to a lesser extent TASK-3 (Clarke et al., 2008; Czirjak and Enyedi, 2006) channels. Our results that mGluRs II-mediated hyperpolarization is insensitive to zinc further confirm that TRESK and TASK-3 channels are not involved. The K_2P channels involved in mGluRs II-induced hyperpolarization in the EC still await further identification.

Signaling mechanisms underlying mGluRs II-induced hyperpolarization in the EC

Our results demonstrate that LY354740-mediated depression of epileptiform activity and hyperpolarization are mediated by mGluRs II because application of the selective mGluRs II antagonist, LY341495, blocked the effects of LY354740. We further demonstrate that G proteins are required for LY354740-induced hyperpolarization because intracellular application of GDP-β-S or GTP-γ-S blocked LY354740-mediated hyperpolarization. Whereas activation of mGluRs II can result in inhibition of AC and PKA pathway, our results demonstrate that this pathway is not required for mGluRs II-mediated hyperpolarization because application of the selective inhibitors for AC and PKA did not alter LY354740-mediated hyperpolarization. The result that application of gallein, a Gβγ inhibitor, significantly reduced LY354740-induced hyperpolarization, suggests that activation of mGluRs II reduces the excitability of entorhinal neurons via Gβγ.

Mechanisms whereby mGluRs II depress epileptiform activity

Whereas activation of mGluRs II has been shown to inhibit epilepsy (Alexander and Godwin, 2006; Caulder et al., 2014; Klodzinska et al., 2000), the underlying cellular and molecular mechanisms have not been determined. Several mechanisms can be proposed to explain mGluRs II-mediated antiepileptic effects in the EC. First, as we have demonstrated previously (Wang et al., 2012), inhibition of glutamate release mediated by mGluRs II contributes to the antiepileptic effects of mGluRs II. Second, mGluRs II-mediated depression of epileptiform activity could be related to the inhibition of the excitability of entorhinal neurons as demonstrated in the present study. Whereas augmentation of GABAergic function could result in depression of epileptiform activity, two lines of evidence demonstrate that this is unlikely to be the mechanism underlying mGluRs II-mediated depression of epilepsy. First, application of LY354740 exerts robust inhibition on epileptiform activity induced by PTX, suggesting that mechanisms other than GABAergic inhibition are involved. Second, as we have demonstrated previously, activation of mGluRs II exerts no effects on GABAergic transmission in the EC (Wang et al., 2012). We therefore conclude that the antiepileptic actions of mGluRs II are mediated by mGluRs II-mediated depression of glutamate release and neuronal excitability in the EC. Because the EC is an indispensable structure involved in the generation and propagation of epilepsy, elucidation of the cellular and molecular mechanisms underlying mGluRs II-induced depression of epileptiform activity would likely shed light on the development of mGluRs II agonists for treating epilepsy.