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. 2014 Apr 23;592(Pt 10):2153–2168. doi: 10.1113/jphysiol.2014.271700

Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus

Rongqing Chen 1, Akihito Okabe 1,2, Haiyan Sun 1, Salim Sharopov 1, Ileana L Hanganu-Opatz 1,3, Sergei N Kolbaev 1, Atsuo Fukuda 4, Heiko J Luhmann 1, Werner Kilb 1,✉
PMCID: PMC4227900  PMID: 24665103

Abstract

While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4–7) rat using field potential recordings. Bath application of 100 μm taurine or 10 μm glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μm 4-aminopyridine in low Mg2+ solution. This proconvulsive effect was prevented by 3 μm strychnine or after incubation with the loop diuretic bumetanide (10 μm), suggesting that it required glycine receptors and an active NKCC1-dependent Cl− accumulation. Application of higher doses of taurine (≥1 mm) or glycine (100 μm) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μm) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.

Introduction

Epileptic seizures are a major neurological disorder with a particular high incidence in children (Sanchez & Jensen, 2001). Human and animal studies clearly identified such early seizures as a risk factor for the manifestation of epilepsy (Holmes, 1997; Khalilov et al. 2003). The pharmacological treatment and, in particular, the refractoriness to pharmacological therapies are considerably different in children as compared to adults (Silverstein & Jensen, 2007), which may relate to the altered properties of the GABAergic system during development (Silverstein & Jensen, 2007). While GABA is the major inhibitory neurotransmitter in the adult CNS, it mediates depolarizing membrane responses in the immature nervous system (Ben Ari et al. 2012). These depolarizing GABAergic responses observed in immature neurons have been proposed to underlie the altered seizure incidence and poor pharmacological control in early postnatal phases (Sanchez & Jensen, 2001; Silverstein & Jensen, 2007). However, depolarizing GABAergic responses do not necessarily mediate excitatory actions (Ben Ari et al. 2012) but can also contribute to inhibition due to membrane shunting (Staley & Mody, 1992), resulting in complex interactions between depolarizing GABAergic membrane responses and neuronal excitability (Valeeva et al. 2010; Kolbaev et al. 2011). Accordingly both, pro- and anticonvulsive effects of the GABAergic system on epileptiform discharges have been described in the immature brain (Khalilov et al. 1997b; Wells et al. 2000; Dzhala & Staley, 2003; Dzhala et al. 2005; Isaev et al. 2005; Richter et al. 2010; Kolbaev et al. 2012).

In addition to GABA, the glycinergic system is crucially involved in the regulation of neuronal excitability. While the role of the glycinergic system was originally attributed to the brain stem and lower brain regions (Betz & Laube, 2006), the expression and functional relevance of glycine receptors have also been established in higher brain areas as the cerebral neocortex or hippocampus (Ito & Cherubini, 1991; Becker et al. 1993; Chattipakorn & McMahon, 2002; Mori et al. 2002). Inhibition of glycine receptors can evoke epileptiform discharges in the adult brain (Pollen & Lux, 1966; Straub et al. 1997), whereas their activation has an anticonvulsive effect in mature rat hippocampus (Peterson & Boehnke, 1989; Chattipakorn & McMahon, 2003; Kirchner et al. 2003). The glycinergic system plays an important role during early cortical development (Flint et al. 1998; Okabe et al. 2004; Nimmervoll et al. 2011). Comparable to the GABAergic system, activation of glycine receptors causes in the immature CNS depolarizing membrane responses (Ito & Cherubini, 1991; Flint et al. 1998; Ehrlich et al. 1999; Kilb et al. 2002; Kilb et al. 2008), generates shunting inhibition (Kilb et al. 2002), and contributes to the generation of neuronal activity (Momose-Sato et al. 2005). As synaptic release of glycine is absent in the immature cortex and taurine is the most abundant agonist of glycine receptors, it was assumed that taurine acts as an endogenous neurotransmitter for this system (Flint et al. 1998). In the immature CNS, glycine receptors may therefore interfere with epileptiform activity in a comparable complex fashion as GABAA receptors. However, to our knowledge no information about the influence of the glycinergic system on epileptiform activity in the immature CNS is available.

Thus, we examined the effect of glycinergic agonists and antagonists on spontaneous discharges in an intact in vitro preparation of the immature [postnatal days (P)4–7] rat corticohippocampal formation (CHF) (Khalilov et al. 1997a) using field potential recordings with tungsten microelectrodes. We show that glycinergic agonists can have dose-dependent, both pro- and anticonvulsive effects, and that a tonic inhibition via glycine receptors is required to suppress epileptiform activity. In addition, we demonstrated that the proconvulsive effect of glycinergic agonists in this intact preparation depends on functional Cl− accumulation.

Methods

Ethical approval

All experiments were conducted in accordance with EU directive 86/609/EEC for the use of animals in research and the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the local ethical committee (Landesuntersuchungsanstalt RLP, Koblenz, Germany). All efforts were made to minimize the number of animals and their suffering.

Preparation

Wistar rat pups of P4–7 were obtained from the local breeding facility and were deeply anaesthetized with enflurane (Ethrane, Abbot Laboratories, Wiesbaden, Germany). After decapitation, the brains were quickly removed and immersed for 2–3 min in ice-cold standard artificial cerebrospinal fluid (ACSF, composition see below). The preparation of the intact CHF, which contained hippocampus, entorhinal cortex and parts of temporal neocortex (Khalilov et al. 1997a; Moser et al. 2006; Luhmann & Kilb, 2012), was performed in oxygenated ice-cold ACSF and lasted about 5 min. After separating the two hemispheres by a scalpel cut through the midline, the frontal cortex, brain stem, cerebellum and all diencephalic structures were removed. The pial membranes were carefully removed. Each CHF was transferred to a fully submerged chamber, superfused with ACSF at 30 ± 1°C at a flow rate of ≈5 ml min−1 (Khalilov et al. 1997a). For the patch clamp experiments 400 μm thick semicoronal slices (tilted between 10° and 45° in a medial direction), including the hippocampus, were cut on a vibratome (HR2; Sigmann Elektronik, Hüffenhardt, Germany). The slices were transferred into an incubation chamber filled with oxygenated ACSF at room temperature where they were allowed to recover for at least 1 h before they were transferred to the recording chamber.

Data acquisition and analysis

Extracellular field potentials were recorded with tungsten microelectrodes (impedance 4–5 MΩ; FHC, Bowdoinham, ME, USA) in the stratum radiatum of the hippocampal CA3 region as described previously (Moser et al. 2006; Luhmann & Kilb, 2012). Signals were amplified by a purpose built amplifier, low-pass filtered at 3 kHz and stored on a PC using an AD/DA board (ITC-16; HEKA, Lamprecht, Germany) and TIDA software (HEKA). Population spikes in epileptiform discharges were identified using either MiniAnalysis program (Synaptosoft, Leonia, NY, USA) or threshold crossing algorithms developed in the Matlab environment (MATLAB R2006a; Mathworks, Natic, MA, USA) and Excel-scripts were used to extract the properties of epileptiform discharges from these identified population spikes. Both ictal- and/or interictal-like activity were used to calculate incidence rates. Postsynaptic field potential responses were recorded in the stratum radiatum of CA3 upon 100 μs pulses of 50–500 μA (A365; WPI, Sarasota, FL, USA) applied via a bipolar tungsten electrode located in the CA1 region.

Whole cell patch clamp recordings were performed as described previously (Achilles et al. 2007) at 30 ± 1°C in a submerged-type recording chamber attached to the fixed stage of a microscope (BX51 WI; Olympus, Hamburg, Germany). Pyramidal neurons in the stratum pyramidale of the CA3 region were identified by their location and morphological appearance in infrared differential interference contrast image. Patch pipettes (5–12 MΩ) were pulled from borosilicate glass capillaries (2.0 mm outside, 1.16 mm inside diameter; Science Products, Hofheim, Germany) on a vertical puller (PP-830; Narishige, Tokyo, Japan) and filled with pipette solution containing (in mm) 80 potassium gluconate, 44 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 Hepes, 2 Na2-ATP, 0.5 Na-GTP (pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose). Signals were recorded with a discontinuous voltage clamp/current clamp amplifier (SEC05L; NPI, Tamm, Germany), low-pass filtered at 3 kHz and stored and analysed using an ITC-16 AD/DA board (HEKA) and TIDA software. In most patch clamp experiments glycine, GABA, muscimol or guanidinoethyl sulphonate (GES) was applied focally to the soma of CA3 pyramidal neurons via a patch pipette for 2–100 ms with a pressure of 0.4 bar using a pressure application system (PDES 02T, NPI or LHDA0533115H; Lee, Westbrook, CT, USA).

Statistics

Data were presented as mean ± s.e.m. For statistical analysis sign-test, Mann–Whitney U test and Fisher exact tests were used (Systat 11; Point Richmond, CA, USA). Significance was assigned at levels of 0.05 (*), 0.01 (**) and 0.001 (***).

Solutions and drugs

Standard ACSF consisted of (in mm) 126 NaCl, 26 NaHCO3, 1.25 NaH2PO5, 1 MgCl2, 2 CaCl2, 2.5 KCl, 10 glucose (pH 7.4, osmolarity 306 mosmol l−1). For low Mg2+ solutions MgCl2 was replaced by 1 mm CaCl2. All solutions were equilibrated with 95% O2/5% CO2 at least 1 h before use. Glycine, strychnine, GABA, muscimol, 4-aminopyridine (4-AP), d-serine, bumetanide, N-[3-(4′-Fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine hydrochloride (ALX 5407), O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-l-serine (ALX 1393) and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide disodium salt hydrate (NBQX) were purchased from Sigma (Taufkirchen, Germany), gabazine and dl-2-amino-5-phosphonopentanoic acid (APV) from Biotrend (Cologne, Germany), and GES from TRC (North York, Canada). GABA, glycine, taurine, d-serine and GES were dissolved in distilled water. Bumetanide, 4-AP, strychnine, gabazine, NBQX, APV, ALX 1393 and ALX 5407 were dissolved in dimethylsulphoxide. All substances were added to the solutions shortly before the experiment. The dimethylsulphoxide concentration of the final solution never exceeds 0.2%.

Results

Effect of taurine on epileptiform activity

In the immature CNS, taurine is proposed to be the major endogenous agonist of glycine receptors (Flint et al. 1998). Hence, we first analysed how taurine affects epileptiform discharges. For this purpose we utilized repetitive epileptiform discharges that occurred with a high temporal precision and very uniform pattern, which enables the precise quantification of pro- and anticonvulsive effects (Kilb et al. 2007). Such repetitive epileptiform discharges could be reliably induced in the CHF of P4–7 rats by bath application of 20 μm 4-AP in low Mg2+ solution (Kilb et al. 2007). Epileptiform activity started with one or few ictal-like discharges that are followed by short repetitive discharges (Fig.1A). These repetitive discharges occurred at a frequency of 0.31 ± 0.02 Hz (n = 108), had an average amplitude of 811 ± 63 μV, and consisted of 7.8 ± 0.3 single spikes that discharged at 17.5 ± 0.68 Hz (Fig.1B).

Figure 1. Effect of taurine on low Mg2+/4-AP-induced epileptiform discharges.

Figure 1

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A, field potential registration illustrating the typical epileptiform activity observed in the presence of 20 μm 4-aminopyridine containing low Mg2+ solution. B, typical repetitive discharge as identified in (A) at higher temporal resolution. C, typical field potential registration illustrating the slightly proconvulsive effect of 0.1 mm taurine. Note the increase in the occurrence of epileptiform discharges. D, bath application of 1 μm taurine decreased the occurrence of epileptiform discharges. E, epileptiform discharges were suppressed in the presence of 5 mm taurine. F, statistical analysis of the taurine effect on the occurrence of epileptiform discharges. Occurrence was normalized to values obtained before taurine application (rel. occurrence). Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. Numbers of quantified experiments are indicated in the diagram. If epileptiform activity was blocked the total number of experiments is shown behind a slash. *P < 0.05 (sign test).

Bath application of 0.1 mm taurine significantly (P < 0.001, sign test) increased the occurrence of repetitive epileptiform discharges to 112 ± 3.2% (n = 37) and the frequency of spikes in a discharge to 107 ± 2.9 % (P = 0.015, sign test), while the number of spikes per discharge (97 ± 4 %) and their amplitude (96.6 ± 3.4) were not significantly altered (Fig.1C and F see also Fig.4B). While 0.2 and 0.5 mm taurine had no effect on epileptiform activity, 1 mm taurine significantly (P = 0.013, sign test) reduced the occurrence to 61 ± 10.9 % (n = 14; Fig.1D and F). Bath application of 2 mm taurine blocked epileptiform activity in six of nine CHF investigated and reduced the occurrence to 15 ± 5.1% in the remaining preparations, while 5 mm taurine completely abolished epileptiform discharges in all nine preparations investigated (Fig.1E and F). To investigate whether the proconvulsive effect of taurine is capable of evoking epileptiform discharges also in the absence of other epileptogenic stimuli we applied taurine in ACSF. Under this condition, bath application of 0.1 mm taurine failed to reliably induce epileptiform activity. In only one of 14 preparations epileptiform activity was observed, indicating that the excitatory effect of 0.1 mm taurine is insufficient to drive reliable epileptiform activity in the immature hippocampus. In summary, these experiments indicate that taurine has a proconvulsive effect at low and an anticonvulsive effect at high concentrations.

Figure 4. Pharmacology of the proconvulsive effect of 0.1 mm taurine.

Figure 4

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A, typical field potential registration illustrating the proconvulsive effect of 0.1 mm taurine in the absence of bumetanide (upper trace), while taurine had no effect on epileptiform discharges after bumetanide incubation (lower trace). B, statistical analysis of the influence of 3 μm gabazine, 3 μm strychnine and 10 μm bumetanide on the proconvulsive effect of 0.1 mm taurine on low Mg2+/4-aminopyridine-induced epileptiform discharges. Values were normalized to control conditions before application of 0.1 mM taurine (rel. response). Note that the proconvulsive taurine effect was abolished in the presence of strychnine and bumetanide. Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. Numbers of experiments are indicated below the boxes. *P < 0.05, ***P < 0.001 (sign test). Bum, bumetanide; GBZ, gabazine; Stry, strychnine.

Taurine is an agonist of both GABAA and glycine receptors (Albrecht & Schousboe, 2005). Therefore we first validated that taurine activated both GABAA and glycine receptors in the immature hippocampus by investigating the effects of the glycinergic antagonist strychnine and the GABAA receptor antagonist gabazine on currents evoked under voltage clamp conditions by focal pressure application of 5 mm taurine on to the soma of CA3 pyramidal neurons in hippocampal slices. These experiments revealed that the taurinergic current of 418 ± 57 pA (n = 15) was attenuated by 62 ± 8.9 % (n = 9) in the presence of 3 μm strychnine and by 33 ± 9.1 % (n = 6) in the presence of 3 μm gabazine (Fig.2A–C). In the combined presence of 3 μm strychnine and 3 μm gabazine taurinergic, responses were virtually abolished (n = 9). Similar responses were obtained if a lower taurine concentration of 0.1 mm was used for focal application. Under this condition the taurinergic current of 11.6 ± 1.5 pA (n = 15) was attenuated by 57 ± 9.7% (n = 9) or 53 ± 10.9% (n = 6) in the presence of 3 μm strychnine or 3 μm gabazine (Fig.2C), respectively. Overall, these results indicate that taurine acts on both glycine and GABAA receptors in the CA3 region of the immature hippocampus.

Figure 2. Effect of glycinergic and GABAergic antagonists on taurine-induced currents.

Figure 2

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A, whole cell patch clamp recording of the inward current induced by focal pressure application of 5 mm taurine. Bath application of 3 μm strychnine massively attenuated the taurine-induced current. The taurine-induced current was virtually abolished in the combined presence of 3 μm strychnine and 3 μm gabazine. B, voltage clamp trace illustrating that 3 μm gabazine had only a small effect on the taurine-induced current. C, statistical analysis of the pharmacological effects of taurinergic currents. Amplitudes were normalized to control values obtained before antagonist application (norm. response ampl.). Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. ***P < 0.001, *P < 0.05 (sign test). Ctrl, control; GBZ, gabazine; Stry, strychnine; Tau, taurine.

Next we used these specific blockers to investigate whether glycine and/or GABAA receptors mediate the anticonvulsive effect of higher taurine concentrations in the immature CHF. Bath application of the glycinergic antagonist strychnine attenuated the anticonvulsive efficacy of taurine. In the presence of 3 μm strychnine, bath application of 5 mm taurine abolished epileptiform activity only in one of seven preparations investigated. In the remaining six preparations the occurrence of epileptiform activity was significantly (P = 0.0313, sign test) reduced by 64 ± 6.9% (Fig.3A and D). The GABAergic antagonist gabazine was much more efficient to suppress the anticonvulsant effect of taurine (Fig.3D). In the presence of 3 μm gabazine, taurine did not block epileptiform activity in any of the 12 preparations investigated, but significantly (P = 0.012, sign test) reduced the occurrence by 38 ± 8% (n = 12). These results demonstrate that both glycine and GABAA receptors contribute to the anticonvulsive effect of taurine.

Figure 3. Pharmacology of the anticonvulsive effect of 5 mm taurine.

Figure 3

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A, in the presence of 3 μm strychnine bath application of 5 mm taurine attenuated low Mg2+/4-aminopyridine-induced epileptiform discharges. B, in the combined presence of 3 μm strychnine and 3 μm gabazine bath application of 5 mm taurine increased the occurrence of epileptiform discharges. C, in the combined presence of 3 μm strychnine, 3 μm gabazine and 300 μm guanidinoethyl sulphonate bath application of 5 mm taurine had no effect on epileptiform discharges. D, statistical analysis of the influence of 3 μm strychnine, 3 μm gabazine and 300 μm guanidinoethyl sulphonate on the effects of 5 mm taurine on low Mg2+/4-aminopyridine-induced epileptiform discharges. Values were normalized to control conditions before application of 5 mM taurine (rel. response). Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. Numbers of experiments are indicated below the boxes. E, bath application of 5 mm taurine attenuated epileptiform discharged induced by the application of 3 μm gabazine. GBZ, gabazine; GES, guanidinoethyl sulphonate; Stry, strychnine.

After inhibition of both GABAA and glycine receptors taurine induced an obvious proconvulsive effect. In the presence of 3 μm strychnine and 3 μm gabazine, bath application of 5 mm taurine significantly (P = 0.013, sign test) increased the occurrence of epileptiform discharges to 181 ± 24.4% (n = 17; Fig.3B and D). To elucidate whether an uptake of taurine via a taurine transporter is involved in this paradoxical action, we performed this experiment after blocking the taurine transporter with GES. The addition of 300 μm GES to 3 μm strychnine and 3 μm gabazine containing solutions virtually abolished this paradoxical proconvulsive effect of 5 mm taurine (103 ± 7%, n = 9; Fig.3C and D).

To support the conclusion that higher taurine concentrations can mediate an anticonvulsive effect via an activation of glycine receptors, we also analysed the effect of taurine on epileptiform activity that was induced in the immature CHF by inhibition of GABAA receptors with 3 μm gabazine (Fig.3E). Bath application of 5 mm taurine significantly (P = 0.007, sign test) reduced the occurrence of gabazine-induced epileptiform activity to 63 ± 9% (n = 15). This effect was not significantly (P = 0.77, U test) altered in the presence of 300 μm GES. Under this condition 5 mm taurine reduced the occurrence of gabazine-induced epileptiform activity to 57 ± 8.4% (n = 12, P = 0.039). In summary, these results indicate that activation of glycine receptors by higher taurine concentrations mediates an anticonvulsive effect in the immature hippocampus.

To investigate whether the proconvulsive effect of low taurine concentrations was also mediated via glycine receptors we blocked GABAA receptors. The proconvulsive effect of 0.1 mm taurine was reduced in the presence of 3 μm gabazine (occurrence: 107 ± 2.6%, n = 10), while it was completely abolished (occurrence: 101 ± 5.1%, n = 12) in the presence of 3 μm strychnine (Fig.4B). In addition, we analysed whether a Cl− accumulation via isoform 1 of the Na+, K+, 2Cl− cotransporter (NKCC1) contributes to the proconvulsive effect of 0.1 mm taurine, using the loop diuretic bumetanide to block this transporter (Russell, 2000). Bath application of 10 μm bumetanide had no significant effect on the occurrence (97 ± 9.2, n = 17, P = 0.063) of low Mg2+/4-AP-induced epileptiform discharges and the frequency (109 ± 4.8%, P = 0.332) and number (97 ± 6.6%, P = 0.63) of spikes per discharge, in accordance with previous results (Kilb et al. 2007). However, in the presence of 10 μm bumetanide the proconvulsive effect of 0.1 μm taurine was completely abolished (Fig.4A and B). Neither the occurrence (99 ± 6%, n = 12, P = 0.774) nor frequency (101 ± 1.4%, P = 0.146) or number of spikes per epileptiform discharges (101 ± 5.4%, P = 0.774) was significantly different from the discharges recorded in the presence of bumetanide. These results indicate that the proconvulsive effect of low taurine concentrations depends on an active Cl− accumulation via NKCC1 and is partially mediated via glycine receptors.

The taurine transport inhibitor guanidinoethyl sulphonate attenuates epileptiform activity

To evaluate whether endogenously released taurine affects epileptiform activity, we blocked taurine uptake in the immature CHF using GES. Bath application of 300 μm GES significantly (P = 0.006, sign test) reduced the occurrence of low Mg2+/4-AP-induced epileptiform activity to 52 ± 11.8 % (n = 12), while other properties were not significantly altered (Fig.5A). As it has been demonstrated that GES can act as a GABAergic agonist (Mellor et al. 2000; Sergeeva et al. 2002), we investigated in whole cell patch clamp recordings from CA3 pyramidal neurons of hippocampal slices whether the effect of GES can be explained by a direct agonistic action on GABAA receptors. Focal application of a short (<10 ms) pulse of 300 μm GES induced a current of –18 ± 4 pA (n = 17). This inward current was virtually abolished (–0.3 ± 0.3 pA, n = 7) in the presence of 3 μm gabazine (Fig.5B), indicating that it was probably caused by direct activation of GABAA receptors. Accordingly, GES had no significant effect on epileptiform activity in the immature CHF if GABAA receptors were blocked. In the presence of 3 μm gabazine, bath application of 300 μm GES had no effect on the occurrence (111 ± 7.8%) and properties of epileptiform activity (Fig.5C). To substantiate the hypothesis that the GES effect can be fully explained by a direct interaction with GABAA receptors, we analysed whether the anticonvulsive effect of 300 μm GES can be mimicked by bath application of the GABAergic agonist muscimol. Bath application of 0.1–3 μm muscimol elicited dose-dependent inward currents with an EC50 of 0.28 μm in CA3 pyramidal neurons of hippocampal slices (Fig.5D). From this dose–response relation we calculated that 0.52 μm muscimol would evoke an inward current equivalent to the current of –46 ± 7.1 pA (n = 9) induced by bath application of 300 μm GES (Fig.5D). Subsequent field potential recordings in the immature CHF demonstrated, that bath application of 0.5 μm muscimol reduced the occurrence of 0 Mg/4-AP-induced epileptiform discharges to 30 ± 6.4% (n = 15; Fig.5E), which is not significantly different from the GES effect (P = 0.097, U test). In summary, these experiments demonstrated that the anticonvulsive GES effect can be fully explained by its agonistic action on GABAA receptors.

Figure 5. Effect of the taurine transport blocker guanidinoethyl sulphonate on epileptiform discharges.

Figure 5

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A, typical field potential registration illustrating that bath application of 300 μm guanidinoethyl sulphonate attenuated low Mg2+/4-aminopyridine-induced epileptiform discharges. B, whole cell voltage clamp recording illustrating that focal application of 300 μm guanidinoethyl sulphonate induced a rapid inward current that was reversibly blocked by the specific GABAA antagonist gabazine. C, field potential registration illustrating that 300 μm guanidinoethyl sulphonate failed to attenuate epileptiform activity in the presence of 3 μm gabazine. D, dose–response curve of inward currents induced by bath application of the GABAA receptor agonist muscimol. The current responses upon muscimol applications were normalized to the inward currents induced by guanidinoethyl sulphonate application in the same neuron (rel. amplitude). Data points in (D) represent mean ± s.e.m. Note that 300 μm guanidinoethyl sulphonate evoked a similar current amplitude as approximately 0.5 μm muscimol (dashed line). E, field potential recording illustrating that bath application of 0.5 μm muscimol evokes an attenuation of epileptiform discharges that is comparable to that evoked by 300 μm guanidinoethyl sulphonate. GBZ, gabazine; GES, guanidinoethyl sulphonate.

Effect of glycine on repetitive epileptiform activity

The results of the previous experiments strongly suggest that a substantial part of the pro- and anticonvulsive effects of taurine was mediated by glycine receptors. To investigate, whether the glycinergic system can indeed directly interfere with epileptiform activity we next analysed how bath-applied glycine modulates repetitive epileptiform discharges. For the analysis of the glycinergic effect on epileptiform activity it has to be considered that bath-applied glycine will be efficiently removed from the interstitial space by glycine transporters (Betz et al. 2006) and that glycine is also a co-agonist of NMDA receptors (Johnson & Ascher, 1987). Therefore, we performed the dose–response analysis in the continuous presence of glycine transporter blockers ALX 1393 and ALX 5407, to inhibit glycine transporter 2 and glycine transporter 1, respectively. In addition, we used 200 μm d-serine in these experiments to maximally activate the glycine binding site of NMDA receptors (Wroblewski et al. 1989). Bath application of 200 μm d-serine had no significant effect on the amplitude of field potential responses evoked by electrical stimulation of the dentate gyrus in the immature CHF (112 ± 6.7% of the control amplitude, n = 10), suggesting that glutamatergic synaptic transmission and excitability was not altered by d-serine in the immature hippocampus. In addition, it did not significantly alter the occurrence (99 ± 2.4; n = 30) of epileptiform discharges and the frequency (101 ± 1.4%) and number (102 ± 2.7%) of spikes within a discharge (Fig.6D). Addition of 0.5 μm ALX 1393 and 0.1 μm ALX 5407 to the d-serine containing solution also did not modify the amplitude of the evoked field potential responses (102 ± 4.3%, n = 10), again indicating that glutamatergic transmission was unaffected. Under this condition neither the occurrence of epileptiform discharges (104 ± 2.1%; n = 30) nor the frequency (100 ± 1.4%) and number (97 ± 3.7%) of spikes within a discharge were significantly affected (Fig.6D).

Figure 6. Effect of glycine on epileptiform activity.

Figure 6

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A, typical field-potential registration illustrating that bath application of 100 μm glycine attenuates epileptiform discharges. B, typical epileptiform discharges as marked in (A) at a higher temporal resolution. Note that the discharges have a similar appearance. C, typical registration illustrating that bath application of 10 μm glycine increases the frequency of epileptiform discharges. D, statistical analyses of the effect of d-serine, a mixture of 0.5 μm ALX 1393 and 0.1 μm ALX 5407, and various glycine concentrations (applied in the continuous presence of d-serine and ALX 1393/ALX 5407) on the occurrence (open symbols), the frequency (grey) and number of spikes (black) in low Mg2+/4-aminopyridine-induced epileptiform discharges. Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. Numbers of experiments are indicated below the boxes. *P < 0.05, **P < 0.01 (sign test). E, typical registration demonstrating that the proconvulsive effect of 10 μm glycine (upper traces) was abolished in the presence of 10 μm bumetanide (lower traces). F, statistical analysis of the bumetanide effect on the proconvulsive action of 10 μm glycine. Bum, bumetanide; Ctrl, control.

Bath application of glycine in the continuous presence of d-serine, ALX 1393 and ALX 5407 had a dose-dependent effect on repetitive epileptiform activity in the immature CHF. Bath application of 100 μm glycine induced a significant (P = 0.0117, sign test) reduction in the occurrence of discharges to 65 ± 8.3% (n = 11, Fig.6A and D), while frequency and number of spikes within a discharge were unaltered (Fig.6B and D). At a concentration of 30 μm, glycine had no significant effect on the occurrence and properties of epileptiform discharges (Fig.6D). In the presence of 10 μm glycine the occurrence of epileptiform discharges was significantly (P = 0.001, sign test) increased to 115 ± 3.8% (n = 15; Fig.6C and D). The number of spikes in an epileptiform discharge was significantly (P = 0.007, sign test) reduced by 8 ± 2.9%, while their frequency was not significantly altered (Fig.6C and D). Glycine at concentrations of 0.1 μm (n = 18), 1 μm (n = 10) and 3 μm (n = 8) had no significant effect on the occurrence and the properties of epileptiform activity (Fig.6D). In summary, these results suggest that the activation of glycine receptors can mediate both pro- and anticonvulsive effects, depending on the concentration.

To analyse whether NKCC1-mediated Cl− accumulation contributes to the proconvulsive effect of 10 μm glycine we again used the loop diuretic bumetanide. In the presence of 10 μm bumetanide the proconvulsive effect of 10 μm glycine was completely abolished. Neither the occurrence of epileptiform discharges (100 ± 4.5%, n = 16, P = 0.804), nor the frequency (100 ± 1.4%, P = 0.21) or number of spikes per epileptiform discharges (101 ± 2.0%, P = 0.455) was significantly different from the control interval in 10 μm bumetanide (Fig.6E and F). These results indicate that the proconvulsive effect of low glycine concentrations depends on an active Cl− accumulation via NKCC1. In addition, we investigated whether low glycine concentrations mediate a proconvulsive effect even in the absence of other epileptogenic stimuli. However, 10 μm glycine failed to induce epileptiform activity (n = 10) if applied in ACSF containing d-serine, ALX1393 and ALX 5407, suggesting that the proconvulsive effect of low glycine concentrations is not strong enough to trigger epileptiform activity, but can only enhance existing epileptogenic stimuli.

Effect of strychnine and gabazine on GABAergic and glycinergic currents

Previous studies have documented that the pharmacology of GABAA and glycine receptor antagonists is different between the immature and the adult nervous system (Tapia & Aguayo, 1998) and that strychnine can block GABAA receptors (Shirasaki et al. 1991). Therefore, we analysed the strychnine sensitivity of GABAA and glycine receptors in the immature hippocampal formation using whole cell patch clamp recordings of CA3 pyramidal neurons in hippocampal slices. Focal application of 20 μm glycine induced a current of –38 ± 5.4 pA (n = 14). Dose–response experiments revealed that this inward current was effectively blocked by low strychnine concentrations with an IC50 of 0.065 μm (Fig.7A and D). The glycinergic current was rather insensitive to gabazine, which blocked this current with an IC50 of 290 μm (Fig.7B and D). Focal application of 20 μm GABA induced a substantially stronger inward current of –695 ± 37.6 pA (n = 8). GABAergic currents were relatively insensitive to bath-applied strychnine, with an IC50 of 41 μm (Fig.7C and D). These results demonstrate that glycine receptors are functionally expressed in CA3 pyramidal cells of the immature hippocampus and demonstrate that strychnine at a concentration of 0.3 μm specifically block glycine receptors.

Figure 7. Effect of glycinergic and GABAergic antagonists on glycinergic currents.

Figure 7

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A, whole cell voltage clamp recording of the inward current induced by focal pressure application of 20 μm glycine. Bath application of 0.3 μm strychnine massively attenuated the glycinergic current. B, current traces illustrating that 3 μm gabazine had no effect on the glycinergic inward current. C, bath application of 3 μm strychnine had no effect on the inward current induced by focal application of 20 μm GABA. D, dose–response curve of the effects of strychnine and gabazine on glycinergic and GABAergic currents. Amplitudes were normalized to control values obtained before antagonist application (norm. response amplitude). Symbols represent mean ± s.e.m. E, current traces illustrating that pharmacologically isolated GABAergic sPSCs (left trace) were completely suppressed in the presence of 1 μm gabazine. F, typical current traces illustrating the dose-dependent reduction of GABAergic sPSC amplitude by strychnine. G, statistical analyses revealing that <1 μm strychnine has no effect of GABAergic sPSC, while they were reduced by ≥1 μm strychnine. Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. Numbers of experiments are indicated below the boxes. *P < 0.05 (sign test). Ctrl, control; GBZ, gabazine; Gly, glycine; Stry, strychnine.

To investigate a possible antagonistic effect of strychnine on phasic GABAA receptor-mediated currents we analysed pharmacologically isolated spontaneous GABAergic postsynaptic currents (GABA-sPSCs). In the continuous presence of 10 μm NBQX and 60 μm APV spontaneous postsynaptic currents with an amplitude of –8.7 ± 0.7 pA (n = 18) occurred with an average frequency of 1.6 ± 0.4 Hz (Fig.7E). These events were completely suppressed in the presence of 1–3 μm gabazine (n = 5; Fig.7E), demonstrating that they were exclusively GABA-sPSCs. Bath application of strychnine led to a dose-dependent reduction in the amplitude of GABA-sPSCs (Fig.7F). While strychnine at concentration <1 μm had no significant effect on GABAergic sPSCs, they were significantly (P < 0.05, sign test) reduced by 26.7 ± 5.5% (n = 7), 53.2 ± 6.3% (n = 7), 79.1 ± 5.5% (n = 7) in the presence of 1 μm, 3 μm and 10 μm strychnine, respectively (Fig.7G). In summary, these results indicate that strychnine at concentrations <1 μm had no effect on synaptic GABAergic inputs.

The glycinergic antagonist strychnine provokes epileptiform activity

The previous experiments illustrate that an activation of glycine receptors has potentially anti- or proconvulsive effects in the immature hippocampus. To unravel the effects of an endogenous activation of glycine receptors, we first analysed whether inactivation of glycine receptors mediates pro- or anticonvulsant effects on the CHF of P4–7 rats. Bath application of 0.3 μm strychnine in ACSF induced in five of 20 preparations epileptiform discharges resembling interictal events (Fig.8A). This epileptiform discharges occurred 29 ± 8 min (n = 5) after wash-in at a frequency of 0.01 ± 0.005 Hz and consisted of 2.2 ± 0.03 spikes with an average amplitude of 226 ± 37 μV at a frequency of 21.7 ± 2.9 Hz (Fig.8D). Increasing the strychnine concentration to 3 μm enhanced the incidence of epileptiform activity to 12 of 16 preparations. This epileptiform activity occurred 46 ± 10 min (n = 12) after wash-in at a frequency of 0.01 ± 0.002 Hz (n = 12) and consisted of short bursts of 4 ± 0.5 spikes (n = 12) with an average amplitude of 1090 ± 143 μV at a frequency of 18.8 ± 1.4 Hz (Fig.8B and D). In contrast, inhibition of GABAA receptors by bath application of 3 μm gabazine induced considerably stronger epileptiform activity than 3 μm strychnine. The gabazine-induced epileptiform discharges occurred with a significantly shorter (P < 0.0001, U test) delay of 3.3 ± 0.5 min after wash-in (n = 20) at a frequency of 0.04 ± 0.01 Hz (n = 47; Fig.8C). These discharges consisted of significantly (P = 0.002, U test) more spikes (7.6 ± 0.6; n = 47), with an amplitude of 1246 ± 87 μV at a comparable frequency of 20.4 ± 0.6 Hz (Fig.8D). Addition of 3 μm gabazine to preparations showing strychnine-induced epileptiform discharges significantly (P = 0.0117, sign test) increased their occurrence by 343 ± 66% (n = 11). In summary, these results suggest that endogenously activated glycine receptors contribute to a persistent inhibitory tone required to suppress epileptiform activity. The inhibitory potential mediated by glycine receptors is, however, considerably weaker than that mediated by GABAA receptors.

Figure 8. Glycinergic antagonists provoke epileptiform discharges.

Figure 8

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A, characteristic field potential recordings showing that bath application of 0.3 μm strychnine provokes epileptiform discharges with few spikes per discharge. B, characteristic field potential recordings showing that bath application of 3 μm strychnine provokes more complex epileptiform discharges. C, bath application of 3 μm gabazine elicited epileptiform discharges with more spikes per discharges. The discharges identified by the arrowheads were displayed at larger temporal resolution on the right side. D, statistical analyses of occurrence of epileptiform events and frequency/number of spikes in each epileptiform events induced by 0.3 μm strychnine (open bars, n = 5), 3 μm strychnine (light grey bars, n = 12) and 3 μm gabazine (dark grey bars, n = 47). Box plots represent upper/lower quartile and median, whiskers indicate maximal and minimal values. *P < 0.05 (U test). Ctrl, control; GBZ, gabazine; Stry, strychnine.

To uncover a possible additional proconvulsive effect of glycine receptor inhibition, we applied strychnine in the presence of 3 μm gabazine. However, these experiments revealed that 3 μm strychnine does not alter the properties of the epileptiform activity induced in the presence of this GABAA receptor antagonist (Fig.9A, B and H). Bath application of 3 μm strychnine affected neither occurrence (98.3 ± 6%; n = 22), nor amplitude (99.8 ± 2.7%), nor frequency (98.4 ± 0.02%), nor number of spikes per discharge (101.1 ± 4%, n = 20). On the other hand, 3 μm strychnine had also no significant effect on the properties of low Mg2+/4-AP-induced repetitive epileptiform discharges, except that the amplitude increased significantly (P = 0.038, sign test) to 123 ± 8.4% (n = 12). The occurrence (94 ± 4.7%), the number of spikes per discharge (123 ± 8.6%) and their frequency (102 ± 3.5%) were not significantly (P > 0.05, sign test) altered (Fig.9C, D, and H). In addition, selective inhibition of glycine receptors by bath application of 0.3 μm strychnine had no significant effect on the properties of ictal-like discharges induced in the presence of low Mg2+ solution (Fig.9E). Occurrence (0.098 ± 0.014 min−1 vs. 0.061 ± 0.08 min−1; n = 12 for both groups), amplitude (580 ± 59 μV vs. 490 ± 70 μV), frequency (15.8 ± 0.7 Hz vs. 16.6 ± 0.4 Hz) and number of spikes per discharge (330 ± 26 vs. 542 ± 49) were not significantly (P > 0.235, U test) different from control experiments on low Mg2+ solution during the same time interval. In contrast, bath application of 3 μm gabazine considerably changed the properties of the 0 Mg2+/4-AP-induced epileptiform discharges. The number of spikes per discharge increased to 155 ± 9.6% (n = 32) and their amplitude to 132 ± 8.3%, while the occurrence of epileptiform discharges decreased to 50 ± 4.8% and the frequency of spikes to 90 ± 2.5% (Fig.9F–H). In summary, these results suggest that an inhibition of glycine receptors is not sufficient to modulate existing epileptiform discharges.

Figure 9. Effect of glycinergic and GABAergic antagonists on epileptiform discharges.

Figure 9

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A, bath application of 3 μm strychnine has no effect on gabazine-induced epileptiform discharges. B, typical discharges under control conditions (B1) and in the presence of 3 μm strychnine (B2) as marked in (A) at higher temporal resolution. C, bath application of 3 μm strychnine has no effect on low Mg2+/4-aminopyridine-induced epileptiform discharges, except of a slight increase in the amplitude. D, typical discharges under control conditions (D1) and in the presence of 3 μm strychnine (D2) as marked in (C) at higher temporal resolution. E, bath application of 0.3 μm strychnine has no effect on the moderate epileptiform activity included in low Mg2+ solution. F, bath application of 3 μm gabazine changes the amplitude and occurrence of low Mg2+/4-aminopyridine-induced epileptiform discharges. G, typical discharges marked in (F) at higher temporal resolution illustrating the increase in amplitude and number of spikes per discharges observed in the presence of 3 μm gabazine. H, statistical analyses of the effect of strychnine and gabazine on epileptiform discharges induced by 3 μm gabazine or low Mg2+/4-aminopyridine. Values were normalized to control conditions before antagonist application (rel. response). Box plots represent upper/lower quartile and median (bold line), whiskers indicate maximal and minimal values. Numbers of experiments are indicated below the boxes. *P < 0.05, **P < 0.01, ***P < 0.001 (sign test). GBZ, gabazine; Stry, strychnine.

Discussion

We used an intact in vitro preparation of the CHF of rats at P4–7 to investigate whether taurine influences the excitability of the immature hippocampus. We quantified in this study only the effect of glycinergic agonists on CA3 region, as it was shown that in the immature hippocampus epileptiform originated mainly from the hippocampus itself (Weissinger et al. 2000) and that epileptiform activity appeared simultaneously in all hippocampal regions in the intact CHF (Moser et al. 2006). The main findings of these experiments can be summarized as follows: (1) Taurine promotes a proconvulsive effect at a concentration of 0.1 mm and an anticonvulsive effect at concentrations ≥1 mm. The pro- and anticonvulsive effects of taurine persist after blockade of GABAA receptors. (2) Glycine at a concentration of 10 μm had a proconvulsive effect, while it had an anticonvulsive effect at a concentration of 100 μm. (3) The proconvulsive effect of glycine and taurine at lower concentration was abolished in the presence of the glycinergic antagonist strychnine or the NKCC1 inhibitor bumetanide. (4) Inhibition of glycine receptors with strychnine-induced epileptiform discharges. We conclude from these experiments: (i) that activation of glycine receptors can promote both pro- and anticonvulsive effects on the immature hippocampus; (ii) that active NKCC1-mediated neuronal Cl− accumulation is required to maintain the proconvulsive effect of glycine receptor activation; and (iii) that a persistent activation of glycine receptors is required to suppress epileptiform activity in the immature hippocampus. In summary, these results demonstrate that the glycinergic system represents an important element for the control of neuronal excitability in the immature hippocampus.

Both taurine and glycine had a proconvulsive effect if applied at low concentrations. As the proconvulsive effect of 0.1 mm taurine was abolished in the presence of strychnine, we conclude that glycine receptors contribute to the proconvulsive taurine effect. This proconvulsive effect of moderate glycine receptor activation most probably reflects an enhanced excitability in the hippocampal circuits caused by the membrane depolarization induced by glycine receptor activation in immature brain (Ito & Cherubini, 1991; Flint et al. 1998; Ehrlich et al. 1999; Kilb et al. 2002, 2008). This membrane depolarization most probably reflects an efflux of Cl− through glycine receptors due to an elevated intracellular Cl− concentration mediated by active Cl− uptake via the NKCC1 (Russell, 2000; Yamada et al. 2004; Achilles et al. 2007; Ben Ari et al. 2012). Accordingly, we revealed that the proconvulsive effects of low taurine and glycine concentrations were completely abolished after incubation with the NKCC1 blocker bumetanide, providing additional evidence that NKCC1-mediated Cl− accumulation support excitatory responses even in an in situ preparation with minimal traumatic injuries. In the present experiments and in accordance with a previous study on intact hippocampal preparations (Kilb et al. 2007), bumetanide did not affect epileptiform activity, while bumetanide mediated an anticonvulsive effect in organotypic slices (Wahab et al. 2011), probably reflecting altered Cl− concentrations and/or extracellular levels of GABAergic agonists in the latter preparation. In the adult hippocampus, taurine mediates a stringent anticonvulsive effect (El Idrissi et al. 2003; Kirchner et al. 2003), due to the lack of depolarizing responses in this age group.

On the other hand, higher concentrations of taurine and glycine also attenuated epileptiform activity in the immature hippocampus. As our experiments revealed that (i) the anticonvulsive effect of taurine was diminished in the presence of strychnine, and (ii) that taurine attenuated epileptiform activity in the presence of a GABAA receptor antagonist, we propose that also a considerable portion of the anticonvulsive taurine effect was mediated via glycine receptors. This anticonvulsive effect evoked by activation of depolarizing glycine receptor most probably indicates that opening of glycine receptors reduce the input resistance of neurons, which will efficiently shunt excitatory synaptic inputs (Staley & Mody, 1992). Such a shunting inhibition by activation of glycine receptors has already been demonstrated in immature neocortical neurons (Kilb et al. 2002). The fact that glycine receptor activation can induce both pro- and anticonvulsive effects, depending on the level of receptor activation, is probably related to the complex interaction between membrane potential and input resistance changes upon activation of ligand-gated Cl– channels (Kolbaev et al. 2011) and/or interactions with additional ion currents mediated, e.g. by persistent Na2+ channels (Valeeva et al. 2010). In any way, the effective attenuation of epileptiform activity by higher concentrations of glycine and taurine demonstrated the anticonvulsive potential of glycine receptor activation already in the immature hippocampus.

During the first postnatal week the expression of glycine receptor subunits α2 and α3 dominates in the hippocampus (Aroeira et al. 2011), which will result in glycine receptors with a relatively low glycine affinity between 70 and 250 μm (Schmieden et al. 1992; Meier et al. 2005). However, alternative splicing can lead to the expression of high-affinity glycine receptor α3L subunit (Eichler et al. 2009), which most probably underlies the effect of low glycine concentrations. Tonic inward currents mediated by these receptors adapted for extrasynaptic signalling may preferably enhance excitability in the immature hippocampus, as has been already shown for tonic GABAergic currents mediated by an α5-subunit containing GABAA receptors (Kolbaev et al. 2012).

While our observations suggest that taurine may be an endogenous agonist of glycine receptors, we cannot provide direct experimental proof. Our experiments with the taurine uptake inhibitor GES showed that this substance had no significant effect on epileptiform activity under conditions when effects on GABAA receptors were prevented. On the other hand, our results also provide evidence that taurine has a proconvulsive action when both GABAA and glycine receptors are blocked. This proconvulsive effect is probably caused by taurine uptake and the resulting cell swelling (Oja & Saransaari, 1996), as it is abolished by preventing taurine uptake via the GES-sensitive taurine transporter. Cell swelling and the resulting decrease in extracellular volume fraction has been shown to decrease seizure thresholds in the immature hippocampus (Kilb et al. 2006).

Finally we were able to demonstrate that inhibition of glycine receptors with low strychnine concentrations induces spontaneous epileptiform discharges. This proconvulsive strychnine effect is most probably directly caused by inhibition of glycine receptor-mediated currents, as our control experiments showed that such low strychnine concentrations had a negligible effect on spontaneous GABAA receptor-mediated currents. We conclude from these observations that a persistent activation of glycine receptors is required to mediate an inhibitory tone that limits excitation and suppresses the generation of epileptiform discharges in the immature hippocampus. However, in accordance with many in vitro and in vivo studies (e.g. Baram & Snead, 1990; Khalilov et al. 1997b; Wells et al. 2000), inhibition of GABAA receptors provoked more pronounced epileptiform discharges that occurred at a substantially shorter latency. The fact that glycine receptors mediate a weaker inhibitory control was also confirmed by our observation that strychnine fails to modulate epileptiform activity induced by different in vitro models. Thus we conclude that the GABAergic system has a dominant role for inhibitory control in the immature hippocampus.

In the mature hippocampus (Kirchner et al. 2003) also observed a dual effect of glycine, showing an initial proconvulsive effect followed by a persistent anticonvulsive action. However, the proconvulsive effect of glycine in their case is most probably mediated by the positive modulation of NMDA receptors. This explanation could not account for our experiments, as we observed that saturation of the glycine binding sites with 200 μm d-serine had no effect on glutamatergic transmission and epileptiform activity.

In summary, our experiments demonstrate that in an intact in vitro preparation of the immature hippocampus the glycinergic system mediate pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress the generation of epileptiform discharges. In contrast to these results in the immature hippocampus, low strychnine concentrations do not induce or modulate epileptiform activity in the adult hippocampus (Kirchner et al. 2003), suggesting that the role of glycine receptors in the control of excitability is developmentally regulated. However, in human epileptic tissue the expression of glycine receptors is upregulated (Eichler et al. 2008), suggesting that they can contribute to inhibition under pathophysiological conditions also in the mature CNS. Interfering with the glycinergic system, in particular by using taurine analogues (Gupta et al. 2005; Oja & Saransaari, 2013), may open a new therapeutic window for pharmacoresistant epilepsies in immature and adult patients.

Acknowledgments

The authors thank Sigrid Stroh-Kaffei and Violetta Steinbrecher for excellent technical assistance.

Glossary

4-AP

4-aminopyridine

ACSF

artificial cerebrospinal fluid

ALX 5407

N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine hydrochloride

ALX 1393

O-[(2-Benzyloxyphenyl-3-flurophenyl)methyl]-l-serine

APV

dl-2-amino-5-phosphonopentanoic acid

CHF

corticohippocampal formation

GBZ

gabazine

GES

guanidinoethyl sulphonate

NBQX

1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide disodium salt hydrate

NKCC1

isoform 1 of the Na+, K+, 2Cl− cotransporter

Key points

  • Taurine has a pro- and anticonvulsive effect on the immature hippocampus, depending on the dose.

  • The taurine effect is mediated by GABAA and glycine receptors.

  • The taurine effect can be partially mimicked by glycine.

  • Inhibition of glycine receptors has a weak proconvulsive effect on the immature hippocampus.

  • We conclude that an endogenous activation of glycine receptors by glycine or taurine contributed to the control of neuronal excitability in the immature hippocampus.

Additional information

Competing interests

None of the authors has any conflict of interest to disclose.

Author contributions

W.K. designed the experiments, R.C., A.O. and S.S. performed and analysed the field potential recordings, A.O., R.C., H.S. and I.L.H.O. performed and analysed the whole cell patch clamp experiments, S.N.K. designed and programmed the analysis routines, W.K., A.F. and H.J.L. wrote the manuscript. All authors have read and approved the final submission.

Funding

The work was supported by the DFG grants to W.K. (KI 835/2-2) and H.L. and a NMFZ grant of the University Mainz to W.K. R.C. and H.S. were graduate students of the neuroscience graduate school at the University of Mainz.

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