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Reduced ictogenic potential of 4-aminopyridine in the hippocampal region in the pilocarpine model of epilepsy
Neuroscience Letters 513 (2012) 124–128
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Reduced ictogenic potential of 4-aminopyridine in the hippocampal region in the pilocarpine model of epilepsy Robert Karl Zahn 1 , Agustin Liotta, Simon Kim, Nora Sandow, Uwe Heinemann ∗ Charité – Universitätsmedizin Berlin, Institute for Neurophysiology, Germany
a r t i c l e
i n f o
Article history: Received 21 November 2011 Received in revised form 25 January 2012 Accepted 28 January 2012 Keywords: 4-Aminopyridine Hippocampal region Epilepsy Pilocarpine
a b s t r a c t It was previously shown that the ictogenic potential of 4-aminopyridine (4-AP) was reduced in the parahippocampal region of kainate treated chronic epileptic rats. In the actual study we investigated the potential of 4-aminopyridine (50 and 100 M) to induce seizure like events (SLEs) in combined entorhinal cortex hippocampal slices from Wistar rats following pilocarpine induced status epilepticus. The potential of 4-AP to induce SLEs in the entorhinal cortex was reduced in the latent period and in slices of chronic epileptic animals with a high seizure incidence in vivo (>2 seizures/24 h). 4-AP induced SLEs in slices from animals with a low incidence of seizures in vivo (<2 seizures/24 h) in a similar manner as compared to controls. The hippocampal formation displayed no SLEs, instead short recurrent epileptiform discharges (REDs) were evoked by application of 4-AP in areas CA3 and CA1. The incidence of REDs was largest in slices from control animals. This study shows that the reduced ictogenic potential of 4-AP is not restricted to kainate treated chronic epileptic animals as it can be found in the pilocarpine model as well. The underlying mechanisms may relate to altered expression and editing of voltage gated potassium channels. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Aminopyridines are potent convulsants in vivo [1,20] as well as in vitro [11,24]. Bath application to brain slices induces seizure-like events (SLEs) [14,24]. 4-AP is a potassium channel blocker, which in low concentrations of 50–100 M affects A-type [7], D-type [18] and delayed rectifier voltage gated potassium (Kv) channels from the Kv1 and Kv3 family [7]. Interestingly, treatment with the powerful convulsant 4-AP failed to induce SLEs in hippocampal tissue from patients with temporal lobe epilepsy [4]. In a previous study, we showed that the ictogenic potential of 4-AP in the parahippocampal region of kainate treated chronic epileptic rats was reduced [24]. The underlying mechanisms are not understood, since a significant downregulation of Kv channels sensitive to low concentrations of 4-AP was not found. We recently described that I400V RNA editing of Kv1.1 generates 4-AP insensitive Kv1 channels and found a 4-fold increase in RNA editing ratios in the entorhinal cortex (EC) of kainate treated chronic epileptic animals when compared to control animals [19]. The reduced
∗ Corresponding author at: Institute of Neurophysiology, Charité – Universitaetsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 450 528 091; fax: +49 30 450 528 962. E-mail address: [email protected] (U. Heinemann). 1 Present address: Center for Musculoskeletal Surgery, Campus-Charité-Mitte, Charité – Universitaetsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.01.071
ictogenic potential of 4-AP in kainate treated animals might have been dependent on seizure frequency. Seizure incidence is generally lower in pilocarpine treated chronic epileptic animals than observed in kainate treated animals in our laboratory [17,24]. In this study we examined the ictogenic potential of 4-AP in slices of pilocarpine treated animals. Particularly strong epileptiform activity is observed during status epilepticus; we consequently hypothesized that sensitivity to 4-AP is strongly reduced in the latent period before spontaneous seizures emerge.
2. Methods 2.1. Animal groups and induction of the status epilepticus Status epilepticus (SE) was induced in 37 adolescent male Wistar rats (125–160 g) 20 min after intraperitoneal (i.p.) injection of methylscopolamine (1 mg/kg; Sigma, St. Louis, MO, USA) by one single i.p. injection of pilocarpine (340 mg/kg; Sigma, St. Louis, MO, USA) and terminated by i.p injection of diazepam (10 mg/kg, Ratiopharm, Ulm, Germany) after 90 min of SE as described previously [17]. Diazpam injections were repeated as needed to terminate SE. Control animals were treated with the same injection-protocol but saline replacing pilocarpine [17]. 21 animals were used 3–9 days after SE for experiments in the latent period and compared with age-matched control adolescent animals (n = 6).
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In the remaining 16 animals the incidence of seizures (stage 4 and 5 according to Racine scale; [15]) was determined by video recording for at least 38 h at 2–2.5 months after SE [24]. Seizures occurred with frequencies of 0.2–14 per 24 h, mean seizure incidence was 3.3 ± 4.2 per 24 h. The control group consisted of age-matched adult animals (n = 10). Before electrophysiological experiments, the seizure incidence of epileptic animals was determined by analyzing video recordings. Animals were subsequently divided into 2 groups: rats with a low incidence of seizures had less than 2 seizures per 24 h (n = 9 animals; named: epileptic, low) and animals with a high incidence of seizures had more than 2 seizures per 24 h (n = 7 animals; named: epileptic, high). All animal procedures were conducted in accordance with the guidelines of the European Communities Council directive 86/609/EEC and were approved by the Regional Berlin Animal Ethics Committee (LaGeTSi No. G 0024/04). 2.2. Slice preparation/measurements of field potentials Rats were anesthetized with diethyl ether before decapitation. The brain was quickly removed and washed in ice-cold (4 ◦ C) artificial cerebrospinal fluid (aCSF) saturated with carbogen gas. Horizontal slices (400 m) containing the dentate gyrus, hippocampus, subiculum, EC, perirhinal cortex and temporal cortex were prepared as described previously using a Vibroslicer (World Precision Instruments, Berlin, Germany [24]). Slices were immediately transferred to a custom made interface chamber maintained at 34 ◦ C and perfused at a rate of 1.5–1.8 ml/min with aCSF saturated with carbogen gas, pH 7.4 containing (in mM): NaCl, 129; NaHCO3 , 21; KCl, 3; NaH2 PO4 , 1.25; CaCl2 , 1.6; MgSO4 , 1.8; glucose, 10; pH 7.4. Extracellular field potential recordings (glass microelectrodes, resistance 5–10 MOhm, filled with 154 mM NaCl) were placed in layer 2/3 and layer 5/6 of the medial entorhinal cortex (MEC), and in the stratum pyramidale of areas CA3 and CA1. As previously reported, single shock stimulation was used to document the quality of slices [17]. Slices with field potential amplitudes larger than 0.5 mV in layer 2/3 of the MEC were accepted for further recordings. The signals were filtered at 1 kHz and stored on hard disk drive with a CED 1401+ interface using Signal and Spike2 software (Cambridge Electronic Design, Cambridge, UK; sampling rate 8 kHz; [17]). 4-AP was first applied at 50 M for 90 min followed by 100 M for additional 90 min in the same slice [24]. Epileptiform activity was analyzed during the last 30 min of 90 min application time for 4-AP at each concentration (Sigma–Aldrich, Deisenhofen, Germany) [24]. Measurements are expressed as mean ± standard deviation. Statistical comparison was performed using dependent or independent nonparametric tests (SPSS 17.0/PASW Statistics 18) as indicated in the result chapter. 3. Results 3.1. Reduced ictogenic potential of 4-AP in the parahippocampal region of pilocarpine treated animals Bath application of 4-AP (50 and 100 M) resulted in ictal and interictal like activity in slices from control animals (Fig. 1A and B). SLEs were restricted to the MEC and absent in areas CA3 and CA1. Interictal like activity was recorded between SLEs (Fig. 1A). The hippocampal formation displayed no seizure like events but instead REDs (Fig. 2). 50% of slices from control adolescent animals displayed SLEs at 50 M 4-AP (n = 6 slices, 6 animals; Fig. 1A and B). 79% of control slices from adult animals showed SLEs after application of 50 M (n = 14 slices, 10 animals). 100 M of 4-AP evoked SLEs in all slices of these control animals (Fig. 3A and B).
Fig. 1. A: Simultaneous recordings of field potentials in areas CA3 and CA1 and in the superficial (II/III) and deep layers (V/VI) of the MEC of an adolescent control animal. Note the display of recurrent epileptiform discharges in area CA3 and CA1 plus seizure like events in entorhinal cortex. B: Expanded display of the indicated SLE (*) with an initial tonic like phase followed by clonic like after-discharges of an adolescent control animal. C–E: Ictal like events in the MEC of the slices from pilocarpine treated animals.
4-AP failed to induce SLEs in the majority of slices from animals in the latent period at both concentrations with a significantly lower incidence compared to control tissue (n = 21 slices, 21 animals; p = 0.025 at 50 M, p = 0.002 at 100 M, Fisher’s exact test). Only 24% of slices displayed SLEs at 50 and 100 M 4-AP (5 of 21 slices). One of these slices showed SLEs at 50 M 4-AP, 5 slices
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Fig. 2. 4-AP induced recurrent epileptiform discharges in area CA3 and CA1. Note the reduced propagation from CA3 to CA1 in the slices of pilocarpine treated animals during latent period and in the slices of animals with high incidence of seizures (B and E). Note also the preserved high frequency of superimposed field potential transients in the slices of animals with a low seizure incidence in vivo.
displayed SLEs at 100 M 4-AP (Figs. 1C and 3A). 4-AP induced SLEs in all slices from chronic epileptic animals with a low seizure incidence (n = 10 slices, 9 animals; Fig. 1D). In this group, 80% of slices showed SLEs at 50 M, the remaining slices displayed SLEs at 100 M 4-AP (Fig. 3B). Slices of chronic epileptic animals with a high incidence of seizures in vivo displayed less frequent SLEs when compared to controls irrespective of the concentration of 4-AP (n = 10 slices, 7 animals; p = 0.011 at 50 M, p = 0.005 at 100 M, Fisher’s exact test; Fig. 1E). 4-AP at 50 M could provoke SLEs in 20% of the slices and at 100 M in 50% of the slices (Fig. 3B). The duration of SLEs was dependent on the concentration of 4AP. It was longer at 100 M compared to 50 M in slices from adult control animals and from epileptic animals with a low incidence of seizures (adult: p = 0.004, n = 14; epileptic, low: p = 0.043, n = 10; Wilcoxon test). We analyzed the mean frequency of field potential transients during the entire SLEs (tonic like and clonic like phase) and the frequency of the tonic like phase. These frequencies showed no significant differences between the groups (see supplementary table). 3.2. Altered recurrent epileptiform discharges in the hippocampal formation in pilocarpine treated animals
Fig. 3. Percentage of slices expressing SLEs during treatment with 4-AP. A: Latent period compared to controls (n = 6 adolescent, 21 latent period; p = 0.025 at 50 M; p = 0.002 at 100 M; Fisher’s exact test). B: Chronic epileptic animals compared to controls (n = 14 adults, 10 epileptic low, 10 epileptic high; p = 0.011 at 50 M, p = 0.005 at 100 M for animals with a high seizure rate in vivo; Fisher’s exact test).
In control animals, REDs propagated from CA3 to CA1 (Fig. 2). Events consisted of slow positive field potential shifts superimposed by faster field potential transients with ripple frequencies of 208.5 ± 23.8 Hz in adolescent control and 224.6 ± 34.17 Hz in adult control animals (see also [16]). A reduced incidence of REDs was found in slices of animals in the latent period (n = 21), compared to controls (n = 6; p = 0.024, Fisher’s exact test). Adolescent control animals displayed propagation from CA3 to CA1 in 83.33% (5 of 6 slices) and adult controls in 91.67% (11 of 12 slices). In slices of pilocarpine treated animals high frequency ripples superimposing REDs were rarely observed and propagation to area CA1 was reduced (Fig. 2). Only 44.4% of slices from animals in the latent period showed propagation from area CA3 to CA1 (4 of 9 slices; p = 0.008, Fisher’s exact
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test). Slices from epileptic animals with a low seizure incidence showed REDs in area CA1 in 50% (4 of 8 slices; p = 0.032, Fisher’s exact test) and those from epileptic animals with a high seizure rate displayed propagation of REDs from area CA3 to CA1 in 37.5% (3 of 8 slices; p = 0.01, Fisher’s exact test). Events were of shorter duration in slices from animals in the latent period compared to controls (p = 0.014 at 50 M, p = 0.026 at 100 M, Mann–Whitney U test). Superimposed fast field potential transients had higher frequencies in slices of chronic epileptic animals with a low seizure incidence in vivo (502.7 ± 211 Hz) when compared to control animals (p = 0.0001, Mann–Whitney U test; Fig. 2D). By contrast REDs in animals with high seizure incidence were less likely to be present with high frequency ripples. For details of epileptiform activity see supplementary table.
4. Discussion This study demonstrates a reduced ictogenic potential of 4-AP in the MEC in the pilocarpine model of epilepsy. The reduced susceptibility of 4-AP may depend on seizure activity in vivo. As reported previously, 4-AP failed to induce SLEs in slices from kainate treated chronic epileptic animals [24]. These animals had a higher seizure rate in vivo compared to the pilocarpine model with a mean seizure incidence of 9.5 per 24 h [5,8,10,12]. The only slice displaying SLEs in the kainate model was obtained from an animal that showed a low incidence of seizures in vivo and a preserved cell number in the entorhinal cortex [24]. Interestingly, brain slices from animals in the latent period after pilocarpine injection showed the lowest susceptibility to 4-AP. Comparisons between the kainate and pilocarpine model are usually restricted. However, we established both animal models in our laboratory and performed experiments over the same time period. Hence age, sex, species or breeder differences could not account for the differences of susceptibility to the ictogenic potential of 4-AP in parahippocampal regions. In vivo seizure incidence also affected the occurrence of REDs in the hippocampus. REDs in chronic animals with low seizure incidence were characterized by superimposed fast field potentials transients, which were not recorded in epileptic animals with high seizure incidence. This may be due to loss of neurons in the hippocampus proper. Alterations in the epileptic parahippocampal region and hippocampal formation may restrict network synchronization in vitro and prevent the incurrence of 4-AP associated SLEs. The underlying mechanisms leading to a reduced susceptibility to 4-AP are not completely understood. A significant downregulation of Kv channels sensitive to low concentrations of 4-AP was not found [24]. Expression of Kv3.4 was reduced in the entorhinal and perirhinal cortex of epileptic animals. Whether heteromeric channels between Kv3.4 and other subunits of voltage gated potassium channels with high affinity to 4-AP exist, is presently unknown. A decreased availability of Kv4.2 channels was found in area CA1, but these subunits are less sensitive to 4-AP and blocked at mM – concentrations [3]. Editing of potassium channels may explain the reduced sensitivity to 4-AP. A 4-fold increase in I400V RNA editing ratios of Kv1.1 generating 4-AP insensitive Kv1 channels was found in the entorhinal cortex of kainate treated chronic epileptic animals when compared to control animals [19]. The reduced susceptibility to 4-AP was also observed by Panuccio et al. in 2011, as they recorded SLEs in 60% of slices from pilocarpine treated epileptic Sprague Dawley rats 4–10 weeks after SE [14]. In their study, the generation of robust ictal like discharges in the epileptic tissue was “favored by decreased hippocampal output with an anti-ictogenic role of CA3, reinforced by EC–subiculum interactions and predominantly driven by amygdala networks” [14]. They reported that the incidence of SLEs in slices of chronic pilocarpine treated rats was increased following
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disruption of hippocampal output. Interestingly, we observed a reduced propagation of REDs from area CA3 to CA1 after SE. Decreased CA3 output should release the entorhinal cortex from hippocampal control of ictal synchronization [2,14]. Onset of SLEs after bath application of 4-AP at 50 M is most commonly within the first 60 min [22,24]. However, it cannot be excluded that in some slices of pilocarpine treated animals, the onset of ictal like events is delayed and SLEs cannot be recorded after 90 min of bath application of each concentration. In contrast to other studies, we did not observe SLEs in areas CA3 and CA1 what may be due to the type of preparation and preserved connectivity of the brain slice. In the actual study, we used non-angled horizontal slices [21,24]. Furthermore, onset and spread of ictal like events depend on species and age [22]. 4-AP induced SLEs in adult rats originate commonly in the perirhinal or entorhinal cortex and propagation into the hippocampal formation is rare [22,24]. In contrast, slices of adolescent rats display SLEs after application of 4-AP in the parahippocampal region and hippocampal formation, propagation into the hippocampal formation is common [22]. The most interesting finding in our study is that seizure activity apparently induces alterations in the epileptic tissue, which are anticonvulsant. These may include editing of potassium channel genes, but other activity dependent alterations in the expression of proteins may also contribute [19] as an activity dependent editing of glycine receptors has been reported [9,13,23]. Upregulation of expression of the GABA synthesizing enzyme glutamatedecarboxylase (GAD) in glutamatergic cells may also have an effect on seizure threshold [6,17]. In conclusion, we could demonstrate here that 4-AP has a reduced ictogenic potential in the pilocarpine model of epilepsy which is dependent on seizure incidence in vivo and likely due to activity dependent changes in neuronal excitability and synaptic interactions. Acknowledgements The authors would like to thank Drs. S. Gabriel and H.-J. Gabriel, Dr. H. Siegmund for excellent technical and computational assistance, Dr. K. Schulze for assistance in SE induction and animal monitoring. The study was supported by SFB TR3 TP C7 and by EpiCure. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2012.01.071. References [1] A. Baranyi, O. Feher, Convulsive effects of 3-aminopyridine on cortical neurones, Electroencephalogr. Clin. Neurophysiol. 47 (1979) 745–751. [2] M. Barbarosie, M. Avoli, CA3-driven hippocampal-entorhinal loop controls rather than sustains in vitro limbic seizures, J. Neurosci. 17 (1997) 9308–9314. [3] C. Bernard, A. Anderson, A. Becker, N.P. Poolos, H. Beck, D. Johnston, Acquired dendritic channelopathy in temporal lobe epilepsy, Science 305 (2004) 532–535. [4] S. Gabriel, M. Njunting, J.K. Pomper, M. Merschhemke, E.R. Sanabria, A. Eilers, A. Kivi, M. Zeller, H.J. Meencke, E.A. Cavalheiro, U. Heinemann, T.N. Lehmann, Stimulus and potassium-induced epileptiform activity in the human dentate gyrus from patients with and without hippocampal sclerosis, J. Neurosci. 24 (2004) 10416–10430. [5] M. Glien, C. Brandt, H. Potschka, H. Voigt, U. Ebert, W. Loscher, Repeated lowdose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy, Epilepsy Res. 46 (2001) 111–119. [6] R. Gutierrez, U. Heinemann, Kindling induces transient fast inhibition in the dentate gyrus—CA3 projection, Eur. J. Neurosci. 13 (2001) 1371–1379. [7] G.A. Gutman, K.G. Chandy, S. Grissmer, M. Lazdunski, D. McKinnon, L.A. Pardo, G.A. Robertson, B. Rudy, M.C. Sanguinetti, W. Stuhmer, X. Wang, International Union of Pharmacology. LIII, Nomenclature and molecular relationships of voltage-gated potassium channels, Pharmacol. Rev. 57 (2005) 473–508.
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[8] J.L. Hellier, P.R. Patrylo, P.S. Buckmaster, F.E. Dudek, Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy, Epilepsy Res. 31 (1998) 73–84. [9] A. Kirchner, J. Breustedt, B. Rosche, U.F. Heinemann, V. Schmieden, Effects of taurine and glycine on epileptiform activity induced by removal of Mg2+ in combined rat entorhinal cortex-hippocampal slices, Epilepsia 44 (2003) 1145–1152. [10] J.P. Leite, N. Garcia-Cairasco, E.A. Cavalheiro, New insights from the use of pilocarpine and kainate models, Epilepsy Res. 50 (2002) 93–103. [11] J. Louvel, M. Avoli, I. Kurcewicz, R. Pumain, Extracellular free potassium during synchronous activity induced by 4-aminopyridine in the juvenile rat hippocampus, Neurosci. Lett. 167 (1994) 97–100. [12] N. Marchi, E. Oby, A. Batra, L. Uva, M. De Curtis, N. Hernandez, A. Van BoxelDezaire, I. Najm, D. Janigro, In vivo and in vitro effects of pilocarpine: relevance to ictogenesis, Epilepsia 48 (2007) 1934–1946. [13] J. Meier, V. Schmieden, Inhibition of alpha-subunit glycine receptors by quinoxalines, Neuroreport 14 (2003) 1507–1510. [14] G. Panuccio, M. D’Antuono, P. de Guzman, L. De Lannoy, G. Biagini, M. Avoli, In vitro ictogenesis and parahippocampal networks in a rodent model of temporal lobe epilepsy, Neurobiol. Dis. 39 (2011) 372–380. [15] R.J. Racine, Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 281–294. [16] J.P. Richter, C.J. Behrens, A. Chakrabarty, U. Heinemann, Effects of 4aminopyridine on sharp wave-ripples in rat hippocampal slices, Neuroreport 19 (2008) 491–496.
[17] N. Sandow, R.K. Zahn, S. Gabriel, U. Heinemann, T.N. Lehmann, Glutamine induces epileptiform discharges in superficial layers of the medial entorhinal cortex from pilocarpine-treated chronic epileptic rats in vitro, Epilepsia 50 (2009) 849–858. [18] J.F. Storm, Potassium currents in hippocampal pyramidal cells, Prog. Brain Res. 83 (1990) 161–187. [19] A.K. Streit, C. Derst, S. Wegner, U. Heinemann, R.K. Zahn, N. Decher, RNA editing of Kv1.1 channels may account for reduced ictogenic potential of 4-aminopyridine in chronic epileptic rats, Epilepsia 52 (2011) 645–648. [20] M. Szente, A. Baranyi, Mechanism of aminopyridine-induced ictal seizure activity in the cat neocortex, Brain Res. 413 (1987) 368–373. [21] E.A. Tolner, C. Frahm, R. Metzger, J.A. Gorter, O.W. Witte, F.H. Lopes da Silva, U. Heinemann, Synaptic responses in superficial layers of medial entorhinal cortex from rats with kainate-induced epilepsy, Neurobiol. Dis. 26 (2007) 419–438. [22] F. Weissinger, K. Buchheim, H. Siegmund, H. Meierkord, Seizure spread through the life cycle: optical imaging in combined brain slices from immature, adult, and senile rats in vitro, Neurobiol. Dis. 19 (2005) 84–95. [23] T.L. Xu, N. Gong, Glycine and glycine receptor signaling in hippocampal neurons: diversity, function and regulation, Prog. Neurobiol. 91 (2010) 349–361. [24] R.K. Zahn, E.A. Tolner, C. Derst, C. Gruber, R.W. Veh, U. Heinemann, Reduced ictogenic potential of 4-aminopyridine in the perirhinal and entorhinal cortex of kainate-treated chronic epileptic rats, Neurobiol. Dis. 29 (2008) 186–200.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Reduced ictogenic potential of 4-aminopyridine in the hippocampal region in the pilocarpine model of epilepsy Robert Karl Zahn 1 , Agustin Liotta, Simon Kim, Nora Sandow, Uwe Heinemann ∗ Charité – Universitätsmedizin Berlin, Institute for Neurophysiology, Germany
a r t i c l e
i n f o
Article history: Received 21 November 2011 Received in revised form 25 January 2012 Accepted 28 January 2012 Keywords: 4-Aminopyridine Hippocampal region Epilepsy Pilocarpine
a b s t r a c t It was previously shown that the ictogenic potential of 4-aminopyridine (4-AP) was reduced in the parahippocampal region of kainate treated chronic epileptic rats. In the actual study we investigated the potential of 4-aminopyridine (50 and 100 M) to induce seizure like events (SLEs) in combined entorhinal cortex hippocampal slices from Wistar rats following pilocarpine induced status epilepticus. The potential of 4-AP to induce SLEs in the entorhinal cortex was reduced in the latent period and in slices of chronic epileptic animals with a high seizure incidence in vivo (>2 seizures/24 h). 4-AP induced SLEs in slices from animals with a low incidence of seizures in vivo (<2 seizures/24 h) in a similar manner as compared to controls. The hippocampal formation displayed no SLEs, instead short recurrent epileptiform discharges (REDs) were evoked by application of 4-AP in areas CA3 and CA1. The incidence of REDs was largest in slices from control animals. This study shows that the reduced ictogenic potential of 4-AP is not restricted to kainate treated chronic epileptic animals as it can be found in the pilocarpine model as well. The underlying mechanisms may relate to altered expression and editing of voltage gated potassium channels. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Aminopyridines are potent convulsants in vivo [1,20] as well as in vitro [11,24]. Bath application to brain slices induces seizure-like events (SLEs) [14,24]. 4-AP is a potassium channel blocker, which in low concentrations of 50–100 M affects A-type [7], D-type [18] and delayed rectifier voltage gated potassium (Kv) channels from the Kv1 and Kv3 family [7]. Interestingly, treatment with the powerful convulsant 4-AP failed to induce SLEs in hippocampal tissue from patients with temporal lobe epilepsy [4]. In a previous study, we showed that the ictogenic potential of 4-AP in the parahippocampal region of kainate treated chronic epileptic rats was reduced [24]. The underlying mechanisms are not understood, since a significant downregulation of Kv channels sensitive to low concentrations of 4-AP was not found. We recently described that I400V RNA editing of Kv1.1 generates 4-AP insensitive Kv1 channels and found a 4-fold increase in RNA editing ratios in the entorhinal cortex (EC) of kainate treated chronic epileptic animals when compared to control animals [19]. The reduced
∗ Corresponding author at: Institute of Neurophysiology, Charité – Universitaetsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 450 528 091; fax: +49 30 450 528 962. E-mail address: [email protected] (U. Heinemann). 1 Present address: Center for Musculoskeletal Surgery, Campus-Charité-Mitte, Charité – Universitaetsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.01.071
ictogenic potential of 4-AP in kainate treated animals might have been dependent on seizure frequency. Seizure incidence is generally lower in pilocarpine treated chronic epileptic animals than observed in kainate treated animals in our laboratory [17,24]. In this study we examined the ictogenic potential of 4-AP in slices of pilocarpine treated animals. Particularly strong epileptiform activity is observed during status epilepticus; we consequently hypothesized that sensitivity to 4-AP is strongly reduced in the latent period before spontaneous seizures emerge.
2. Methods 2.1. Animal groups and induction of the status epilepticus Status epilepticus (SE) was induced in 37 adolescent male Wistar rats (125–160 g) 20 min after intraperitoneal (i.p.) injection of methylscopolamine (1 mg/kg; Sigma, St. Louis, MO, USA) by one single i.p. injection of pilocarpine (340 mg/kg; Sigma, St. Louis, MO, USA) and terminated by i.p injection of diazepam (10 mg/kg, Ratiopharm, Ulm, Germany) after 90 min of SE as described previously [17]. Diazpam injections were repeated as needed to terminate SE. Control animals were treated with the same injection-protocol but saline replacing pilocarpine [17]. 21 animals were used 3–9 days after SE for experiments in the latent period and compared with age-matched control adolescent animals (n = 6).
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In the remaining 16 animals the incidence of seizures (stage 4 and 5 according to Racine scale; [15]) was determined by video recording for at least 38 h at 2–2.5 months after SE [24]. Seizures occurred with frequencies of 0.2–14 per 24 h, mean seizure incidence was 3.3 ± 4.2 per 24 h. The control group consisted of age-matched adult animals (n = 10). Before electrophysiological experiments, the seizure incidence of epileptic animals was determined by analyzing video recordings. Animals were subsequently divided into 2 groups: rats with a low incidence of seizures had less than 2 seizures per 24 h (n = 9 animals; named: epileptic, low) and animals with a high incidence of seizures had more than 2 seizures per 24 h (n = 7 animals; named: epileptic, high). All animal procedures were conducted in accordance with the guidelines of the European Communities Council directive 86/609/EEC and were approved by the Regional Berlin Animal Ethics Committee (LaGeTSi No. G 0024/04). 2.2. Slice preparation/measurements of field potentials Rats were anesthetized with diethyl ether before decapitation. The brain was quickly removed and washed in ice-cold (4 ◦ C) artificial cerebrospinal fluid (aCSF) saturated with carbogen gas. Horizontal slices (400 m) containing the dentate gyrus, hippocampus, subiculum, EC, perirhinal cortex and temporal cortex were prepared as described previously using a Vibroslicer (World Precision Instruments, Berlin, Germany [24]). Slices were immediately transferred to a custom made interface chamber maintained at 34 ◦ C and perfused at a rate of 1.5–1.8 ml/min with aCSF saturated with carbogen gas, pH 7.4 containing (in mM): NaCl, 129; NaHCO3 , 21; KCl, 3; NaH2 PO4 , 1.25; CaCl2 , 1.6; MgSO4 , 1.8; glucose, 10; pH 7.4. Extracellular field potential recordings (glass microelectrodes, resistance 5–10 MOhm, filled with 154 mM NaCl) were placed in layer 2/3 and layer 5/6 of the medial entorhinal cortex (MEC), and in the stratum pyramidale of areas CA3 and CA1. As previously reported, single shock stimulation was used to document the quality of slices [17]. Slices with field potential amplitudes larger than 0.5 mV in layer 2/3 of the MEC were accepted for further recordings. The signals were filtered at 1 kHz and stored on hard disk drive with a CED 1401+ interface using Signal and Spike2 software (Cambridge Electronic Design, Cambridge, UK; sampling rate 8 kHz; [17]). 4-AP was first applied at 50 M for 90 min followed by 100 M for additional 90 min in the same slice [24]. Epileptiform activity was analyzed during the last 30 min of 90 min application time for 4-AP at each concentration (Sigma–Aldrich, Deisenhofen, Germany) [24]. Measurements are expressed as mean ± standard deviation. Statistical comparison was performed using dependent or independent nonparametric tests (SPSS 17.0/PASW Statistics 18) as indicated in the result chapter. 3. Results 3.1. Reduced ictogenic potential of 4-AP in the parahippocampal region of pilocarpine treated animals Bath application of 4-AP (50 and 100 M) resulted in ictal and interictal like activity in slices from control animals (Fig. 1A and B). SLEs were restricted to the MEC and absent in areas CA3 and CA1. Interictal like activity was recorded between SLEs (Fig. 1A). The hippocampal formation displayed no seizure like events but instead REDs (Fig. 2). 50% of slices from control adolescent animals displayed SLEs at 50 M 4-AP (n = 6 slices, 6 animals; Fig. 1A and B). 79% of control slices from adult animals showed SLEs after application of 50 M (n = 14 slices, 10 animals). 100 M of 4-AP evoked SLEs in all slices of these control animals (Fig. 3A and B).
Fig. 1. A: Simultaneous recordings of field potentials in areas CA3 and CA1 and in the superficial (II/III) and deep layers (V/VI) of the MEC of an adolescent control animal. Note the display of recurrent epileptiform discharges in area CA3 and CA1 plus seizure like events in entorhinal cortex. B: Expanded display of the indicated SLE (*) with an initial tonic like phase followed by clonic like after-discharges of an adolescent control animal. C–E: Ictal like events in the MEC of the slices from pilocarpine treated animals.
4-AP failed to induce SLEs in the majority of slices from animals in the latent period at both concentrations with a significantly lower incidence compared to control tissue (n = 21 slices, 21 animals; p = 0.025 at 50 M, p = 0.002 at 100 M, Fisher’s exact test). Only 24% of slices displayed SLEs at 50 and 100 M 4-AP (5 of 21 slices). One of these slices showed SLEs at 50 M 4-AP, 5 slices
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Fig. 2. 4-AP induced recurrent epileptiform discharges in area CA3 and CA1. Note the reduced propagation from CA3 to CA1 in the slices of pilocarpine treated animals during latent period and in the slices of animals with high incidence of seizures (B and E). Note also the preserved high frequency of superimposed field potential transients in the slices of animals with a low seizure incidence in vivo.
displayed SLEs at 100 M 4-AP (Figs. 1C and 3A). 4-AP induced SLEs in all slices from chronic epileptic animals with a low seizure incidence (n = 10 slices, 9 animals; Fig. 1D). In this group, 80% of slices showed SLEs at 50 M, the remaining slices displayed SLEs at 100 M 4-AP (Fig. 3B). Slices of chronic epileptic animals with a high incidence of seizures in vivo displayed less frequent SLEs when compared to controls irrespective of the concentration of 4-AP (n = 10 slices, 7 animals; p = 0.011 at 50 M, p = 0.005 at 100 M, Fisher’s exact test; Fig. 1E). 4-AP at 50 M could provoke SLEs in 20% of the slices and at 100 M in 50% of the slices (Fig. 3B). The duration of SLEs was dependent on the concentration of 4AP. It was longer at 100 M compared to 50 M in slices from adult control animals and from epileptic animals with a low incidence of seizures (adult: p = 0.004, n = 14; epileptic, low: p = 0.043, n = 10; Wilcoxon test). We analyzed the mean frequency of field potential transients during the entire SLEs (tonic like and clonic like phase) and the frequency of the tonic like phase. These frequencies showed no significant differences between the groups (see supplementary table). 3.2. Altered recurrent epileptiform discharges in the hippocampal formation in pilocarpine treated animals
Fig. 3. Percentage of slices expressing SLEs during treatment with 4-AP. A: Latent period compared to controls (n = 6 adolescent, 21 latent period; p = 0.025 at 50 M; p = 0.002 at 100 M; Fisher’s exact test). B: Chronic epileptic animals compared to controls (n = 14 adults, 10 epileptic low, 10 epileptic high; p = 0.011 at 50 M, p = 0.005 at 100 M for animals with a high seizure rate in vivo; Fisher’s exact test).
In control animals, REDs propagated from CA3 to CA1 (Fig. 2). Events consisted of slow positive field potential shifts superimposed by faster field potential transients with ripple frequencies of 208.5 ± 23.8 Hz in adolescent control and 224.6 ± 34.17 Hz in adult control animals (see also [16]). A reduced incidence of REDs was found in slices of animals in the latent period (n = 21), compared to controls (n = 6; p = 0.024, Fisher’s exact test). Adolescent control animals displayed propagation from CA3 to CA1 in 83.33% (5 of 6 slices) and adult controls in 91.67% (11 of 12 slices). In slices of pilocarpine treated animals high frequency ripples superimposing REDs were rarely observed and propagation to area CA1 was reduced (Fig. 2). Only 44.4% of slices from animals in the latent period showed propagation from area CA3 to CA1 (4 of 9 slices; p = 0.008, Fisher’s exact
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test). Slices from epileptic animals with a low seizure incidence showed REDs in area CA1 in 50% (4 of 8 slices; p = 0.032, Fisher’s exact test) and those from epileptic animals with a high seizure rate displayed propagation of REDs from area CA3 to CA1 in 37.5% (3 of 8 slices; p = 0.01, Fisher’s exact test). Events were of shorter duration in slices from animals in the latent period compared to controls (p = 0.014 at 50 M, p = 0.026 at 100 M, Mann–Whitney U test). Superimposed fast field potential transients had higher frequencies in slices of chronic epileptic animals with a low seizure incidence in vivo (502.7 ± 211 Hz) when compared to control animals (p = 0.0001, Mann–Whitney U test; Fig. 2D). By contrast REDs in animals with high seizure incidence were less likely to be present with high frequency ripples. For details of epileptiform activity see supplementary table.
4. Discussion This study demonstrates a reduced ictogenic potential of 4-AP in the MEC in the pilocarpine model of epilepsy. The reduced susceptibility of 4-AP may depend on seizure activity in vivo. As reported previously, 4-AP failed to induce SLEs in slices from kainate treated chronic epileptic animals [24]. These animals had a higher seizure rate in vivo compared to the pilocarpine model with a mean seizure incidence of 9.5 per 24 h [5,8,10,12]. The only slice displaying SLEs in the kainate model was obtained from an animal that showed a low incidence of seizures in vivo and a preserved cell number in the entorhinal cortex [24]. Interestingly, brain slices from animals in the latent period after pilocarpine injection showed the lowest susceptibility to 4-AP. Comparisons between the kainate and pilocarpine model are usually restricted. However, we established both animal models in our laboratory and performed experiments over the same time period. Hence age, sex, species or breeder differences could not account for the differences of susceptibility to the ictogenic potential of 4-AP in parahippocampal regions. In vivo seizure incidence also affected the occurrence of REDs in the hippocampus. REDs in chronic animals with low seizure incidence were characterized by superimposed fast field potentials transients, which were not recorded in epileptic animals with high seizure incidence. This may be due to loss of neurons in the hippocampus proper. Alterations in the epileptic parahippocampal region and hippocampal formation may restrict network synchronization in vitro and prevent the incurrence of 4-AP associated SLEs. The underlying mechanisms leading to a reduced susceptibility to 4-AP are not completely understood. A significant downregulation of Kv channels sensitive to low concentrations of 4-AP was not found [24]. Expression of Kv3.4 was reduced in the entorhinal and perirhinal cortex of epileptic animals. Whether heteromeric channels between Kv3.4 and other subunits of voltage gated potassium channels with high affinity to 4-AP exist, is presently unknown. A decreased availability of Kv4.2 channels was found in area CA1, but these subunits are less sensitive to 4-AP and blocked at mM – concentrations [3]. Editing of potassium channels may explain the reduced sensitivity to 4-AP. A 4-fold increase in I400V RNA editing ratios of Kv1.1 generating 4-AP insensitive Kv1 channels was found in the entorhinal cortex of kainate treated chronic epileptic animals when compared to control animals [19]. The reduced susceptibility to 4-AP was also observed by Panuccio et al. in 2011, as they recorded SLEs in 60% of slices from pilocarpine treated epileptic Sprague Dawley rats 4–10 weeks after SE [14]. In their study, the generation of robust ictal like discharges in the epileptic tissue was “favored by decreased hippocampal output with an anti-ictogenic role of CA3, reinforced by EC–subiculum interactions and predominantly driven by amygdala networks” [14]. They reported that the incidence of SLEs in slices of chronic pilocarpine treated rats was increased following
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disruption of hippocampal output. Interestingly, we observed a reduced propagation of REDs from area CA3 to CA1 after SE. Decreased CA3 output should release the entorhinal cortex from hippocampal control of ictal synchronization [2,14]. Onset of SLEs after bath application of 4-AP at 50 M is most commonly within the first 60 min [22,24]. However, it cannot be excluded that in some slices of pilocarpine treated animals, the onset of ictal like events is delayed and SLEs cannot be recorded after 90 min of bath application of each concentration. In contrast to other studies, we did not observe SLEs in areas CA3 and CA1 what may be due to the type of preparation and preserved connectivity of the brain slice. In the actual study, we used non-angled horizontal slices [21,24]. Furthermore, onset and spread of ictal like events depend on species and age [22]. 4-AP induced SLEs in adult rats originate commonly in the perirhinal or entorhinal cortex and propagation into the hippocampal formation is rare [22,24]. In contrast, slices of adolescent rats display SLEs after application of 4-AP in the parahippocampal region and hippocampal formation, propagation into the hippocampal formation is common [22]. The most interesting finding in our study is that seizure activity apparently induces alterations in the epileptic tissue, which are anticonvulsant. These may include editing of potassium channel genes, but other activity dependent alterations in the expression of proteins may also contribute [19] as an activity dependent editing of glycine receptors has been reported [9,13,23]. Upregulation of expression of the GABA synthesizing enzyme glutamatedecarboxylase (GAD) in glutamatergic cells may also have an effect on seizure threshold [6,17]. In conclusion, we could demonstrate here that 4-AP has a reduced ictogenic potential in the pilocarpine model of epilepsy which is dependent on seizure incidence in vivo and likely due to activity dependent changes in neuronal excitability and synaptic interactions. Acknowledgements The authors would like to thank Drs. S. Gabriel and H.-J. Gabriel, Dr. H. Siegmund for excellent technical and computational assistance, Dr. K. Schulze for assistance in SE induction and animal monitoring. The study was supported by SFB TR3 TP C7 and by EpiCure. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2012.01.071. References [1] A. Baranyi, O. Feher, Convulsive effects of 3-aminopyridine on cortical neurones, Electroencephalogr. Clin. Neurophysiol. 47 (1979) 745–751. [2] M. Barbarosie, M. Avoli, CA3-driven hippocampal-entorhinal loop controls rather than sustains in vitro limbic seizures, J. Neurosci. 17 (1997) 9308–9314. [3] C. Bernard, A. Anderson, A. Becker, N.P. Poolos, H. Beck, D. Johnston, Acquired dendritic channelopathy in temporal lobe epilepsy, Science 305 (2004) 532–535. [4] S. Gabriel, M. Njunting, J.K. Pomper, M. Merschhemke, E.R. Sanabria, A. Eilers, A. Kivi, M. Zeller, H.J. Meencke, E.A. Cavalheiro, U. Heinemann, T.N. Lehmann, Stimulus and potassium-induced epileptiform activity in the human dentate gyrus from patients with and without hippocampal sclerosis, J. Neurosci. 24 (2004) 10416–10430. [5] M. Glien, C. Brandt, H. Potschka, H. Voigt, U. Ebert, W. Loscher, Repeated lowdose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy, Epilepsy Res. 46 (2001) 111–119. [6] R. Gutierrez, U. Heinemann, Kindling induces transient fast inhibition in the dentate gyrus—CA3 projection, Eur. J. Neurosci. 13 (2001) 1371–1379. [7] G.A. Gutman, K.G. Chandy, S. Grissmer, M. Lazdunski, D. McKinnon, L.A. Pardo, G.A. Robertson, B. Rudy, M.C. Sanguinetti, W. Stuhmer, X. Wang, International Union of Pharmacology. LIII, Nomenclature and molecular relationships of voltage-gated potassium channels, Pharmacol. Rev. 57 (2005) 473–508.
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