Impaired function of GABAB receptors in tissues ... - Wiley Online Library

Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

FULL-LENGTH ORIGINAL RESEARCH

Impaired function of GABAB receptors in tissues from pharmacoresistant epilepsy patients *Laura A. Teichgra¨ber, yThomas-Nicolas Lehmann, zHeinz-Joachim Meencke, xTorsten Weiss, *Robert Nitsch and *Rudolf A. Deisz *Center for Anatomy, Institute for Cell Biology and Neurobiology, Charite´–Universita¨tsmedizin Berlin, Berlin, Germany; yDepartment of Neurosurgery, Charite´–Universita¨tsmedizin Berlin, Berlin, Germany; zEpilepsy Center Berlin-Brandenburg, Berlin-Lichtenberg, Germany; and xCenter for Anatomy, Institute for Integrative Neuroanatomy, Charite´–Universita¨tsmedizin Berlin, Berlin, Germany

SUMMARY Purpose: Effects of pre- and postsynaptic c-aminobutyric acid B (GABAB) receptor activation were characterized in human tissue from epilepsy surgery. Methods: Slices of human cortical tissue were investigated in a submerged-type chamber with intracellular recordings in layers II/III. Parallel experiments were performed in rat neocortical slices with identical methods. Synaptic responses were elicited with single or paired stimulations of incrementing intervals. Results: Neurons in human epileptogenic tissue exhibited usually small inhibitory postsynaptic potentials (IPSP) mediated by GABAB receptor, verified by the sensitivity to the selective antagonist CGP 55845A. The IPSPB conductance averaged 5.8 nS in neurons from epileptogenic tissues and 15.9 nS in neurons from nonepileptogenic tissues (p < 0.0001). Application of baclofen caused small conductance increases in human neurons, which were linearly related to IPSPB

c-Aminobutyric acid (GABA), the key inhibitory transmitter in the mammalian central nervous system (CNS), exerts its effects by activating two types of receptors, termed GABAA and GABAB. The GABAA receptors are a pentameric arrangement of 17 possible subunits (see Mçhler et al., 1997) constituting an anion channel

Accepted February 5, 2009; Early View publication April 27, 2009. Address correspondence to Rudolf A. Deisz, Center for Anatomy, Institute for Cell Biology and Neurobiology, Charit–Universittsmedizin Berlin, 10115 Berlin, Germany. E-mail: [email protected] Wiley Periodicals, Inc. ª 2009 International League Against Epilepsy

conductances. Paired-pulse stimulation revealed constant synaptic responses in human temporal lobe epilepsy (TLE) slices at all interstimulus intervals (ISIs). Pharmacologically isolated IPSPA in the human tissue exhibited a small paired-pulse depression (average 10% at 500 ms ISI). Bicuculline-induced paroxysmal depolarization shifts (PDSs) were transiently depressed by 24% in human TLE tissue; and by 74% in rat neocortical slices (200 ms ISI; p = 0.015). The depressions of bicuculline-induced PDSs were antagonized by CGP 55845A in both species. Staining for GABAB receptors revealed significantly smaller numbers of immunopositive dots in human epileptogenic neurons versus human control neurons. Discussion: The small IPSPB, baclofen-conductances, and paired-pulse depression of PDSs and IPSPs in human TLE tissue indicate a reduced density of post- and presynaptic GABAB receptors. The reduced efficacy of presynaptic GABAB receptors facilitates the occurrence of repetitive synaptic activity. KEY WORDS: Pharmacoresistance, Epilepsy, GABA.

(Thompson et al., 1988; Kaila et al., 1993). The GABAB receptors, originally cloned by Kaupmann et al. (1997), are heterodimers of two distinct subunits (Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999) coupling to effector systems by means of G proteins (see Misgeld et al., 1995; Deisz, 1997). A wealth of evidence indicates that impairment of inhibition mediated by GABAA receptors plays a pivotal role in the initiation and spread of epileptiform activity. Numerous investigations have demonstrated that an experimental decrease in GABAA conductance by receptor antagonists causes epileptiform activity in the mammalian neocortex in vitro (e.g., Gutnick et al., 1982; Deisz &

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1698 L. A. Teichgra¨ber et al. Prince, 1987; Chagnac-Amitai & Connors, 1989; Sutor & Luhmann, 1998). Moreover, aberrant GABAA receptors are expressed during epilepsy development and throughout the course of the disease (Brooks-Kayal et al., 1998). The role of GABAB receptors in epileptic disorders, however, is less clear. GABAB receptors have been implicated in primary generalized epilepsies (e.g., Marescaux et al., 1992; Vergnes et al., 1997), yet their role in focal epilepsies is controversial. GABAB receptor antagonists have marginal effects on excitatory postsynaptic potentials and properties of cortical neurons (Deisz et al., 1997). However, paroxysmal depolarization shifts (PDSs) induced by GABAA antagonists are augmented by the GABAB antagonist CGP 35348 (Karlsson et al., 1992; Sutor & Luhmann, 1998), but abolished by CGP 35348 when elicited with near-threshold intensities (Sutor & Luhmann, 1998). Blockade of GABAB receptors also facilitates the occurrence of audiogenic seizures in susceptible rats in vivo (Vergnes et al., 1997). These data from antagonist applications suggest that GABAB receptors modulate normal excitatory synaptic responses only to a very limited extent, and that the impact of inhibition by GABAB receptors becomes observable only under certain pathophysiological conditions. However, the GABAB agonist baclofen is also proconvulsant (VanRijn et al., 1987; Mott et al., 1989), compatible with a predominance of disinhibition by baclofen through depression of GABA release by presynaptic GABAB receptors (Deisz & Prince, 1989). In a model of temporal lobe epilepsy (TLE), termed selfsustained limbic status epilepticus, postsynaptic GABAB responses are virtually absent in the CA1 area of rat hippocampus (Mangan & Lothman, 1996). In this model, GABAA responses are fairly robust and display little paired-pulse depression (Mangan & Lothman, 1996; Wu & Leung, 1997), attributed to impairment of presynaptic GABAB function (Mangan & Lothman, 1996). Also at the mossy fiber–CA3 synapses, a GABAB receptor modulation of release was greatly reduced after status epilepticus, induced either by pilocarpine or perforant path stimulation (Chandler et al., 2003). This absence of pre- and postsynaptic GABAB responses in epileptic rats is complemented by generalized seizure activity in GABAB1 receptor knockout mice (Prosser et al., 2001; Schuler et al., 2001). However, the functional evidence toward a deficit of GABAB function is contrasted by the variable increases and decreases of in situ hybridization signals in various areas of human hippocampus from epilepsy surgery. For instance, the hybridization signal for GABAB1a is decreased in area CA1, but increased in the dentate gyrus (Princivalle et al., 2003). The hybridization signals for GABAB1b and GABAB2 receptor subunits [Correction made after online publication 20 May 2009: GABAB1b and receptor subunits GABAB2 changed to GABAB1b and GABAB2 receptor subunits] are increased in area CA1; in the dentate gyrus, however, GABAB1b signal is decreased, whereas the GABAB2 Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

signal is increased. Relating the mRNA levels to neuronal density revealed a marked and consistent increase of mRNA for all isoforms in all hippocampal areas (Princivalle et al., 2003). In human neocortical tissue from epilepsy surgery the presence of GABAB receptor–mediated inhibition has been established (McCormick, 1989), yet the magnitude appeared rather small (Deisz, 1999b). Here we report on experiments designed to evaluate the properties of GABAB receptor–mediated events in human neocortex from epilepsy surgery. The data obtained indicate a reduced effectiveness of pre- and postsynaptic GABAB receptors, which facilitates repetitive synaptic activity.

Materials and Methods Preparation of human and rodent neocortical slices Human neocortical tissues were obtained from 140 patients undergoing surgical treatment of pharmacoresistant epilepsy or removal of tumors. Every patient gave written informed consent for the scientific use of the tissue, and the procedures were approved by the local ethics committee (30.9.97), adhering to the Declaration of Helsinki. The presurgical evaluation of the patients was carried out according to the guidelines of the German Chapter of the International League Against Epilepsy. Resected tissue comprised the anterior part (about 2 cm) of the temporal lobe [129 resections(r)] or part of the frontal lobe (11 r). The frontopolar part of the temporal lobe pole (approximately 10% of the resected tissue) was cut out by the neurosurgeon and assigned for the studies presented herein; the main part was reserved for standard neuropathological examination. According to the main clinical symptoms, the tissues were grouped into seven categories: (1) TLE with Ammon's horn sclerosis (AHS; n = 71 r), (2) TLE without AHS (n = 16 r), (3) tumors (n = 19 r), (4) cortical dysplasias (n = 10 r), (5) lesions (n = 8 r), (6) frontal lobe (n = 11 r), and (7) ‘‘control’’ tissue (n = 5 r). The latter tissue was resected from tumor patients without pharmacoresistant epilepsy (four patients without any seizures and one patient, who experienced one first seizure, 1 month before the operation). The tissues were immediately transferred into cold (about 5C) modified artificial cerebrospinal fluid (ACSF, equilibrated with 95% O2 and 5% CO2) and transported to the laboratory within 20 min in a Dewar container. The tissue was weighed in some cases (average 2.3 € 1.4 g; n = 57 r) before it was cut into smaller blocks. Individual blocks were mounted on a Vibratome (TPI, St. Louis, MO, U.S.A.) and cut into slices, perpendicular to the cortical surface (nominal thickness 400 lm). The slices were stored at room temperature in ACSF equilibrated with 95% O2 and 5% CO2. For some experiments, male Wistar rats (body weight 100–160 g) were anesthetized with ether and decapitated, as approved by the Berlin animal health protection agency (T 026/96). A block of tissue containing the somatosensory

1699 Impairment of GABAB Receptors in Human TLE cortex was removed, mounted on a Vibratome, and cut into coronal slices (nominal thickness 400 lm). Because the temporal lobe has no anatomic equivalent in rodents we chose the somatosensory cortex because it is a physiologically well-characterized area of isocortex. Storage of the rat slices, recording, and stimulation were identical to that used for the human slices. Solutions and drugs Normal ACSF, containing (in mM): NaCl 124, KCl 5, NaH2PO4 1.25, MgSO4 2, CaCl2 2, NaHCO3 26, and glucose 10, was equilibrated with 95% O2/5% CO2 (pH was 7.4, 32C). For the tissue transport, we used a modified ACSF containing (in mM): NaCl 70, KCl 2.5, NaH2PO4 1.25, MgSO4 7, CaCl2 0.5, NaHCO3 26, sucrose 75, and glucose 25 (pH 7.4 after equilibration with 95% O2/5% CO2). The substances used for the ACSF were of analytical grade (Merck, Darmstadt, Germany). D())-2-amino5-phosphonovaleric acid (D-APV, Tocris Neuramin, Bristol, United Kingdom) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris Neuramin) were bathapplied at final concentrations of usually 20 and 10 lM, to reduce synaptic responses mediated by N-methyl-Daspartate (NMDA) and a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) receptors, respectively. The GABAA receptor antagonist ())-bicuculline methiodide (Sigma-Aldrich, Taufkirchen, Germany) was applied at 2 lM. The GABAB receptor agonist baclofen was applied at 2 lM, and the antagonist CGP 55845A {[3[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino]-2-(S)-hydroxypropyl] (phenylmethyl) phosphinic acid hydrochloride} (Froestl et al., 1992) at 2 lM. In a few experiments CGP 55845A was applied at 4 lM causing no noticeably different effects. Therefore, the data were pooled and are uniformly referred to as 2 lM. All drugs were dissolved in H2O, stored in aliquots at )20C, and added to the ACSF to give the desired concentration. Electrophysiologic recordings Individual slices were transferred to a submerged-type chamber (Deisz, 1999a) for intracellular recordings in layer II/III neurons using sharp microelectrodes. The chamber (volume approximately 500 ll) was continuously perfused at about 5 ml/min with ACSF, equilibrated with 95% O2/5% CO2. The temperature of the bath was held constant at 32C with a control unit (SCTC 20E, npi, Tamm, Germany). Electrodes were pulled from filamented capillaries (1.2 mm outer diameter and 0.8 mm inner diameter; Hilgenberg, Malsfeld, Germany) with a Brown Flaming puller (P 87, Sutter Instruments, Novato, CA, U.S.A.) to resistances of 80–120 MX when filled with 1 M K+-acetate and 1 mM KCl, titrated to pH 7.2. Electrodes were connected to a current–voltage clamp amplifier (SEC 10L, npi electronic, Tamm, Germany). Extracellular stimulation was applied in layer I, 0.5; Rm 38.8 € 21.4 MX; p > 0.5; n = 52). With sufficient depolarization most neurons fired APs at fairly regular intervals and low frequency; the AP amplitudes averaged 92.8 € 8.6 mV in human (n = 302) and 92.7 € 7.1 mV in rat neurons (n = 52; p > 0.5). We, therefore, assume that most neurons were regularly firing pyramidal cells, rather than interneurons or burst-firing neurons (McCormick et al., 1985). A marked difference to rat cortical neurons was the fairly linear current–voltage relationship in the hyperpolarizing direction and higher Rm of most human neurons in epileptogenic tissues (see Table 1). Synaptic components in human cortical neurons Synaptic responses in neurons from human epileptogenic tissues differed markedly from that of rodent neurons. Increasing the stimulus intensity (2–20 V, 2 V increments) often elicited abruptly increasing depolarizations of about 20 mV, rather than the more gradual increase observed in rodent cortex. Altering the prevailing membrane potential by current injection revealed a strong voltage dependence of synaptic response (Fig. 1), that is, hyperpolarizations greatly increased the amplitudes such that the peak amplitudes almost attained the same membrane potential. The mechanisms underlying these large early synaptic responses will be presented elsewhere

1701 Impairment of GABAB Receptors in Human TLE Table 1. The table gives the number of resections (r) and neurons (n) investigated in each group with K-acetate electrodes Group

r

n

1. TLE with AHS 2a. TLE wo AHS 2b. TLE wo AHS 3. Tumor 4. Dysplasia 5. Lesions 6. Frontal lobe 7. Control

71 15 1 19 10 8 11 5

167 27 7 40 19 11 23 8

Em (mV) )71.2 )71.5 )73.1 )70.7 )71.0 )73.6 )72.6 )71.7

± ± ± ± ± ± ± ±

4.8 5.1 2.3 4.8 4.8 5.2 3.3 3.6

Rm (MX) 45.2 41.0 24.8 44.2 49.4 48.9 33.6 29.6

± ± ± ± ± ± ± ±

25.2+ 24.3 5.3 21.9+ 25.0+* 25.8+ 26.2 10.3

g IPSP-B (nS) 5.9 6.5 17.3 4.4 5.7 5.4 6.6 15.9

± ± ± ± ± ± ± ±

6.4***+++ 6.2**+ 17.4 5.3***+++ 6.0**+ 7.2*+ 8.3*+ 10.8

The mean values (±SD) of resting membrane potentials (Em), neuronal input resistance (Rm) and IPSPB conductance (gIPSP-B) are given. The levels of significance are indicated as one symbol for p < 0.05, two symbols for p < 0.01, and three symbols for p < 0.001. The asterisks denote the p-values versus control and the + denotes p-values versus group 2b. Membrane potentials were not different between the groups (each p > 0.05); therefore, the significance levels have been omitted. Please note, 7 n from 1 r from the temporal lobe epilepsy (TLE) without Ammon’s horn sclerosis (AHS) group exhibited properties close to control and, therefore, are presented as a separate group (2b)

(Deisz RA, Lehmann T-N, Dehnicke C, Nitsch R, unpubl. data). In brief, the apparent reversal near 20-ms poststimulus (time to maximal IPSPA in rodent cortex) averages about )60 mV in human epileptogenic cortex (Deisz et al., 1998), rather than near )70 mV as in adult rodent cortex (e.g., Luhmann & Prince, 1991). We refer to these responses as depolarization shifts (DSs), since the peak of the depolarization was usually devoid of the high-frequency barrage of APs characteristic of the paroxysmal depolarization shift (PDS) in the penicillin-treated rodent cortex (Gutnick et al., 1982). These depolarizations were followed by relatively small and late hyperpolarizations at resting Em, the focus of the present study. To ascertain the involvement of GABAB receptors in this late hyperpolarization (time to peak near 170 ms), the GABAB receptor antagonist CGP 55845A (2 lM) was applied. Em, AP amplitude, and Rm [Correction added after online publication 20 May 2009: Rm changed to Rm] were not significantly affected by CGP 55845A (p > 0.1). The slight hyperpolarization was virtually eliminated by CGP 55845A in all neurons tested (Fig. 1A) and hence represents a GABAB receptor–mediated IPSP (IPSPB). The elimination of the IPSPB by CGP 55845A is particularly obvious from the change in firing after the stimulus. The neurons that exhibited in control ACSF a pause in firing (about 250 ms) after the orthodromic stimulation, generated in the presence of CGP 55845A unimpeded APs (Fig. 1A). At resting Em, the IPSPB amplitude of human epileptogenic neurons decreased on average from )1.5 € 1.2 mV to +1.3 € 1.6 mV (n = 14 n, 10 r; p < 0.0001), that is, the polarity reversed. To account for the variability of Em, the amplitudes of the IPSPB were also compared at a given Em. At )70 mV, the amplitudes decreased on average from )2.3 € 1.9 mV to +1.1 € 2.0 mV (n = 14 n, 10 r; p < 0.001). In the presence of CGP 55845A, the apparent reversal potential at 170 ms exhibited a depolarizing shift (see Fig. 1B)

from )80.8 € 5.6 to )63.4 € 12.5 mV (n = 14 n, 10 r; p < 0.001). This shift in EIPSP-B is probably due to the fact that after reduction of the IPSPB conductance, the apparent reversal potential is governed by the tail of the GABAA response (see Deisz et al., 1997). In any case, even with depolarizations to near-firing threshold ()60 mV), no obvious IPSPB remained (0.2 € 1.4 mV vs. )4.4 € 2.4 mV in control; p < 0.0001). To test a possible curtailing of the synaptic responses by GABAB receptors (Karlsson et al., 1992; Kantrowitz et al., 2005), we also evaluated the effects of CGP 55845A (2 lM) on the amplitude and time-course of synaptic potentials at various stimulus intensities (2–20 V in 2 V increment). At most, stimulus intensities (8–20 V) CGP 55845A had no consistent effect on the amplitude of the synaptic responses (Fig. 2A1; n = 10 n, 6 r). At the stimulus intensity of 6 V, however, CGP 55845A reduced synaptic responses in some neurons (seven of 10 n, 6 r) on average by 52% (p = 0.005, paired t test, Fig. 2A2). This depression of synaptic responses might be due to an elevation of extracellular GABA following elimination of GABA feedback on its own release. The time-course of the synaptic responses was unaffected; single exponential fits to the decay phase of synaptic responses (between 20 and 80 ms poststimulus) elicited at intermediate stimulus intensities revealed time constants of decay between 40 and 150 ms in control conditions. Addition of CGP 55845A caused a slight decrease of the decay time constant from 45.4 € 15.6 ms to 35.6 € 11.1 ms, which did not reach significance (n = 10 n, 6 r; p = 0.06; see Fig. 2A). Properties of the IPSPB The amplitude of the IPSPB was rather small in epileptogenic tissue, averaging )0.56 € 1.8 mV (at Em; n = 163 n) in neurons from TLE patients with AHS, considerably smaller than in human controls ()2.0 € 1.5 mV, Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

1702 L. A. Teichgra¨ber et al.

Figure 1. Blockade of the late hyperpolarization by CGP 55845A. (A) Traces Em during orthodromic stimulation (indicated by the arrows, intensity 10 V) at rest and during current injection (as indicated in the figure) in control condition (A1) and during the application of 4 lM CGP 55845A (A2). Both plates are recordings of the same neuron [temporal lobe epilepsy (TLE) without Ammon’s horn sclerosis (AHS) specimen]. During the application of CGP 55845A the slight hyperpolarization at resting Em (indicated by dashed line) inverts to a depolarization at the time that the IPSPB has its peak amplitude in control conditions. (B) Plot of the average IPSPB amplitudes in control condition and in the presence of CGP 55845A versus the membrane potential before stimulation. The graph shows the amplitudes near 170 ms, corresponding to the peak of the IPSPB in control conditions from 14 neurons (10 resections). Membrane potentials within 1 mV were selected from families of potential changes induced by current injections (0.05 nA increments). The vertical bars denote the standard error of the mean (SEM) in this and all subsequent figures. Note the unmasking of a depolarizing response in the presence of CGP 55845A with a reversal potential near )55 mV. Epilepsia ILAE Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

1703 Impairment of GABAB Receptors in Human TLE

Figure 2. Effects of CGP 55845A on synaptic responses. (A) Traces of synaptic potentials elicited at high (A1, 14 V) and low (A2, 6 V) stimulus intensities. The traces obtained in control condition and in the presence of CGP 55845A (indicated by arrows) have been superimposed to facilitate comparison. The average resting membrane potential during these series was )73.4 mV in control and )73.2 mV in the presence of CGP 55845A. Note the elimination of the slight hyperpolarization elicited at the higher stimulus intensity by CGP 55845A. Neuron from temporal lobe epilepsy (TLE) with Ammon’s horn sclerosis (AHS) specimen. (B) The mean values of the amplitudes of synaptic responses in control and CGP 55845A (n = 10 n, 6 r each) versus the stimulus intensity have been plotted. The vertical bars denote the standard error of the mean (SEM). Epilepsia ILAE

n = 8 n, 5 r, p = 0.024) or rat neurons ()2.6 € 2.3 mV, n = 43). To account for differences in membrane potential, the amplitudes were also determined at )60 mV (€1 mV, obtained by current injection). In TLE with AHS tissues an average amplitude of )2.4 € 3.2 mV (n = 151 n, 64 r) was obtained, significantly smaller than in human controls ()5.6 € 3.9 mV, n = 8, 5 r, p = 0.008) or rat neurons ()7.8 € 3.7 mV, n = 41). A detailed account of the different pathologic groups will be given below. To evaluate the mechanisms underlying the difference in amplitudes, we estimated reversal potentials and synaptic conductance (see Materials and Methods), as illustrated in Fig. 3. In Fig. 3B1, the traces from a neuron (TLE with AHS tissue) show only marginal IPSPB amplitudes at all potentials; the corresponding plot estimating the synaptic conductance (Fig. 3B2) yields 2.9 nS in this case. Traces from a neuron with a high IPSPB conductance are shown in Fig. 3A1, and the linear regression shown in Fig. 3A2 yields a conductance of 32 nS (tumor tissue without epilepsy, one seizure). The IPSPB conductance in neurons from human epileptogenic cortices averaged 5.8 € 6.4 nS (n = 282 n, 134 r), considerably lower than in cortical neurons from human controls (15.9 € 10.8 nS, n = 8 n, 5 r; p < 0.0001), Wistar rat: (20.0 € 16.9 nS, n = 49), Sprague-Dawley rat (18 nS, Luhmann & Prince, 1991), cat (19 nS, Connors et al., 1988), or guinea pig (22 nS; Deisz & Prince, 1989). The apparent reversal potential of the IPSPB was similar in epileptogenic ()76.6 € 8.2 mV; n = 248 n) and control tissue ()82.7 € 4.5 mV; n = 6 n; p = 0.053). It should be emphasized that part of this difference may be due to the smaller IPSPB conductance. At the reduced IPSPB conductance, the other concurrent conductances (resting conductance and/or tail of GABAA conductance) would have more impact on the apparent reversal potential. Despite this marked difference in conductance, the time-course of the IPSPB was comparable between the two human groups. Near half maximal stimulus intensity, the decay time constant averaged 131.6 € 38.9 ms (n = 20, 17 r) in human epileptogenic and 128.8 € 20.2 ms (n = 8 n, 5 r) in human control tissue (p = 0.8). The IPSPB time constants obtained in rat cortical neurons (n = 14) were indistinguishable from the values determined in human controls (p > 0.05). This similarity of the time courses in human epileptogenic, human control, and rat cortical neurons indicates that gross changes in kinetics are unlikely to contribute to the smaller IPSPB conductance in epileptogenic tissues. Baclofen effects on human cortical neurons The small apparent IPSPB conductance in human epileptogenic neurons could be due to either a reduced GABA release or a reduced efficacy or density of

Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x


Figure 3. Estimate of IPSPB conductance in human neurons from control and epileptogenic neocortex. (A1) Traces of membrane potential changes (resting Em )77.1 mV) during current injections (from top to bottom +0.3 to )0.5 nA) and orthodromic stimulation (16 V). Note the pronounced IPSPB near 170-ms poststimulus. This neuron was recorded in a control tissue (tumor specimen with one seizure 5 weeks before operation). (A2) Plot of the membrane potentials attained before stimulation (open squares) and at 170 ms poststimulus (filled circles) during current injections from the neuron shown in A1. The slopes correspond to a conductance of 40.5 nS before and 72.2 nS at 170 ms poststimulus; the difference yields an apparent IPSPB conductance of 31.7 nS. (B1) Superimposed traces of membrane potential changes from a neuron [temporal lobe epilepsy (TLE) with Ammon’s horn sclerosis (AHS) specimen, resting Em )72.3 mV] obtained by injecting currents of incremental amplitudes (from top to bottom +0.3 nA to )0.5 nA). During each current injection an orthodromic stimulus (20 V) was applied. (B2) Plot of the membrane potentials attained before (open squares) and 170 ms after the orthodromic stimulus (filled circles) during current injections. The slopes correspond to a conductance of 19.3 nS before and 22.2 nS at 170 ms poststimulus; the difference yields an IPSPB conductance of 2.9 nS. Epilepsia ILAE

postsynaptic receptors. To distinguish between these alternatives, we applied the selective GABAB receptor agonist baclofen (2 lM, about half-maximal concentration; Howe et al., 1987). Baclofen caused small changes in Em, between +0.7 and )5.6 mV, and on average Em increased from )69.8 € 4.2 mV to )71.2 € 4.7 V (p < 0.01, n = 19 n, 12 r) in epileptogenic tissues. Neuronal input resistance decreased, on average, from 40.7 € 18.1 MX to 35.3 € 16.3 MX (p < 0.0001, n = 19 n, 12 r). Estimating Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

the baclofen-induced conductance yielded 13.6 nS (Fig. 4C) for the neuron from the control group shown in Fig. 3A. For comparison, the neuron illustrated in Fig. 3B from epileptogenic tissue yielded a baclofen-induced conductance of 3.3 nS (data not shown). Such small baclofeninduced conductances were obtained in most neurons from epileptogenic tissue, on average 4.1 € 3.1 nS (n = 19 n, 12 r), about half of the IPSP conductance (6.8 € 6.1 nS, n = 19, 12 r), when one neuron from a


Figure 4. Comparison of IPSPB conductance and baclofeninduced conductance. (A) Plot of the membrane potentials (from the neuron illustrated in Fig. 3A) attained during various current injections before (open squares) and during the application of baclofen (filled diamonds). The slopes correspond to a conductance of 40.5 nS before and 54.1 nS during the application of baclofen. The difference yields a baclofen-induced conductance of 13.6 nS. (B) Plot of the IPSPB conductances versus baclofen-induced conductances from different neurons. The slope of the regression line is 1.9, indicating a 2-fold higher IPSPB conductance compared to baclofen-induced conductance (R = 0.94). The two data points at the top right are from the neuron illustrated in Fig. 3A (indicated by con). The other point (indicated by sc) is from a special case of temporal lobe epilepsy (TLE) without Ammon’s horn sclerosis (AHS) tissue, exhibiting control-like IPSPB conductance (see text for details). Epilepsia ILAE

peculiar case is omitted (see below). Two neurons from control tissues had baclofen-induced conductances of 3 and 13 nS. Interestingly, the neuron with the largest baclofen-induced conductance (21.2 nS; IPSPB conductance 36 nS) was from a tissue (TLE without AHS patient) in which seven neurons had an average IPSPB conductance similar to control values (17.3 nS, see below). If this neuron is considered a control, the average baclofen-induced conductance (12.4 € 9.2 nS, n = 3) becomes significantly different from the epileptogenic tissue group (p = 0.004) and is about half of the IPSPB conductance of these neurons (24.3 € 18.1 nS). The arguments for regarding this tissue as ‘‘control-like’’ will be given below. These data indicate that responses to a constant exogenous baclofen concentration in human epileptogenic tissue are on average smaller than those of nonepileptogenic human tissue. To test whether a reduced baclofen sensitivity accounts for the small IPSPB conductance in a given human neuron, the two parameters were correlated. If the variation in IPSPB conductance was due to differences in transmitter release, constant exogenous baclofen should induce constant conductances. If, however, the variation in IPSPB conductance were due to variation in receptor function or density, the two parameters should correlate. As shown in Fig. 4B, neurons exhibiting relatively high IPSPB conductances also had large baclofen-induced conductances and vice versa (R = 0.9; p < 0.0001). The linear relationship between the two parameters and the slope of the regression (1.9) indicate that the IPSPB conductance is twice the conductance induced by a half-maximal baclofen concentration. Secondly, because IPSPB conductances correlate with the baclofen-induced conductances, a reduced density or function of GABAB receptors in human neurons can be inferred. The paucity of human control neurons led us to compare IPSPB- and baclofen-induced conductances also in the rat cortex. To this end we applied 2 lM baclofen to rat cortical neurons under identical experimental conditions. These neurons had an average IPSPB conductance of 25.2 € 18.6 nS, and the baclofen-induced conductance averaged 9.6 € 4.7 nS (n = 11). Both IPSPB- and baclofen-induced conductances of rat neurons were significantly larger than in the human epileptogenic tissue (p = 0.007 and p = 0.0005, respectively).

Presynaptic GABAB effects at excitatory terminals The small postsynaptic GABAB receptor–mediated effects in the human epileptogenic tissue raised the possibility that presynaptic GABAB receptor mechanisms may also be attenuated. We, therefore, investigated whether GABAB receptors at excitatory (Howe et al., 1987; Connors et al., 1988) and/or inhibitory terminals (Howe et al., 1987; Deisz & Prince, 1989; Deisz, 1999a) are impaired in human epileptogenic tissue. Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x


Figure 5. Effects of baclofen on synaptic responses. (A) K+acetate–filled electrode recordings. Superimposed traces of Em changes following orthodromic stimuli (A1 low, A2 high intensity as indicated) in control conditions and during the application of 2 lM baclofen. The membrane potential before stimulation is given below the traces. (B) Cs+-acetate–filled electrode recordings similar to A to antagonize the postsynaptic GABAB receptor–mediated increase in K+ conductance. Both neurons were recorded in slices from temporal lobe epilepsy with Ammon’s horn sclerosis specimen. (C) Plot of the data illustrated in A and B. The baclofen-induced depression of synaptic responses has been expressed as percent of control. The depression averaged 68% with K+-containing electrodes (stimulus intensity 4–8 V, average 6.2 V. p = 0.0001, n = 17 n, 12 r) and 74% with Cs+-containing electrodes (p = 0.0001, n = 8). At high-stimulus intensities the amplitudes were also significantly reduced, but to a lesser extent (18 V: 22%, p = 0.002, n = 17 n, 12 r with K+- and 22.4%, p = 0.04, n = 8 with Cs+containing electrodes). Epilepsia ILAE Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

To this end, we evaluated the effects of 2lM baclofen (the half maximal concentration for decreasing glutamate release, Pende et al., 1993) on synaptic responses of human neurons. Synaptic responses were elicited with various stimulus intensities (2–20 V, 2 V increments, three times each) before and during the application of 2lM baclofen (Fig. 5A). Baclofen reduced the amplitudes of postsynaptic potentials (PSPs) at all stimulus intensities, particularly at low-stimulus intensities (Fig. 5A1). The smallest discernible PSPs elicited with stimulus intensities between 4 and 8 V (mean stimulus intensity 6.2 V; average amplitude 5.1 mV) were reduced by 68% (p = 0.0001; n = 17 n; 12 r). At higher stimulus intensities (>10 V), the depression was much smaller (Fig. 5A2), averaging 22% at, for example, 18 V (p = 0.002; n = 17). Interestingly, only three neurons exhibited a marked (>50%, mean 66%) depression at higher stimulus intensities, as anticipated from the concentration employed and published dose–response curves (EC50 of 1 lM; Howe et al., 1987). The average depression in all neurons (22%) near the half-maximal concentration was smaller than published values in the rat neocortex (50% depression of amplitude at 1 lM) and hence might indicate that presynaptic GABAB receptors at glutamatergic terminals were impaired. The comparison with published data may be questionable, due to different methods. Nevertheless, the marked depression of synaptic responses in a minority of neurons suggests that the baclofen-induced depression may be more pronounced in the healthy human cortex than observed in most epileptogenic tissues. We considered the possibility that the postsynaptic baclofen-induced conductance contributes to a depression of synaptic responses. To ascertain whether the reduction of PSPs was mediated by presynaptic effects at glutamatergic terminals, we antagonized the postsynaptic GABAB receptor–mediated increase in K+ conductance by using Cs+-containing electrodes (Desarmenien et al., 1984). The most obvious difference to K+-containing electrodes was a complete lack of IPSPB. Application of 2lM baclofen caused no detectable changes in membrane potential or membrane resistance (p > 0.05; n = 9 n), indicating an effective blockade of postsynaptic GABAB receptor–mediated conductance. However, baclofen still induced a significant depression of PSPs at low- and high-stimulus intensities (Fig. 5B), averaging 74% and 22.4% (p = 0.0001 and 0.04; n = 8 n), respectively. The baclofen-induced depression of PSPs recorded with Cs+-filled electrodes was not different from that with K+- containing electrodes (p > 0.1 for both stimulus intensities). This similarity of data (Fig. 5C) indicates that elimination of postsynaptic GABAB conductance with intracellular Cs+ has no effect on the magnitude of PSP depression, that is, the baclofen effects were mediated presynaptically.

1707 Impairment of GABAB Receptors in Human TLE Temporal consequences of impaired GABAB receptors The impairment of presynaptic GABAB receptors might alter the magnitude of subsequent synaptic responses, considering the long-lasting depressant effects of presynaptic GABAB-receptor activation (Deisz & Prince, 1989). This possibility was tested in the human epileptogenic cortex with a paired-pulse paradigm. Although synaptic responses were relatively large and often displayed ‘‘all or none’’ properties with slight increases in the stimulus intensity, the paired stimulation elicited virtually constant synaptic responses (Fig. 6) in most (68 of 70) neurons. To test whether stimulus intensity affects paired-pulse behavior, paired stimulation was carried out at two or three intensities in 25 neurons (19 r). At low-stimulus intensities, when evoked GABA release is unlikely, the constancy of responses (Fig. 6A1) was anticipated from previous evidence on rodent cortex (Deisz, 1999a). At higher stimulus intensities (14–20 V), when GABA is released, as evidenced by the small CGP 55845A sensitive component, the DS amplitudes are still constant (Fig. 6A2). The averages of these measurements, plotted in Fig. 6B for low (B1) and high (B2) stimulus intensities, show a lack of time dependence. The constancy of DS amplitudes in the human epileptogenic cortex is in sharp contrast to the refractoriness of PDSs at stimulus intervals below approximately 1 s in the rodent penicillin model of epilepsy (Gutnick et al., 1982). This refractoriness might be due to the activation of GABAB receptors at excitatory terminals (Howe et al., 1987; Connors et al., 1988), decreasing release during subsequent stimuli. Alternatively, the failure of N-methylD-aspartate (NMDA) receptor–mediated components to follow higher frequencies (Sutor & Hablitz, 1989) might limit the frequency of PDSs. We first tested the frequency dependence of bicuculline-induced PDSs in rat neocortical neurons. Bicuculline application (2 lM) invariably induced typical PDSs. Paired stimulation of such PDSs revealed a consistent depression at ISI between 200 and 500 ms (Fig. 7A); in some neurons the depression lasted up to 1 s. The second PDS was maximally reduced at 200 ms (on average by 74.0 € 17.3%), and, at 500 ms ISI, the depression still averaged 43% (n = 17), consistent with previous data using penicillin (Gutnick et al., 1982). As shown in Fig. 7B, application of CGP 55845A virtually eliminated the transient failure of PDSs, resulting in essentially constant PDSs at all ISIs. The changes in amplitude were significant (p < 0.0001 at 100 ms and p = 0.0033 at 400 ms). The average values of this series are shown in Fig. 10. The comparison of the DSs of human epileptogenic cortex with the bicuculline-induced PDSs may not be valid. To directly compare the temporal properties of bicuculline-induced PDSs we also applied bicuculline (2 lM) to slices from human epileptogenic tissues. Bicuculline increased the amplitude of the synaptic responses,

on average from 13.4 € 5.6 mV to 23.6 € 7.3 mV (p = 0.0002; n = 25) at unaltered Em (control, )71.8 € 4.9 mV; bicuculline, )72.1 € 4.6 mV; p = 0.62). Interestingly, in the presence of bicuculline the IPSPB was more pronounced and elicited at lower stimulus intensities (compare Fig. 8A1, B1). These changes were quantified in 14 neurons yielding an IPSPB conductance of 10.7 € 9.1 nS before and 23.1 € 16.5 nS (p = 0.0012) during the application of bicuculline, despite unaltered or even reduced stimulus intensity (average: 12 V and 8 V, respectively). Paired stimuli revealed that a second PDS was typically only slightly reduced (Fig. 9A), although some neurons exhibited a marked depression (Fig. 9B). On average, bicuculline-induced PDSs in human epileptogenic tissue were depressed by 27.5 € 33% (200 ms; n = 17 n, 9 r), much less than in the rat neocortex (74%; p < 0.0001; see Fig. 10). A postsynaptic mechanism for this transient failure appears unlikely, since some neurons with a large postsynaptic IPSPB conductance exhibited little depression of the second PDSs and, conversely, some neurons with a marginal IPSPB exhibited a pronounced depression. If the postsynaptic IPSPB conductance contributes to the depression of PDSs the magnitude and/or time course of the two parameters should vary correspondingly. However, neither the decay time constants nor magnitudes of IPSPB correlated with the transient suppression of bicuculline-induced PDSs (n = 19; R2: 0.048 and 0.027, respectively; data not shown), indicating that the postsynaptic effects do not determine the paired-pulse behavior, that is, the latter are independent and presumably governed by presynaptic mechanisms. Addition of CGP 55845A not only eliminated the paired-pulse depression (Fig. 9B,C), but also caused a significant prolongation of the decay phase (Fig. 8B) of bicuculline-induced PDSs (tau, 42.5 ms in bicuculline and 65.8 ms in bicuculline + CGP 55845A; n = 6 n; p = 0.038). These data indicate that the commonly observed DSs of human epileptogenic cortex without bicuculline differ fundamentally from bicucullineinduced PDSs (see Discussion). Nevertheless, the smaller magnitude of the transient suppression of bicucullineinduced PDSs in epileptogenic human neocortex versus rat neocortex (see Fig. 10), together with the sensitivity to CGP 55845A, indicates that the presynaptic GABAB receptors are impaired.

Presynaptic GABAB effects at inhibitory terminals in human epileptogenic tissues To evaluate the properties of presynaptic GABAB mechanisms at inhibitory terminals, the paired-pulse paradigm was again applied. Due to the DSs in many of the neurons, hyperpolarizing IPSPs could not be clearly resolved below firing threshold. Therefore, paired-pulse depression (PPD) was determined in the presence of the Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x


Figure 6. Time-dependent properties of synaptic responses. (A) Superimposed traces of synaptic responses evoked by paired-pulse stimulations with increasing interstimulus intervals (ISIs). The panel illustrates traces elicited at low (A1, 6 V) and at high (A2, >14 V) stimulus intensity. Part of the stimulus paradigm is schematically indicated at the bottom of panel A2. Both recordings were from the same neuron [temporal lobe epilepsy (TLE) with Ammon’s horn sclerosis (AHS) specimen]. (B) Plot of subsequent synaptic responses (expressed as percentage of the first amplitude) versus the ISI. B1 shows the mean percentage values of measurements at low intensity stimulation (3– 8 V, mean 5.2 ± 1.3 V, n = 29 n, 24 r). B2 shows the mean values at high stimulus intensity (12–20 V, mean 15.4 ± 2.6 V, n = 29 n, 22 r). The first synaptic responses averaged 9.2 ± 4.7 mV at low and 19.7 ± 4.3 mV at high stimulus intensity. The vertical bars denote standard error of the mean (SEM), if exceeding the symbol size. The legend to panel B1 applies also to panel B2. Please note the gap in the ordinate. Epilepsia ILAE

AMPA and NMDA receptor antagonists CNQX (10 lM) and D-APV (20 lM), respectively. Under these conditions, paired stimuli at incremental intervals revealed a small and brief PPD (see Fig. 11). On average, the second IPSPA was reduced by 10.7 € 11% and 7.0 € 7.7% at 500 and 1,000 ms, respectively (n = 19 n, 16 r). The time constant of the PPD decay ranged from 22 ms to approximately Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

1,700 ms. The mean value of the decay time constants from all neurons was 337 € 453 ms and 190 € 123 ms after exclusion of two neurons exceeding a 1 s time constant. By comparison, under the same experimental conditions, PPD in rat neurons averaged 44.6 € 21% and 36.7 € 24% at 500 and 1,000 ms, respectively (n = 18, p < 0.001). The time constant of PPD averaged


Figure 7. Paired-pulse behavior of bicuculline-induced hyperexcitability. (A) Traces from a rat cortical neuron in the presence of 2 lM bicuculline, illustrating the transient depression of paroxysmal depolarization shifts (PDSs). (B) Traces from the same neuron as shown in panel A in the presence of bicuculline and 2 lM CGP 55845A. Note the constant amplitude of PDSs in the presence of bicuculline and CGP 55845A. Epilepsia ILAE

282 € 96 ms in rat neurons. In the presence of CGP 55845A, IPSPA amplitudes were constant at all ISIs, except for a brief, apparent depression partly due to the superposition of temporally close IPSPs (compare Fig.11A and B). The weaker PPD suggests an impairment of presynaptic GABAB receptors at GABAergic terminals. Immunocytochemical localization of GABAB receptors The data described so far indicate a reduced function or density of GABAB receptors in human epileptogenic tissues. To test our hypothesis, immunocytochemistry was carried out on three sections from different epilepsy surgery tissues and two sections from a human control tissue (gyrus orbitalis of the frontal lobe). The immunosignal for GABAB receptors in human control tissue was quite pronounced in many neurons (Fig. 12B). At high magnification (100·) a dense clustering of immunopositive dots was detected at the soma and proximal dendrites (Fig. 12C), representing accessible GABAB receptors. In sections from human epileptogenic tissue,

Figure 8. Effects of 2 lM bicuculline on synaptic responses of human cortical neurons. (A) Traces of synaptic responses elicited with low (A1, 4 V) and high stimulus intensity (A2, 16 V). (B) Traces from the same neuron in the presence of bicuculline. (B1) Note, bicuculline converts the PSP into a typical PDS and produces a late hyperpolarization compared to panel A1. (B2) Application of bicuculline and 2 lM CGP 55845A [Correction made after online publication 20 May 2009: m changed to M] has no obvious effect on the amplitude and initial decay of the depolarization shift (DS), in this particular neuron. Note the elimination of the IPSPB in the presence of CGP 55845A compared to panel B1. Epilepsia ILAE

immunostaining was very faint (Fig. 12E) and high magnification revealed only a low number of immunopositive dots at the soma and proximal dendrites (Fig. 12F). Some of these appeared to be adjacent to the just discernible neuronal surface, as if they represent dots of presynaptic terminals. As a first approach we counted the dots from 30 neurons in each of the three sections from epileptogenic tissues. Semiquantitative analysis revealed that some of the human epileptogenic neurons exhibited relatively large number of dots; however, the majority exhibited rather low numbers of dots, that is, 74 of 90 neurons had less than 30 dots (see Fig. 12H). On average, neurons from human epileptogenic tissues had 19.6 € 10.7 dots. The counting of immunodots in the human control tissue was much more difficult due to the high density of dots in some neurons. In fact 88 of 90 neurons had more than 30 immunodots on average (73.8 € 24.9, p < 0.0001). Interestingly, the distributions are quite different: human control neurons exhibit an even distribution of counts of immunodots and human epileptogenic neurons exhibit a skewed distribution toward lower values (cf. Fig. 12G, H). We tentatively assume a decrease of GABAB receptors in a large fraction of neurons, rather than a preferential loss of those neurons exhibiting a high density of GABAB Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x


Figure 10. Plot of the mean amplitudes of the slow component of synaptic responses (expressed as percentage of the first amplitude) versus the interstimulus interval from rat cortical neurons (n = 15; open circles) and from human neurons from epileptogenic tissues (n = 31; filled squares) in the presence of bicuculline. The original traces for the averages of human neurons are illustrated in Fig. 9. The vertical bars denote standard error of the mean (SEM). Epilepsia ILAE

Figure 9. Temporal properties of bicuculline-induced paroxysmal depolarization shifts (PDSs) of human cortical neurons. (A) Superimposed traces of bicuculline-induced PDSs during paired stimulation with increasing interstimulus intervals, representing the commonly observed small depression of subsequent PDSs. (B) Traces from another neuron under the same experimental conditions as in A. Note the marked depression of subsequent depolarizations, comparable to rat neurons. (C) Traces from the same neuron as shown in B in the presence of bicuculline (2 lM) and CGP 55845A (2 lM). Note the constancy of responses during the presence of CGP 55845A, comparable to the behavior of responses from other neurons without application of drugs (cf. Fig. 6). All neurons were from temporal lobe epilepsy with Ammon’s horn sclerosis tissues. Epilepsia ILAE

receptors. In any case, the predominance of neurons exhibiting small numbers of GABAB receptor–immunopositive dots in human epileptogenic cortex is consistent with the electrophysiologic findings. Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

Correlation of the IPSPB with clinical categories The data presented so far indicate that neurons from human epileptogenic cortical tissue exhibit on average small IPSPB conductances. To test whether this fact is common to all clinically defined groups (see Materials and Methods), we compared the IPSPB conductances of these groups. All groups of epileptogenic cortex exhibited, on average, much lower IPSPB conductances (between 4.4 and 5.7 nS, see Table 1) than the control group (15.9 nS), with p-values between 0.02 and 0.0001 (ANOVA post hoc). Data from one tissue of the TLE without AHS group were omitted from this analysis. In this tissue we recorded seven neurons, three of which exhibited high IPSPB conductances. The average IPSPB conductance from this tissue was much larger (average 17.3 nS), significantly different from the rest of the group (p = 0.008) and indistinguishable from the control (p = 0.85). The part of this tissue we investigated may have been normal with respect to GABAB function. In this case, the focus might have been in the large volume of the tissue (typically 90%) used for the routine pathological examination, or unfortunately even outside the resected tissue, since this patient did not become seizure-free. A total of 14 neurons (5%) from pathological tissues had IPSPB conductances >19 nS, that is, values twice the SD outside the mean. These neurons were from group 1 (7 n, 6 r), group 2 (4 n, 2 r, three from the patient mentioned above), group 3 (1 n), group 5 (1 n), and group 6 (1 n). These neurons with high IPSPB

1711 Impairment of GABAB Receptors in Human TLE conductances appear to contradict our hypothesis of low IPSPB conductance in epileptogenic tissue. However, the relative occurrence of these values is rather small in all groups of epileptogenic cortex: group 1, 7 of 158 neurons (4.4%); group 2, 4 of 29 (13%, or 5% if the neurons from the above patient are disregarded); group 3, 1 of 39 (2.6%); and group 5, 1 of 11 neurons (9%). Therefore, neurons with a high IPSPB conductance are usually below 10% in all pathological groups. In the control group, however, four of eight neurons exhibited such high values, and the average is close to established values from the neocortex of other mammals.

Discussion

Figure 11. Paired-pulse behavior of pharmacologically isolated inhibition. (A1) Traces of synaptic responses elicited with pairs of stimuli have been superimposed. 10 lM CNQX and 20 lM D-APV have been applied to pharmacologically isolate inhibition. The Em was depolarized by a current injection of 0.2 nA. The interstimulus interval (ISI) was increased from 50 ms to 1,500 ms in this series, but only every fourth trace has been plotted. The second IPSPA is only slightly smaller than the control response. (A2) Traces from the same neuron during the application of CGP 55845A [temporal lobe epilepsy with Ammon’s horn sclerosis specimen]. (B) Plot of the paired-pulse behavior of pharmacologically isolated IPSPs (n = 19, 16 r). The amplitudes of the second IPSPs have been expressed as the percentage of the first response and plotted versus the ISI (filled squares). For comparison, the data from rat cortical neurons have been added (open symbols). Note the smaller paired-pulse depression in human epileptogenic tissue, indicating impaired function of presynaptic GABAB receptors at GABAergic terminals. Epilepsia ILAE

Our data from human cortical neurons demonstrate remarkably small GABAB receptor–mediated effects in the majority of neurons, indicating a deficit of postsynaptic receptors as well as reduced effects at presynaptic GABAB receptors of glutamatergic and GABAergic terminals. Before discussing the implications of reduced GABAB effects for hyperexcitability, possible sources of error should be considered. To name a few, the human tissue may have (1) suffered considerable hyperexcitability for variable periods, (2) been exposed to various anticonvulsants, and (3) suffered individual traumas and/or ischemias during tissue transfer and slicing. In addition, most of the tissues were from standard resections with some ambiguity concerning the presence of a seizure onset zone. Typically only tissues from frontal lobe (group 6) had been preoperatively characterized with implanted grid electrodes to detect the zone of seizure onset. Despite the limitations, our data indicate that neurons from human control tissue have a significantly larger IPSPB conductance than neurons from epileptogenic tissues, implying that our methods per se are unlikely to account for the small IPSPB conductance in the majority of neurons. Considering the diversity of pathologies, which exceed the simplifying categories used, the consistent and comparable impairment of GABAB receptor function in a vast majority of epileptogenic tissues from all pathological groups was a surprise. The similarity of data from frontal lobe (with preoperatively established hyperexcitability) and the other pathologic groups, in particular, indicates that reduced GABAB receptor function represents a common feature of human epileptogenic cortex. Reduction of postsynaptic GABAB receptor function Given the conclusion that the effects described do relate to the pathophysiology of epilepsy, neurons from various pathologies exhibit a significantly smaller IPSPB conductance (average 5.8 € 6.4 nS, n = 282, when 7 neurons from 1 patient are excluded) compared to those from control tissue (15.9 nS, n = 8, p < 0.0001). Numerous Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x


Figure 12. Immunocytochemistry of c-aminobutyric acid (GABA)B receptors. (A) Low power photomicrograph of human control tissue section (Cresyl violet staining). (B) Low power photomicrograph of an adjacent tissue section stained with GABAB antibodies. (C) High power photomicrograph of a neuron (layer II/III) with GABAB-receptor immunostaining. Note the marked staining with immunopositive dots; the diffuse staining represents dots out of focus. (D) Low power photomicrograph of a section from human epileptogenic cortex (Cresyl violet staining). (E) Low power photomicrograph of the adjacent tissue section stained with GABAB antibodies. (F) High power photomicrograph of the same tissue section, illustrating the few immunopositive dots of a presumed pyramidal neuron. (G) Histogram of the number GABAB immunopositive dots per neuron in human control cortex (gyrus orbitalis of the frontal lobe). (H) Histogram of number of GABAB immunopositive dots per neuron in human epileptogenic cortex. Epilepsia ILAE Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

1713 Impairment of GABAB Receptors in Human TLE pre- and postsynaptic mechanisms may cause a decrease of the IPSPB conductance. The linear relation between the IPSPB conductance and the conductance induced by a half-maximal baclofen concentration (see Fig. 4B), indicates that the decrease in IPSPB conductance is due to an impaired function or density of postsynaptic GABAB receptors. If the variation in IPSPB conductance was due to differences in GABA release, baclofen should induce an approximately constant conductance. Moreover, the slope of the relation (1.9) implies that physiological GABA release induces a 2-fold higher conductance; hence, insufficient GABA release is unlikely to underlie the small IPSPB conductance. A decrease of postsynaptic GABAB responsiveness could be due to several effects: (1) a decreased number of functional receptors, (2) changes in G-protein signaling, and/or (3) impaired effectors. Considering the intricacies of GABAB receptors, changes at the receptor level are more likely. Functional receptors are heterodimers of GABAB1a/b and GABAB2 subunits, and the receptor trafficking depends on the GABAB2 subunit (Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999). In addition, agonist binding takes place at the GABAB1 subunit and G-protein interaction at the GABAB2 subunit through a coil–coil interaction (Pagano et al., 2001). Despite these numerous possibilities contributing to malfunction of GABAB receptors, our immunohistochemical evidence indicates a marked reduction of immunopositive dots in neurons of human epileptogenic tissue compared to human control tissue, that is, reduced density of receptors. This reduction corresponds to our functional evidence of a reduced IPSPB conductance in the majority of neurons. From the average immunodots per neuron (control 73.8, epileptogenic tissues 19.6), a decrease of about 73%, and from the conductance values (control 15.9 nS, epileptogenic tissues 5.8 nS) a decrease by 64% is obtained. The presence of a few neurons with relatively large numbers of immunodots corresponds to those rare neurons exhibiting an almost normal IPSPB conductance in human epileptogenic tissues. Interestingly, a decreased GABAB receptor 1a/b subtype immunostaining has also been reported in the dentate gyrus of epilepsy patients (Munoz et al., 2002). Binding studies of tritiated GABA to GABAB receptors also found a marked decrease of radiolabeling in area CA1, CA2, CA3, hilus, and dentate gyrus of human epileptogenic hippocampus; however, when normalized to the number of cells, an apparent increase of GABAB receptor labeling per neuron was obtained (Billinton et al., 2001). Essentially similar conclusions were reached in further studies on human hippocampus employing in situ hybridization techniques (Furtinger et al., 2003; Princivalle et al., 2003). Perhaps the considerable postmortem delay of the control tissues of the latter studies, of up to 36 h (Furtinger et al., 2003) may cause a larger decrease

of GABAB receptors compared to epilepsy tissues. In any case, the reduced immunosignal in human hippocampus (Munoz et al., 2002) corresponds to immunostaining data from a mouse model of TLE (Straessle et al., 2003) as well as functional evidence from a rat model of TLE indicating reduced GABAB receptors (Mangan & Lothman, 1996). The consequence of impaired postsynaptic GABAB receptors in human epileptogenic tissue would be a prolongation of DSs due to an insufficient curtailing of the falling phase once the duration of DSs exceeds about 100 ms (see Fig. 2), corresponding to the prolongation of 4-aminopyridine–induced giant synaptic responses by CGP 55845A (Kantrowitz et al., 2005). At first glance, the lack of effect of CGP 55845A on the time course of individual DSs appears to be at odds with the prolongation of bicuculline-induced hyperexcitability in the rat neocortex by GABAB antagonists (Karlsson et al., 1992; Sutor & Luhmann, 1998). The DSs of human epileptogenic tissue have a fast decay (time constant 45. 4 ms), unaffected by CGP 55845A. The bicuculline-induced PDS of human tissues has a similar decay time constant (42.5 ms), however, is significantly increased by CGP 35348A (to 65.8 ms). Therefore, we propose two reasons for the lack of CGP 55845A effects on the DS: (1) DSs are too short to be affected by alterations of the IPSPB and (2) the GABAB receptors are reduced in human epileptogenic tissue; hence, CGP 55845A is bound to have smaller effects. Reduced GABAB receptor function at glutamatergic terminals Presynaptic GABAB receptors on glutamatergic terminals are well established in the mammalian neocortex (Howe et al., 1987; Connors et al., 1988) and the prototype agonist baclofen causes a half-maximal reduction of EPSPs at about 1 lM and a near-complete depression of EPSPs and IPSPs at 100 lM (Howe et al., 1987). In the human epileptogenic neocortex, however, baclofen (2 lM) decreased synaptic responses by only about 20% at comparable stimulus intensities, indicating a reduced efficacy of presynaptic GABAB receptors. This effect was mostly due to a presynaptic action of baclofen, because abolition of the postsynaptic GABAB-mediated K+ conductance with Cs+-containing electrodes (Desarmenien et al., 1984) had no effect on the magnitude of PSP depression (Fig. 5). This decrease in presynaptic GABAB receptor function has two conceivable consequences: first, an increase in the magnitude of synaptic activity, because a given steady state level of GABA would have less effect on release. Second, the effect could cause an altered temporal pattern of synaptic activity because any event releasing GABA would have a decreased effectiveness in depressing subsequent responses. The latter is particularly obvious from the transient failure of bicuculline-induced Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

1714 L. A. Teichgra¨ber et al. PDSs in the rodent neocortex (Fig. 7) lasting up to 1 s, consistent with the original report (Gutnick et al., 1982). The elimination of the transient PDS failure by CGP 55845A (Fig. 7) demonstrates the crucial involvement of presynaptic GABAB receptors, whereas the large variation in the PPD between human neurons (Fig. 9A vs. 9B) indicates that the function of presynaptic GABA receptors is variably attenuated. Our observations of CGP 55845A antagonizing the transient depression of PDSs appears to be at odds with a previous report indicating a complete failure of threshold PDSs after addition of CGP 35348 (Sutor & Luhmann, 1998). The authors proposed that CGP 35348 antagonizes presynaptic GABAB receptors at GABAergic terminals and that the elimination of this negative feedback (Deisz & Prince, 1989) increases GABA release. In turn, the elevated GABA would displace bicuculline from its binding to GABAA sites and abolish the PDSs, according to this hypothesis (Sutor & Luhmann, 1998). However, CGP 35348 has only small effects compared to CGP 55845A on presynaptic receptors at GABAergic terminals in the neocortex (Deisz et al., 1997; Deisz, 1999a) and other terminals (Guyon & Leresche, 1995). We envisage a different scenario of conceivable and/or established effects to account for the failure of PDSs observed by Sutor and Luhmann (1998). First, the blockade of GABAB receptors by CGP 35348 of upstream neurons in the presence of bicuculline would tend to further increase GABA release. Secondly, the presence of bicuculline and CGP 35348 reduces the GABA buffering by both GABAA and GABAB receptors, thereby further increasing available GABA. Third, elevated GABA could in turn more readily overcome the diffusional constraints of saturable GABA uptake (Deisz et al., 1984; Hablitz & Lebeda, 1985), similar to facilitated recruitment of GABAB receptors following blockade of GABA uptake (Isaacson et al., 1993). Finally, the excess of GABA at glutamatergic terminals could abolish PDSs by maximally activating presynaptic GABAB receptors, which are little affected by CGP 35348 in the neocortex (Deisz et al., 1997). The differences between the PDS of the bicuculline model and the DS in the human epileptogenic cortex may shed light on a key issue of epilepsy. In the rodent bicuculline model an augmented GABA release, as in the human tissue (see Fig. 8), can effectively activate functional presynaptic GABAB receptors (see Fig. 7). This feedback can limit the maximal frequency of excess activity in the healthy cortex, or even cause cessation of such activity (Sutor & Luhmann, 1998). Reduction of this feedback allows the unimpeded generation of PDSs at high frequencies (Figs. 7, 9). During the DS in the human cortex, when normal amounts of GABA are released, the buffering of extracellular GABA by GABAA receptors is preserved, and uptake limits the spill-over of GABA to impaired presynaptic GABAB receptors. Therefore, the DSs exhibit no Epilepsia, 50(7):1697–1716, 2009 doi: 10.1111/j.1528-1167.2009.02094.x

PPD. In essence, the bicuculline model of acute hyperexcitability has a preserved frequency limitation of excess synaptic activity via presynaptic GABAB receptors and this is the very mechanism impaired in human epileptogenic tissue. Reduced GABAB receptor function at GABAergic terminals In addition to impaired GABAB receptors at excitatory terminals, those on GABAergic terminals were also affected in human focal tissue, as indicated by the relatively small PPD of pharmacologically isolated IPSPs (see Fig. 11). A comparable effect has been reported in various animal models (Buhl et al., 1996; Haas et al., 1996; Mangan & Lothman, 1996). At first sight, the absence of PPD in human TLE tissue, that is, a constant inhibition at higher frequencies, seems difficult to equate with a use-dependent depression of inhibition as a major factor for the initiation of epileptiform activity (Prince et al., 1992). But GABAA receptor–mediated inhibition is depolarizing in some neurons from human epileptogenic tissue (Deisz et al., 1998), hence the impaired frequency limitation of inhibition would allow for repetitive GABAA depolarizations. Interestingly, depolarizing GABAA responses in human subiculum neurons have been proposed to underlie interictal activity (Cohen et al., 2002). If such GABAA depolarizations were the only cause, interictal activity should rapidly dwindle due to the frequency dependence of GABA release through presynaptic GABAB receptors in the neocortex (Deisz & Prince, 1989) and hippocampus (Davies & Collingridge, 1993). We, therefore, propose that interictal activity in cortical tissue is due to a combination of GABAA depolarization and impaired presynaptic GABAB receptors. However, the disinhibition concept of Prince et al. (1992) remains valid. The focal processes delineated above may proceed undetected in spatially confined arrays of neurons at subclinical levels. The presumably intact GABAB receptors outside the focal epileptogenic areas would cause a frequency-dependent disinhibition and facilitate the recruitment of larger arrays of neurons, and thus may increase confined subclinical focal activity above the detection level. The reduced function of presynaptic GABAB receptors in the neocortex corresponds to the decrease of metabotropic glutamate receptors in human hippocampus in cases of TLE with AHS. The function of metabotropic glutamate receptors is reduced only in the TLE with AHS group; in a lesion group this function is preserved (Dietrich et al., 1999). Our data from human neocortex from the two corresponding groups, however, suggest that both groups have a comparable attenuation of postsynaptic GABAB receptor function in the neocortex (see Table 1). Considering possible mechanisms for this decline, processes similar to the ‘‘baclofen tolerance’’ of

1715 Impairment of GABAB Receptors in Human TLE patients receiving intrathecal baclofen for the treatment of spasticity of spinal origin may be involved. Comparable to the downregulation of GABAB-binding density during intrathecal administration of baclofen in the rat spinal cord (Kroin et al., 1993), the ongoing neuronal activity in focal cortical areas may release excess GABA causing, for example, a decreased expression or augmented internalization of GABAB receptors. To conclude, our data indicate that both postsynaptic as well as presynaptic GABAB receptors are impaired in tissue slices from pharmacoresistant epilepsy patients. The key question is whether the decrease in GABAB-receptor function is the cause or the consequence of epilepsy. The data from the hippocampus of rodent epilepsy models (Mangan & Lothman, 1996) and after status epilepticus (Chandler et al., 2003) indicate a decreased function of GABAB receptors after seizures, that is, it is a consequence of epilepsy. Our data, indicating a comparable reduction of GABAB-receptor function in the human neocortex from various human pathologic groups, corroborate and extend this suggestion. On the other hand, and additionally, our data also confirm evidence from GABAB1 knockout mice (Prosser et al., 2001; Schuler et al., 2001), indicating that impaired GABAB function can cause epilepsy. This is obvious from our finding that reduced GABAB-receptor function (particularly at presynaptic sites) would facilitate occurrence of high frequency excess activity, and hence promote the onset and perpetuation of epilepsy. Therefore, reductions of GABAB receptors may be both consequence and cause of epilepsy, that is, they can be regarded as a vicious circle.

Acknowledgments Early experiments of this study were supported by a grant from the DFG (De 419/3-1). We are indebted to Dr. Froestl, Novartis, for the gift of CGP 55845A and to Prof. R. Veh for his help with immunohistochemistry and providing two human control sections. We are grateful to Dr. S. Gigout and S. Wierschke for comments on the manuscript. Part of the data has been presented in abstract form (Deisz et al., 2002). We confirm that we have read the Journal`s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Disclosure: None of the authors has any conflicts of interest to disclose.

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