Experimental Neurology 253 (2014) 1–15

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Spreading depression triggers ictaform activity in partially disinhibited neuronal tissues Marius Eickhoff a,1, Stjepana Kovac b,c,1, Parviz Shahabi d,1, Maryam Khaleghi Ghadiri e, Jens P. Dreier f, Walter Stummer e, Erwin-Josef Speckmann a,g, Hans-Christian Pape a, Ali Gorji a,b,d,e,g,⁎ a

Institute of Physiology I, University of Münster, Münster, Germany Department of Neurology, University of Münster, Münster, Germany UCL Institute of Neurology, University College London, London, UK d Shefa Neuroscience Research Center, Tehran, Iran e Klinik und Poliklinik für Neurochirurgie, University of Münster, Münster, Germany f Centre for Stroke Research Berlin, Charité University Medicine Berlin, Germany g Epilepsy Research Center, University of Münster, Münster, Germany b c

a r t i c l e

i n f o

Article history: Received 16 August 2013 Revised 19 November 2013 Accepted 16 December 2013 Available online 22 December 2013 Keywords: Seizure attacks Spreading depression Refractory epilepsy Migraine aura Cerebrovascular disorders

a b s t r a c t There is unequivocal electrophysiological evidence that spreading depression (SD) can trigger epileptiform field potentials. In vitro experiments on human brain tissues indicated that γ-aminobutyric acid (GABA)-mediated inhibition prevented this process. Intra- and extracellular recordings of bioelectrical activities were performed in the rodent neocortex, hippocampus and amygdala after perfusion of low concentrations of the GABAA antagonist bicuculline and induction of SD by KCl application. Induction of SD in combined amygdala–hippocampus–cortex slices pre-treated with low concentration of bicuculline triggered epileptiform burst discharges in cortical as well as subcortical brain structures. Propagation of SD significantly depolarized the membrane, decreased the amplitude and duration of action potentials (APs) and after-hyperpolarization as well as the neuronal membrane input resistance and the amplitude of threshold potentials. Ten to twenty minutes after induction of SD, the pattern of APs changed from regular firing to a series of APs riding on an underlying paroxysmal depolarization shift before the appearance of typical ictaform activities. Changes of characteristic features of APs occurred after SD persisted during the appearance of epileptiform activities. These results indicate that SD increases neuronal excitability and facilitates synchronization of neuronal discharges in the presence of partial disinhibition of neuronal tissues. Our findings might explain the occurrence of seizures in neurological disorders with partial impairment of inhibitory tone, such as brain ischemia and epilepsy. © 2013 Elsevier Inc. All rights reserved.

Introduction Spreading depolarization (SD) is a pronounced depolarization of neurons and glia that spreads slowly across the brain tissue followed by a massive redistribution of ions between intracellular and extracellular compartments (Leao, 1944; Somjen, 2001). SD belongs to the domain of the pathophysiology of the brain and has been linked to various neurological disorders, including migraine with aura, cerebrovascular diseases, head injury, transient global amnesia, and epilepsy (Gorji, 2001; Lauritzen et al., 2011). There is unequivocal evidence that SD occurs in the brain of patients with aneurismal subarachnoid hemorrhage, delayed ischemic stroke after subarachnoid hemorrhage, malignant hemispheric stroke, spontaneous intracerebral hemorrhage or traumatic brain injury (Dreier, 2011). ⁎ Corresponding author at: Epilepsy Research Center, Universität Münster, RobertKoch-Strasse 27a, D-48149 Münster, Germany. Fax: +49 251 8355551. E-mail address: [email protected] (A. Gorji). 1 The first three authors contributed equally to this article. 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.12.008

The close kinship between SD and experimental epileptic activity stimulated extensive investigation into the mutual relationship of these two phenomena in the last 65 years. Several characteristics of SD onset, propagation, and termination are similar to the activity observed during seizure episodes (Gorji, 2001; Somjen, 2001). Cooccurrence of SD and epileptic activity has been observed in a variety of in vitro and in vivo experimental models (Koroleva and Bures, 1983; Van Harreveld and Stamm, 1953) and in human neocortical slices (Avoli et al., 1991; Gorji and Speckmann, 2004). Despite plenty of evidence for its existence, the relationship between SD and epileptic activity is complex and remains poorly characterized. Epileptiform field potentials (EFP) and SD may occur in an alternating fashion (Mody et al., 1987; Avoli et al., 1991). EFP can precede SD in in vitro epilepsy models in human and rodent brains, induced by electrical stimulation or low magnesium (Mody et al., 1987; Avoli et al., 1991; Gorji et al., 2001). In a similar fashion, ictal epileptic field potentials were demonstrated in the front of SD in patients with acute brain injury (Fabricius et al., 2008). Moreover, SD can be initiated in a susceptible area by a single discharge of an epileptic focus termed spike-triggered SD (Koroleva

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and Bures, 1983). Repeated SD may enhance epileptic activities (Gorji and Speckmann, 2004) probably by selective suppression of γaminobutyric acid (GABA)ergic function (Kruger et al., 1996). Another form of co-occurrence of SD and epileptiform activity is spreading convulsion (Van Harreveld and Stamm, 1953). Such spreading convulsions are characterized by EFP on the final shoulder of the large slow potential change of spreading depolarization. This means that a run of EFP is recorded during the period which normally shows the spreading depression of spontaneous activity (Leao, 1944; Whieldon and Van Harreveld, 1950). Recent investigations demonstrated the occurrence of spreading convulsions in the human brain. This phenomenon was observed in subdural recordings in patients suffering from aneurismal subarachnoid hemorrhage in vivo and neocortical slices from patients with intractable temporal lobe epilepsy in vitro. The in vitro results suggested that partial GABA-mediated inhibition protects from triggering of epileptiform potentials by SD (Dreier et al., 2012). How these findings translate into cellular mechanisms underlying SD and more importantly how partial inhibition, suspected to be a key regulator of the interplay between SD and epileptiform activity, contributes to SD-linked epileptoform activity, remain to be determined. We aim to elucidate neuronal mechanism of SD-triggering EFP and their progression to ictaform activity in partially disinhibited cortical and subcortical tissue of rat brain.

Material and methods Slice preparation Adult Wistar rats (250–280 g) were decapitated under deep methohexital anesthesia and the brains were quickly transferred to ice-cold (4 °C) artificial cerebrospinal fluid (ACSF). After the cerebellum was removed and after dividing the two cerebral hemispheres, combined amygdala–hippocampus–cortex slices, comprising the temporal cortex, the perirhinal cortex, the entorhinal cortex, the subiculum, the

dentate gyrus, the hippocampus as well as the amygdala (500 μm), were cut in a nearly horizontal plane. A maximum of two different slices from each side containing the abovementioned structures were collected and stored at 28 °C. Storage solution (ACSF) contained (in mM) NaCl, 124; KCl, 4; CaCl2, 1.0; NaH2PO4, 1.24; MgSO4, 1.3; NaHCO3, 26; and glucose, 10 (pH 7.4), always gassed with 95% O2 and 5% CO2 for N1 h. The calcium concentration in the storage solution was reduced (1 mM) to keep the viability of neurons during the preparation (Tanaka et al., 2008). Thirty minutes after incubation, CaCl2 concentration was increased to 2.0 mM. For recording, each slice was placed on a transparent membrane in the interface recording chamber, and continuously perfused (1.5–2 ml/min) with carbogenated ACSF at 32 °C (the optimal temperature for keeping the normal synaptic and cellular functions in interface chamber; Javedan et al., 2002). To assure oxygen supply, a warmed and humidified gas mixture of 95% O2 and 5% CO2 was evaporated over the surface of the slices.

Electrophysiological recordings Intracellular recordings were performed in the fifth layer of the temporal neocortex, hippocampal CA1 area (stratum pyramidale), or the lateral amygdala (LA) using sharp microelectrodes filled with 2 mol/l potassium methylsulfate. Two hundred millisecond square positive and negative current pulses were injected into the neurons, intending to determine the neuronal input resistance (Rn) and discharge patterns (0.1–0.2 nA). To evaluate the effect of changes in the membrane potentials on cellular and synaptic properties (Shu et al., 2006), a constant positive or negative current was injected to the cells to set the membrane potentials to −40 mV or to −75 mV, respectively. The reference electrode and the connection to the microelectrode were symmetric Ag– Ag–Cl bridges. The microelectrodes were selected to have a resistance between 80 and 160 MΩ. Extracellular field potentials were recorded simultaneously close to the intracellular recording microelectrode using glass microelectrodes filled with 150 mmol/l NaCl and with a resistance of 2–10 MΩ. The potential of the intracellular electrode was

Table 1 Characteristics of membrane potential changes in somatosensory neocortical slices before and after induction of cortical spreading depression (SD). Characteristics of membrane potential changes in the fifth layer of somatosensory neocortical neurons 5 min before and 45 min after induction of SD at the resting membrane potential (RMP; A) and after continuous injection of a constant positive or negative current to depolarize (−40 mV, B) or hyperpolarize(−75 mV, C) the membrane as well as after positive current pulses (D). THP, the threshold potential; APs, action potentials; AHP, after-hyperpolarization; ISI, inter-spike interval. Values represent mean ± SEM. A

RMP (mV)

THP (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Before SD After SD P-value

−55.6 ± 0.4 −52.9 ± 2.7 0.004a

3.9 ± 0.1 2.9 ± 0.2 b0.001a

58.5 ± 1.4 60.1 ± 1.6 0.451

1.5 ± 0.2 0.7 ± 0.1 b0.001a

4.5 ± 0.3 2.7 ± 0.2 b0.001a

79.3 ± 5.9 48.7 ± 9.6 b0.001a

B

THP (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

Before SD After SD P-value

3.4 ± 0.2 3.1 ± 0.7 0.555

54.6 ± 1.9 39.2 ± 1.6 0.004a

11.8 ± 2.1 6.3 ± 1.2 0.313

4.7 ± 0.4 1.1 ± 0.1 b0.001a

122.2 ± 8.6 12.5 ± 2.2 b0.001a

160.3 ± 30.1 670.0 ± 122.9 b0.001a

C

THP (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

Before SD After SD P-value

3.9 ± 0.4 5.4 ± 0.3 0.012a

68.9 ± 1.3 55.2 ± 0.9 0.007a

4.2 ± 0.8 2.2 ± 0.4 0.030a

3.8 ± 0.6 1.7 ± 0.2 0.194

195.6 ± 49.3 21.7 ± 1.7 0.456

83.0 ± 37.0 230.1 ± 10.2 0.146

D

0.1 nA

Before SD After SD P-value a

Indicates significance.

0.2 nA

THP (mV)

APsamp (mV)

ISI (min−1)

THP (mV)

APsamp (mV)

ISI (min−1)

9.5 ± 2.7 17.1 ± 1.5 0.039a

71.1 ± 6.1 64.1 ± 12.9 0.594

105.8 ± 17.3 59.0 ± 8.9 0.137

15.1 ± 3.0 29.8 ± 4.0 0.02a

61.9 ± 3.8 71.8 ± 1.2 0.197

109.2 ± 15.3 41.3 ± 8.5 0.017a

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Fig. 1. Spreading depolarization (SD) triggers ictaform activity in partially disinhibited neocortical tissues. Membrane potential (MP) changes of a neocortical cell (somatosensory cortex; treated by bicuculline) during a cortical SD event along with extracellular field potential recording (FP) near the impaled neuron. After application of KCl in layers I–II of the neocortex, neurons (typical regular spiking cells) depolarized first gradually and slightly, then abruptly at very nearly the same point of time at which the negative extracellular fluctuation started. The recovery from a SD event was usually followed by overshooting of the control level in both intra- and extracellular recordings. The membrane potential fluctuations were recorded from layer V of the somatosensory neocortex. Representative traces of intracellular recordings 5 min before (A), 20 s (B), 10 min (C), 20 min (D), and 30 min (E) after initiation of SD are shown. Note the brief burst of high-frequency spikes recorded during an early period of the DC deflection (B) and development of interictal and ictal activities (C–E) within 30 min after SD induction. Negative voltage is represented by a downward deflection in extracellular FP recording.

referred to an extracellular microelectrode to ensure control of the true membrane potential during large shifts of the extracellular potential. Extracellular recordings were obtained using a custom made differential amplifier and the membrane potential fluctuations were obtained using a home-made active bridge mode amplifier (Gorji et al., 2011; Sachs et al., 2007). Traces were digitized by a Digidata 1200 (Axon Instruments, CA, USA) and the data were collected and analyzed by Axoscope 10 (Axon Instruments, CA, USA). The amplitudes of action potentials (APs) were measured from the resting membrane potential (RMP) baseline to the peak of the APs. The duration of the APs was measured as the half-amplitude width. The amplitudes of after-hyperpolarizations (AHP) were measured from RMP to the peak. Rn was calculated by Ohm's law from the ratio of the voltage deflection versus the injected current. Intracellular recording data acceptable for inclusion in the study met the following criteria: recording stability without any sign of injury discharges, and

membrane potential more negative than −45 mV with a deviation of less than 5% during the first 15 min of recordings. Induction of SD A glass electrode filled with 3 M KCl was fixed in a special holder connected with plastic tube to a pressure injector. The tip of the electrode was inserted either into the temporal cortex (layers I–II), the subiculum, or the centromedial amygdala of combined amygdala–hippocampus– cortex slices. An amount of KCl sufficient to induce SD was applied via high-pressure pulse (tip diameter, 2 μm; injection pressure, 0.5–1.0 bar applied for 200–300 ms, two separate injections, 1–3 nl per pulse, 2–5 mm apart from nearby recording electrodes). Amplitudes and duration of SD were analyzed. Duration of DC potential fluctuation width was measured at its half-maximal amplitude. EFP induced by SD were analyzed according to their amplitude, duration and repetition rate.

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Fig. 2. The membrane potential fluctuations recorded from layer V of the somatosensory neocortex before and after induction of cortical spreading depression (SD). Intracellular injection of a constant positive or negative current was used to investigate how burst characteristics and after-potentials change at depolarized (−40 mV) and hyperpolarized (−75 mV) states of the membrane and were compared with the values observed at the resting membrane potential (upper traces). SD was elicited by KCl injection in layers I–II of the neocortex. Traces were selected from intracellular activities 5 min before (left) and 30 min after (right) induction of SD.

Statistical analysis Results are given as mean ± S.E.M. Data were statistically processed using paired Student's t-test with two group comparisons, and analysis of variance test (ANOVA) with multiple group comparisons. Significance was assumed if the probability values were less than 0.05. Experiments were approved by the Bezirksregierung Münster (Bezirksregierung Münster, AZ: 50.0835.1.0, G79/2002). Results Field potentials were recorded in control condition for at least 15 min before superfusion was switched to bicuculline (1.25 μM) up to the end of the experiment. Bicuculline at this concentration did not induce any EFP after superfusion for 360 min in the neocortex,

hippocampus or LA (n = 12). Intracellular recordings along with DC potential recordings were obtained from 36 neurons in the neocortex, hippocampus and LA. After a minimum of 15 min of intracellular recordings under administration of bicuculline, SD was induced by local application of KCl. In keeping with previous observation (Dreier et al., 2012; Gorji et al., 2001), local application of KCl in the brain slices washed with ACSF did not induce any EFP (n = 6). In addition, local application of saline in slices treated with bicuculline at concentration of 1.25 μM neither induced DC negative shift nor changed the characteristic features of neuronal activities (n = 6). SD-related activity in the neocortex Intracellular recordings were obtained from 12 cells (classified as typical regular spiking cells; McCormick et al., 1985) in the neocortex

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Table 2 Characteristics of membrane potential changes in the hippocampus before and after induction of spreading depression (SD). Characteristics of membrane potential changes in hippocampal CA1 pyramidal neurons 5 min before and 45 min after induction of SD at the resting membrane potential (RMP; A) and after continuous injection of a constant positive or negative current to depolarize (−40 mV, B) or hyperpolarize(−75 mV, C) the membrane as well as after positive current pulses (D). THP, the threshold potential; APs, action potentials; AHP, after-hyperpolarization; ISI, inter-spike interval. Values represent mean ± SEM. A

RMP (mV)

Dep (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

Before SD After SD P-value

−52.8 ± 0.4 −51.4 ± 0.5 0.020a

3.9 ± 0.1 3.2 ± 0.2 b0.001a

62.0 ± 1.0 58.0 ± 0.9 0.006a

1.2 ± 0.1 0.5 ± 0.1 0.004a

4.7 ± 0.4 2.6 ± 0.2 b0.001a

78.7 ± 3.9 51.3 ± 2.2 b0.001a

349.0 ± 61.7 530 ± 70.4 0.032

B

THP (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

Before SD After SD P-value

3.6 ± 0.2 4.6 ± 0.2 0.002a

58.3 ± 1.2 55.7 ± 2.4 0.298

11.4 ± 0.8 14.7 ± 0.8 0.024a

4.2 ± 0.2 – –

78.9 ± 2.9 98.2 ± 5.8 0.002a

430.6 ± 92.6 241.0 ± 139 0.324

C

THP (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

Before SD After SD P-value

4.2 ± 0.2 4.2 ± 0.2 0.9

60.0 ± 1.5 69.5 ± 0.9 0.007a

16.7 ± 1.1 12.0 ± 1.0 0.133

2.6 ± 0.1 3.7 ± 0.2 0.194

88.3 ± 5.8 73.4 ± 4.1 0.092

135.2 ± 36.1 247.5 ± 12.5 0.120

D

0.2 nA

Before SD After SD P-value a

0.3 nA

THP (mV)

APsamp (mV)

ISI (min−1)

THP (mV)

APsamp (mV)

ISI (min−1)

10.4 ± 0.5 22.6 ± 5.5 0.02a

54.9 ± 3.1 65.8 ± 12.0 0.51

116.5 ± 11.9 58.9 ± 10.7 0.04a

13.4 ± 2.4 18.1 ± 0.8 0.031a

50.0 ± 3.1 62.7 ± 2.7 0.01a

59.4 ± 3.0 44.2 ± 5.8 0.259

Indicates significance.

(Table 1A). During SD waves, the membrane potentials changed drastically. After induction of SD (11 ± 2.3 mV, 86 ± 11 s), alterations in the membrane potentials usually began with a short and small hyperpolarization, followed by a depolarization. Neocortical neurons depolarized first gradually and slightly. This depolarization

at early stages was followed by a brief burst of high frequency spikes (Fig. 1B). Then the neurons depolarized abruptly 2 to 5 s after the onset of negative extracellular DC deflection. At the peak of the negative DC shift, the mean RMP reached − 2 ± 0.6 mV. Depolarization of the membrane potential was followed by a repolarization which

Fig. 3. Group of bars represents the mean ± S.E.M. of the amplitude of threshold potentials, duration of paroxysmal depolarization shift (PDS), amplitude of epileptiform field potentials (EFP) and duration of EFP, measured 12, 20 and 30 min after induction of SD. *Significant at p b 0.001 determined by ANOVA test.

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Fig. 4. Typical discharge patterns of cortical neurons (typical regular spiking cells) induced by intracellular injection of square positive current pulses (200 ms; 0.1 and 0.2 nA). Current injection was performed 5 min before as well as 15 and 30 min after induction of cortical spreading depression (SD). Note the higher frequency of action potentials with shorter interspike interval after induction of SD. The membrane potential fluctuations were recorded from layer V of the somatosensory neocortex.

often passed over to a transient hyperpolarization of smaller amplitude (Fig. 1). After 8.0 ± 1.6 min of the absence of APs, the activity reappeared as single, paired or triplet spikes (Fig. 1C; Table 1A). Propagation of SD reduced RMP to − 52.9 ± 2.7 mV compared with the period prior to SD (− 55.6 ± 2.7 mV; p = 0.004). SD decreased Rn, the threshold potential (THP) and the duration of APs as well as the amplitude and duration of AHP (all p b 0.001; Table 1A). After SD, APs had a lower amplitude as well as lower and shorter AHP with a higher frequency when neurons depolarized to − 40 mV (p b 0.001; Fig. 2, Table 1B). Hyperpolarization of neurons to − 75 mV after SD led to smaller and shorter APs and larger THP (p b 0.001; Fig. 2, Table 1C). After 19 ± 1.6 min of SD induction, the pattern of AP activity changed from regular firing to a series of APs riding on an underlying paroxysmal depolarization shift (PDS, Fig. 1D). PDS always outlasted the firing and thereby the primary burst. The primary burst was immediately followed by a short series of 2–8 afterdischarges at 20–30 Hz, with a duration of 30–40 ms each and 150–430 ms overall. These afterdischarges rode on the tail of the PDS or the primary burst, each discharge starting another depolarizing wave. The overall event ended upon return of the membrane potential to the pre-PDS baseline potential. Thirty minutes after SD, typical ictaform activity appeared. During this type of activity, at the end of the afterdischarges, and sometimes after a delay of 20–100 ms, a train of rhythmic bursts appeared, each of which was comprised of a depolarization with a few APs. The amplitude of THP and the duration of PDS were significantly enhanced and inter-spike interval (ISI) was decreased, measured 15, 20 and 30 min after induction of SD (Fig. 1 and Table 2). A significant enhancement of the amplitude, duration and frequency of EFP was observed, measured 15, 20 and 30 min after SD (Fig. 3). The response to intracellular current pulses injection revealed a higher frequency of APs and THP with a shorter ISI after SD induction (Fig. 4 and Table 1D). SD-related activity in the hippocampus Intracellular recordings were obtained from 12 pyramidal neurons (classified as typical regular spiking cells; Madison and

Nicoll, 1984) in hippocampal CA1 area (Table 2A). After induction of SD (12 ± 2.6 mV, 98 ± 14 s), RMP changed with a relatively short and small hyperpolarization, followed by a large depolarization. Depolarization of the membrane potential at early stages was followed by a brief burst of high frequency spikes (Fig. 5B). Then the neurons depolarized abruptly at roughly the same time at which the negative extracellular DC wave started. At the peak of the negative DC shift, the mean RMP reached + 3 ± 0.7 mV. Depolarization of the membrane potential was followed by a repolarization which was typically succeeded by a transient 10–20 mV hyperpolarization (Fig. 5). During the transient hyperpolarization phase, spikelets of small amplitude (9.6 ± 3.6 mV) occurred at a frequency of 4 Hz. Spikelets were replaced by the full-blown APs after 5 ± 1.9 min, coincidently with the end of the hyperpolarization phase of SD. SD reduced RMP to 51.4 ± 0.5 mV compared with the control period (−52.8 ± 0.4 mV, p = 0.02; Table 2A). SD decreased Rn, THP and the amplitude and duration of AHP and APs (all p b 0.001, Table 2A). The APs, depolarized to − 40 mV, had a larger THP as well as longer APs and AHP duration in cells affected by SD (p b 0.002; Fig. 6, Table 2B). Hyperpolarization of neurons to −75 mV after induction of SD led to enhancement of the amplitude of APs (p b 0.007; Fig. 6, Table 2C). After 10 ± 1.9 min of SD induction, PDS appeared which was accompanied by EFP (Fig. 5C). PDS outlasted the firing and thereby the primary burst. The primary burst was immediately followed by a short series of 1–3 afterdischarges at 10–20 Hz, lasting 10–30 ms each and 70–100 ms overall. Twenty minutes after SD, typical ictaform activity appeared. During ictaform activity, at the end of the afterdischarges, and sometimes after a delay of 20–80 ms, a train of rhythmic bursts appeared, each of which comprised of a depolarization with a few APs. The amplitude of THP and the duration of PDS were significantly increased and ISI was decreased after SD induction (Fig. 6). An increase in the amplitude, duration and frequency of EFP was observed within 30 min after SD induction (p b 0.001; Fig. 7). The response to intracellular current pulses revealed a higher frequency of APs with a shorter ISI after SD (Fig. 8 and Table 2D).

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Fig. 5. Spreading depolarization (SD) triggers ictaform activity in partially disinhibited hippocampal tissues. Membrane potential (MP) changes of a hippocampal cell (CA1 area; treated by bicuculline) during a SD event along with extracellular field potential recording (FP) near the impaled neuron. After application of KCl in the subiculum, neurons depolarized first gradually and slightly, then abruptly at very nearly the same point of time at which the negative extracellular fluctuation started. The recovery from a SD event was usually followed by overshooting of the control level in both intra- and extracellular recordings. Representative traces of intracellular recordings 5 min before (A), 30 s (B), 10 min (C) and 30 min (D) after induction of SD are shown. Note the brief burst of high-frequency spikes recorded during an early period of the DC deflection (B) and development of interictal and ictal activities (C–D) within 30 min after SD induction. Negative voltage is represented by a downward deflection in extracellular FP recording.

SD-related activity in LA Recordings of RMP were obtained from 12 cells (classified as projection neurons; Pape et al., 1998) in LA (Table 3A, Fig. 9A). After SD (8 ± 1.6 mV, 84 ± 10 s), RMP was characterized by a short and small hyperpolarization, followed by a large depolarization. Depolarization of the membrane potential at early stages was followed by a brief burst of high frequency population spikes (Fig. 9 B). Then the neurons depolarized abruptly at roughly the same time at which the negative DC fluctuation began. At the peak of the negative DC wave, the mean RMP reached −4 ± 0.4 mV. Depolarization of the membrane potential was followed by a repolarization (Fig. 9). During the repolarization phase, spikelets of small amplitude (8.3 ± 2.3 mV) occurred at a frequency of 3 Hz. Spikelet pattern of activity was subsided by the full-

blown APs after 7 ± 1.2 min, nearly at the end of the repolarization phase of SD. SD significantly depolarized the membrane to − 54.1 ± 1.2 mV compared to the period prior to SD (− 60.3 ± 0.5 mV; p b 0.001, Table 3A). SD decreased Rn, THP, the amplitude and duration of APs as well as AHP and increased AP frequency (all p b 0.001, Table 3A). The APs, depolarized to − 40 mV, had larger THP, AHP and APs as well as longer AHP after SD (p b 0.001; Fig. 10, Table 3B). Hyperpolarization of neurons to − 75 mV after SD did not change the characteristic features of the intracellular recordings (data are not shown, Table 3C). After 12 ± 0.9 min of SD induction, PDS appeared which was accompanied by EFP (Fig. 9C). The primary burst was immediately followed by a short series of 1–3 afterdischarges at 30–40 Hz, lasting 20–50 ms each and 100–150 ms overall. Twenty minutes after SD,

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Fig. 6. The membrane potential fluctuations recorded from hippocampal CA1 area before and after induction of spreading depression (SD). Intracellular injection of a constant positive or negative current was used to investigate how burst characteristics and after-potentials change at depolarized (−40 mV) and hyperpolarized (−75 mV) states of the membrane and was compared with the values observed at the resting membrane potential (upper traces). SD was elicited by KCl injection in the subiculum. Traces were selected from intracellular activities 5 min before (left) and 30 min after (right) induction of SD.

typical ictaform activity appeared. During ictaform activity, at the end of the afterdischarges, and sometimes after a delay of 20–80 ms, a train of rhythmic bursts appeared, each of which comprised of a depolarization with a few APs. The amplitude of THP and the duration of PDS were significantly enhanced and ISI was decreased 30 min after SD (Fig. 11). Field potentials showed a significant increase in the amplitude and duration of EFP (Figs. 9 and 11). The injection of current pulses displayed a higher frequency of APs with a shorter ISI and a larger THP after SD (Fig. 12 and Table 3D).

Discussion The present study shows that partial disinhibition of brain tissues by application of a GABAA antagonist promotes triggering of EFP by SD. Induction of SD in combined amygdala–hippocampus–cortex slices pre-treated with low concentration of the GABAA antagonist bicuculline triggered ictal activities in cortical as well as in subcortical brain structures. In keeping with our data, partial decrease of GABAA inhibitory tone facilitated the co-occurrence of SD and EFP in human tissues

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Fig. 7. Group of bars represents the mean ± S.E.M. of the amplitude of threshold potentials, duration of paroxysmal depolarization shift (PDS), amplitude of epileptiform field potentials (EFP), duration of EFP and frequency of EFP (/1 min), measured 12, 20 and 30 min after induction of SD. *Significant at p b 0.001 determined by ANOVA test.

resected during epilepsy surgery (Dreier et al., 2012). GABAA as well as GABAB receptors, although not directly involved in the initiation and propagation of SD, play a role in maintaining the state of excitation of the neuronal networks, and if this is interrupted may strengthen the late excitatory period following the depression phase of SD (Berger et al., 2008; Ghadiri et al., 2012). Partial impairment of GABAAmediated inhibition is believed to increase the susceptibility of neuronal tissues to both EFP and SD (Psarropoulou and Avoli, 1993). General principle of SD-triggering EFP Application of low concentrations of bicuculline (1–2.5 μM) led to an increase in the amplitude of excitatory postsynaptic potentials, but it had little overall effect on the early GABAA mediated component of the inhibitory postsynaptic potentials (IPSP; Scanziani et al., 1991). Low concentrations of bicuculline, however, resulted in a striking 2- to 3-fold increase in the amplitude of the late component of the IPSP (Knowles et al., 1984; McCormick, 1989). This large, late IPSP component was K+-mediated, blocked by the GABAB receptor antagonists

and sensitive to the NMDA receptor antagonists. Inhibition of this late component of IPSP by application of GABAB antagonists resulted in intense and sustained epileptic discharges (Scanziani et al., 1991). Negative DC deflection of SD is accompanied by a massive influx of Cl− (Müller, 2000; Somjen, 2001), which may reduce GABAB currents (Lenz et al., 1997). Therefore, GABAB conductance may be inhibited by massive entrance of Cl− (Lenz et al., 1997) during SD, compromise cellular functioning affected by low concentration GABAA antagonist and may contribute to ictal activity. The late IPSP component evoked in low concentrations of bicuculline is also sensitive to NMDA receptor antagonists (Scanziani et al., 1991). A small decrease in the efficacy of the inhibitory system can lead to NMDA receptor-mediated synchronized afterdischarges, which might play an important role in the susceptibility to epileptogenesis (Luhmann and Prince, 1990). On the other hand, blocking of NMDA receptors reduced the number of the evoked APs in the presence of low concentration of bicuculline (Hwa et al., 1991). Elevated extracellular K+ causes the release of glutamate and enhances NMDA currents during SD initiation. Neuronal tissues release glutamate and aspartate to the extracellular

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Fig. 8. Typical discharge patterns of hippocampal neurons induced by intracellular injection of square positive current pulses (200 ms; 0.1 and 0.2 nA). Current injection was performed 5 min before as well as 15 and 30 min after induction of cortical spreading depression (SD). Note the higher frequency of action potentials with shorter inter-spike interval after induction of SD.

space during propagation of SD (Van Harreveld and Kooiman, 1965) and activation of NMDA receptors is a prerequisite of SD initiation and propagation (Gorji et al., 2001). Enhancement of NMDA receptor-mediated activity via induction of SD may promote instability in partially disinhibited neuronal network and make it more susceptible to development of epileptiform discharges.

In our study, the amplitude and duration of AHP were reduced after SD propagation in both cortical and subcortical structures, which was probably due to a decrease of outward K+ conductance. Enhancement of extracellular K+ occurred during initiation of SD, which should decrease the driving force for K+ efflux, and decreased the size of AHP (Viana et al., 1993). Reduction of the amplitude of AHP

Table 3 Characteristics of membrane potential changes in the amygdala before and after induction of spreading depression (SD). Characteristics of membrane potential changes in lateral amygdala neurons 5 min before and 45 min after induction of SD at the resting membrane potential (RMP; A) and after continuous injection of a constant positive or negative current to depolarize (−40 mV, B) or hyperpolarize (−75 mV, C) the membrane as well as after positive current pulses (D). THP, the threshold potential; APs, action potentials; AHP, after-hyperpolarization; ISI, inter-spike interval. Values represent mean ± SEM. A

RMP (mV)

Dep (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

Before SD After SD p-Value

−60.3 ± 0.5 −54.1 ± 1.2 b0.001a

5.5 ± 0.2 3.3 ± 0.1 b0.001a

63.7 ± 2.0 53.9 ± 1.1 b0.001a

1.3 ± 0.2 0.6 ± 0.1 b0.001a

5.6 ± 0.1 2.8 ± 0.2 b0.001a

98.4 ± 5.0 37.2 ± 3.3 b0.001a

164.1 ± 40.2 227.2 ± 45.1 b0.04a

B

RMP (mV)

Dep (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

3.7 ± 0.2 4.6 ± 0.2 b0.001a

63.7 ± 2.2 49.8 ± 1.6 b0.001a

22.9 ± 0.7 23.7 ± 0.3 0.471

2.5 ± 0.8 5.1 ± 0.4 b0.001a

37.6 ± 2.2 83.9 ± 5.9 b0.001a

183.3 ± 43.3 305.0 ± 22.1 0.031a

Dep (mV)

APsamp (mV)

APsdur (ms)

AHPamp (mV)

AHPdur (ms)

Freq (min−1)

4.7 ± 0.3 4.6 ± 0.2 0.138

63.7 ± 2.2 68.8 ± 1.3 0.133

29.9 ± 0.6 33.7 ± 0.3 0.276

1.6 ± 0.7 2.1 ± 0.6 0.194

57.6 ± 2.2 63.9 ± 5.9 0.254

123.2 ± 33.1 145.4 ± 19.1 0.125

Before SD After SD p-Value C

RMP (mV)

Before SD After SD p-Value D

0.1 nA

Before SD After SD p-Value a

Indicates significance.

0.2 nA

THP (mV)

APsamp (mV)

ISI (min−1)

THP (mV)

APsamp (mV)

ISI (min−1)

10.9 ± 2.0 14.8 ± 2.1 0.041a

46.1 ± 1.1 51.7 ± 2.2 0.771

129.9 ± 29.6 58.4 ± 8.9 0.08

12.8 ± 3.1 22.8 ± 2.3 0.035a

68.3 ± 7.7 54.1 ± 3.9 0.58

43.7 ± 2.2 26.4 ± 3.2 0.01a

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Fig. 9. Spreading depolarization (SD) triggers ictaform activity in partially disinhibited amygdala tissues. Membrane potential changes (MP) of an amygdala projection neuron (the lateral amygdala; treated by bicuculline) during a SD event along with extracellular field potential recording (FP) near the impaled neuron. After application of KCl in the centromedial amygdala, neurons depolarized first gradually and slightly, then abruptly at very nearly the same point of time at which the negative extracellular fluctuation started. Representative traces of intracellular recordings 5 min before (A), 10 s (B), 15 min (C) and 30 min (D) after induction of SD are shown. Note the brief burst of high-frequency spikes recorded during an early period of the DC deflection (B) and development of interictal and ictal activities (C–D) within 30 min after SD induction. Negative voltage is represented by a downward deflection in extracellular FP recording.

after propagation of SD was reported earlier (Ghadiri et al., 2012). The AHP is functionally inhibitory and provides a strong negative feedback control on the activity of the neuron (Madison and Nicoll, 1984). A reduction in the AHP would be expected to increase neuronal excitability and facilitates the NMDA receptor-mediated response (Disterhoft et al., 2004). A reduction in the amplitude of the AHP may serve as a general mechanism of increased excitability, which facilitates

activity-dependent synaptic modifications (Baldissera and Gustafsson, 1974). Transition from the ictal to the interictal state is accompanied by the disappearance of the AHP (Madison and Nicoll, 1984). A further increase in excitability may be caused by SD suppressive effect of GABAergic synaptic plasticity (Haghir et al., 2009; Kruger et al., 1996). Increasing extracellular K+ during SD shifts the GABA reversal potential in a depolarizing direction (Thompson and Gähwiler,

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M. Eickhoff et al. / Experimental Neurology 253 (2014) 1–15

Fig. 10. The membrane potential fluctuations recorded from the lateral amygdala before and after induction of spreading depression (SD). Intracellular injection of a constant positive or negative current was used to investigate how burst characteristics and after-potentials change at depolarized (−40 mV) state of the membrane and was compared with the values observed at the resting membrane potential (upper traces). SD was elicited by KCl injection in the centromedial amygdala. Traces were selected from intracellular activities 5 min before (left) and 30 min after (right) induction of SD from an amygdala projection neuron.

1989), which exploits the transmembrane potassium gradient to extrude Cl− from cells (Misgeld et al., 1986). Further alterations in cellular and synaptic excitation–inhibition ratio induced by SD in partially disinhibited neuronal tissues may favor network synchrony and thus facilitate ictal discharges (Stief et al., 2007). In spite of several similarities (Somjen, 2001), there are some differences between SD induced by high K+ (normoxic SD) and hypoxic SD. Synaptic failure occurs minutes before the onset of hypoxic SD, whereas in normoxic SD synapses continue to function until depolarization inactivates ion channels. There are also differences in the pharmacology of the two conditions as well as in the vulnerability to neuronal injury, cell release of inorganic phosphate, the shortfall in oxidative energy and lactic acid production (Bureš et al., 1974; Somjen, 2001). Regionally specific differences In our study, each SD episode was heralded by brief spontaneous population spikes. However, this period was significantly shorter with lower rate of spikes in the amygdala. Before the initiation of the SD, the membrane potential began to depolarize gradually and slightly, indicating a small inward membrane current. This inward current initiates brief high frequency spikes. During this brief period, the Na+ pump of neurons is stimulated by the increased extracellular K+ and pumps the Na+ into the cell (Sugaya et al., 1975). A dual mechanism controls

spike frequency adaptation in LA pyramidal neurons. In contrast to hippocampal and neocortical cell types, which predominantly express either the voltage-gated potassium current or the slow AHP, LA pyramidal neurons express both types of current, which together control spike frequency adaptation. By contrast, the repetitive discharge properties in cortical pyramidal neurons are largely controlled by the voltage-gated potassium current (Bekkers and Delaney, 2001) and in hippocampal pyramidal neurons are largely determined by the slow AHP (Madison and Nicoll, 1984). The medium AHP, mediated by activation of small conductance (SK) channels, controls AP firing frequency in a number of cell types including hippocampal and cortical pyramidal neurons (Pedarzani et al., 2001). By contrast, in LA neurons, blocking the medium AHP has no effect either on discharge frequency or on spike frequency adaptation (Faber and Sah, 2005). During SD, the membrane potential shifted from a negative value toward zero. The depolarization could change the kinetics of the voltage-sensitive channels, for example by inactivating the sodium channels, and depress cellular activity (Somjen, 2001). During the repolarization phase of SD, spikelets of small amplitude were observed in the partially disinhibited hippocampus and amygdala, but not in the neocortex, which subsided by the full-blown APs after a few minutes. This may be due to the slow inactivation of Na+ channels after prolonged membrane depolarization by SD (Ulbricht, 2005). Slow inactivation of Na+ channels plays a role in regulating

M. Eickhoff et al. / Experimental Neurology 253 (2014) 1–15

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Fig. 11. Group of bars represents the mean ± S.E.M. of the amplitude of threshold potentials, duration of paroxysmal depolarization shift (PDS), amplitude of epileptiform field potentials (EFP) and duration of EFP, measured 12, 20 and 30 min after induction of SD. *Significant at p b 0.001 determined by ANOVA test.

excitability (Ruff et al., 1988), such as by modulating burst discharges (Elinder and Arhem, 1997). Differences in Na+ channel density as well as in their regulation in physiological (Xia et al., 2003) and pathophysiological (such as hypoxia, neuronal injury, and epilepsy; Zhang

et al., 2001) conditions between the neocortex, hippocampus and amygdala have been reported. Spikelets, however, may originate from dendritic spikes, ectopic spikes, or spikes in an electrically coupled neuron (Avoli et al., 1998).

Fig. 12. Typical discharge patterns of the lateral amygdala projection neurons induced by intracellular injection of square positive current pulses (200 ms; 0.1 and 0.2 nA). Current injection was performed 5 min before as well as 15 and 30 min after induction of cortical spreading depression (SD). Note the higher frequency of action potentials with shorter inter-spike interval after induction of SD.

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Clinical impacts of SD-triggering EFP Partial impairment of GABAA receptor mediated inhibition in the peri-infarct zone as well as in remote sites of ischemic lesions was reported in a rat model of transient middle cerebral artery occlusion (Neumann-Haefelin and Witte, 2000). Both ipsi- and contralateral to the cortical ischemic lesion an increase of neuronal excitability and a decrease of GABAergic inhibition were found. This was associated with a down-regulation of GABAA receptor binding (Frauenknecht et al., 2009) and an altered composition of GABA receptors by different subunits (Witte and Stoll, 1997). The mean GABAA phasic inhibition was transiently decreased at 7-days post-stroke in mice (Clarkson et al., 2010). However, this study revealed an enhancement of GABAA tonic inhibition mediated by extrasynaptic GABAA receptors. In clinical epilepsy, a partial decrease in the efficacy of GABAergic inhibition is clearly defined (Lloyd et al., 1986: Sherwin and van Gelder, 1986). Previous investigations demonstrated a co-occurrence of SD and EFP in patients suffering from subarachnoid hemorrhage (Dreier et al., 2012). SD triggered EFPs is likely to contribute to epileptogenesis in ischemia. In addition, the occurrence of SD in the brain of the patients suffering from epilepsy may enhance impairment of inhibitory tone which in turn may lead to seizures. Acknowledgments This work was supported by SFB Tr3 D10, DFG DR 323/6-1, BMBF Center for Stroke Research Berlin 01 EO 0801 and BMBF BCCN 01GQ1001C-B2 and by Shefa Neuroscience Center/Thesis-12507B. References Avoli, M., Drapeau, C., Louvel, J., Pumain, R., Olivier, A., Villemure, J.G., 1991. Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro. Ann. Neurol. 30, 589–596. Avoli, M., Methot, M., Kawasaki, H., 1998. GABA-dependent generation of ectopic action potentials in the rat hippocampus. Eur. J. Neurosci. 10, 2714–2722. Baldissera, F., Gustafsson, B., 1974. Firing behaviour of a neurone model based on the afterhyperpolarization conductance time course and algebraical summation. Adaptation and steady state firing. Acta Physiol. Scand. 92, 27–47. Bekkers, J.M., Delaney, A.J., 2001. Modulation of excitability by alpha-dendrotoxinsensitive potassium channels in neocortical pyramidal neurons. J. Neurosci. 21, 6553–6560. Berger, M., Speckmann, E.J., Pape, H.C., Gorji, A., 2008. Spreading depression enhances human neocortical excitability in vitro. Cephalalgia 28, 558–562. Bureš, J., Burešová, O., Křivánek, J., 1974. The mechanism and applications of Leao's spreading depression of electroencephalographic activity. Academia, Prague. Clarkson, A.N., Huang, B.S., Macisaac, S.E., Mody, I., Carmichael, S.T., 2010. Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke. Nature 468, 305–309. Disterhoft, J.F., Wu, W.W., Ohno, M., 2004. Biophysical alterations of hippocampal pyramidal neurons in learning, ageing and Alzheimer's disease. Ageing Res. Rev. 3, 383–406. Dreier, J.P., Major, S., Pannek, H.W., Woitzik, J., Scheel, M., Wiesenthal, D., Martus, P., Winkler, M.K., Hartings, J.A., Fabricius, M., Speckmann, E.J., Gorji, A., 2012. Spreading convulsions, spreading depolarization and epileptogenesis in human cerebral cortex. Brain 135 (Pt 1), 259–275. Dreier, J.P., 2011. The role of spreading depression, spreading depolarization and spreading ischemia in neurological disease. Nat. Med. 17, 439–447. Elinder, F., Arhem, P., 1997. Tail currents in the myelinated axon of Xenopus laevis suggest a two-open-state Na channel. Biophys. J. 73, 179–185. Faber, E.S., Sah, P., 2005. Independent roles of calcium and voltage-dependent potassium currents in controlling spike frequency adaptation in lateral amygdala pyramidal neurons. Eur. J. Neurosci. 22, 1627–1635. Fabricius, M., Fuhr, S., Willumsen, L., Dreier, J.P., Bhatia, R., Boutelle, M.G., Hartings, J.A., Bullock, R., Strong, A.J., Lauritzen, M., 2008. Association of seizures with cortical spreading depression and peri-infarct depolarizations in the acutely injured human brain. Clin. Neurophysiol. 119, 1973–1984. Frauenknecht, K., Plaschke, K., Sommer, C., 2009. Transient oligemia is associated with long-term changes in binding densities of cortical inhibitory GABAA receptors in the rat brain. Brain Res. 1271, 95–102. Ghadiri, M.K., Kozian, M., Ghaffarian, N., Stummer, W., Kazemi, H., Speckmann, E.J., Gorji, A., 2012. Sequential changes in neuronal activity in single neocortical neurons after spreading depression. Cephalalgia 32, 116–124. Gorji, A., 2001. Spreading depression: a review of the clinical relevance. Brain Res. Brain Res. Rev. 38, 33–60. Gorji, A., Speckmann, E.J., 2004. Spreading depression enhances the spontaneous epileptiform activity in human neocortical tissues. Eur. J. Neurosci. 19, 3371–3374.

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