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Gabapentin prevents cortical spreading depolarization-induced disinhibition
Accepted Manuscript Gabapentin prevents cortical spreading depolarization-induced disinhibition Masoud Mesgari, Johanna Krüger, Christopher Theo Riemer, Maryam Khaleghi Ghadiri, Stjepana Kovac, Ali Goji PII: DOI: Reference:
S0306-4522(17)30567-5 http://dx.doi.org/10.1016/j.neuroscience.2017.08.009 NSC 17958
To appear in:
Neuroscience
Received Date: Revised Date: Accepted Date:
28 March 2017 28 July 2017 3 August 2017
Please cite this article as: M. Mesgari, J. Krüger, C.T. Riemer, M.K. Ghadiri, S. Kovac, A. Goji, Gabapentin prevents cortical spreading depolarization-induced disinhibition, Neuroscience (2017), doi: http://dx.doi.org/10.1016/ j.neuroscience.2017.08.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gabapentin prevents cortical spreading depolarization-induced disinhibition
Masoud Mesgari1, Johanna Krüger 1, Christopher Theo Riemer 1, Maryam Khaleghi Ghadiri2, Stjepana Kovac3, Ali Goji1-5* 1
Epilepsy Research Center, Westfälische Wilhelms-Universität Münster, Germany
2
Department of Neurosurgery, Westfälische Wilhelms-Universität Münster, Germany
3
Department of Neurology, Westfälische Wilhelms-Universität Münster, Germany
4
Shefa Neuroscience Research Center, Khatam-Alanbia Hospital, Tehran, Iran
5
Department of Neuroscience, Mashhad University of Medical Sciences, Mashhad, Iran
*Corresponding authors: Prof. Ali Gorji, MD Epilepsy Research Center University of Münster, Robert-Koch-Straße 45 D-48149 Münster, Germany Tel.: +49 251 8355564 Fax: +49 251 8347479 E-mail: [email protected]
1
Abstract Cortical spreading depolarization (CSD) has an important role in brain diseases such as stroke, subarachnoid haemorrhage, migraine with aura, and epilepsy. Several anti-epileptic drugs (AEDs) are used to treat paroxysmal brain diseases and are thus known to suppress CSD. One of these AEDs is gabapentin (GBP) which has been traditionally used for treatment of some CSDrelated neurological diseases. We applied intra- and extracellular recordings to investigate the effect of CSD on inhibitory post synaptic potentials (IPSPs) and synaptic properties of rodent neocortex after application of GBP. Application of GBP after CSD increased the amplitude of IPSPs. In addition, GBP inhibited induction of long-term potentiation after CSD. These data support an effect of GBP on GABA-mediated inhibition in the late hyperexcitable phase of CSD. Modulation of synaptic properties and post-CSD GABAergic function are likely GBP´s mechanisms of action in CSD-related disorders. These mechanisms could be targeted for further drug discovery in CSD-related diseases. Key words: Anticonvulsive, Seizure, Epilepsy, Stroke, Spreading depolarization
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Introduction
Gabapentin (GBP) is widely used for the treatment of excess hyperexcitability, such as is seen in epilepsy (Calabresi et al., 2007). As mirrored in its name, GBP was initially synthetized in an attempt to enhance GABAergic function, yet more direct evidence for this is lacking. GBP has been shown to bind to the alpha 2 delta subunit of voltage gated calcium channels, a subunit which is closely linked to receptor trafficking, localization, and biophysical properties of the channels (Marais et al., 2001; Calabresi et al., 2007). Cortical spreading depolarization (CSD) has been linked to a plethora of neurological disorders with very robust data confirming a role in stroke, subarachnoid haemorrhage (SAH), migraine with aura and epilepsy (Dreier et al., 2012; Gorji, 2001). Thus, CSD represents the neurophysiological signature of a continuum of diseases ranging from paroxysmal benign diseases, such as migraine with aura, to diseases with a high mortality, such as stroke and subarachnoid haemorrhage (Dreier and Reiffurth, 2015). CSD is associated with a synchronized massive depolarization wave of neurons and glial cells at the velocity of 3-5 mm/min in the cerebral neocortex, which is immediately followed by a long lasting depression of cellular activity (Leao, 1944). More, recent evidence suggests that cellular and synaptic hyperexcitability occurs in the late phase after CSD. This late hyperexcitability seen after CSD can promote seizure activity if GABAergic tone is decreased, i.e. in partially disinhibited brain tissue (Berger et al., 2008; Eickhoff et al., 2014). In humans this late increase in excitability during SD was shown in patients with subarachnoid haemorrhage (Dreier et al., 2012). GBP has been shown to suppress cortical susceptibility to CSD ( Hoffmann et al., 2010), but whether it has an effect on cortical electrophysiological properties once CSD has occurred, is unclear. More importantly, how CSD cellular and synaptic hyperexcitability is influenced by GBP remains unknown. It has been shown that CSD modulated GABA-mediated inhibition and influenced neural network excitability in rodent and human brain (Dreier et al., 2012; Mesgari eta al., 2015). We thus aimed to explore the effects of GBP on (i) CSD induced modulation of GABAergic tone as measured by post-CSD inhibitory post-synaptic potentials (IPSPs) and on (ii) synaptic plasticity as explored by long-term potentiation (LTP). These mechanisms likely explain GBP´s overall inhibitory effect on CSD-induced hyperexcitability. Material and Methods 3
All experimental procedures were in accordance with the guiding principles for the care and use of animals in the University of Münster, Germany (50.0835.2.0/ A-18/2006). Slices were prepared from adult Wistar rats (male, 10–12 week old) in 500-µm thickness. The techniques for auditory thalamocortical brain slice preparation have been described in detail elsewhere (Broicher et al., 2010; Fig. 1). It has been demonstrated that these in vitro preparations contain functionally intact thalamocortical connections from the medial geniculate and the primary auditory cortex (Cruikshank et al., 2002). Briefly, the brain was removed under deep isoflurane anesthesia. Brains were placed into a vibratome and superfused with artificial cerebrospinal fluid (ACSF; 4 °C). The ACSF contained (in mmol/l): NaCl 124, KCl 4, CaCl2 1.0, NaH2PO4 1.24, MgSo4 1.3, NaHCO3 26, and glucose 10. The ACSF was continuously equilibrated with 5 % CO2 in O2, stabilizing the pH at 7.35–7.4. Brain slices were pre-incubated at 28 °C for 60 min and after 30 min CaCl2 was elevated to 2.0 mmol/l. Then, slices were transferred to an interface recording chamber and superperfused with ACSF at 32°C. Intracellular recordings were performed in the fourth layer auditory cortex pyramidal cells. Extracellular DC potential was recorded in the auditory cortex and the hippocampal CA1 region. The technique for intracellular recording is described in detail elsewhere (Ghaffarian et al., 2016). Briefly, intracellular recordings were performed with sharp microelectrodes filled with 2 mol/l potassium methylsulphate (connected to a 2 mol/l KCl solution-bridge through a ceramic junction; 60–100 MΩ). A bipolar extracellular stimulating electrode was placed in the thalamocortical projections in rostral position with respect to the cortex and IPSPs were evoked (Cruikshank et al., 2002). Only the monosynaptic compounds of the IPSPs were analyzed. The traces were digitized by Digidata 1200 (Axon Instruments, CA, USA) and the data were collected and analyzed using Axoscope 10.3 (Axon Instruments, CA, USA). For intracellular recording, the amplitude and duration of IPSPs were measured. For LTP induction, a bipolar platinum stimulus electrode was placed in the thalamocortical projection to apply electrical stimuli. A borosilicate glass microelectrode filled with ACSF and placed in the third layer of the auditory cortex to record field excitatory synaptic potentials (fEPSP). For control experiments, ACSF solution was used whereas, GBP (100 µM; purchased from Fluka, St. Louis/MO, USA) was added to the ACSF 30 min prior to tetanic stimulus in the GBP group. Amplitudes had to be stable (maximum difference of 10%) for at least 45 min before induction of a tetanic stimulus (10 trains of four pulses at 100 Hz, 200 ms apart) was delivered. 4
LTP was inuced 30 min after CSD induction and recordings were continued for 60 min after LTP. CSD was triggered by 3 M KCl application through a glass electrode inserted into the temporal cortex (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). Data are expressed as mean ± SD. One way ANOVA and Mann–Whitney rank sum test were used for comparisons between all data. Significance was established when the probability values were below 0.05. Results Effect of GBP on CSD-induced IPSPs Regular spikes were obtained from the auditory cortex layer IV pyramidal neurons by intracellular recordings (Fig. 1 A, lower panel). Furthermore, to investigate the effect of CSD on IPSPs, thalamocortical projections were stimulated. The synaptic delay of IPSPs evoked by thalamocortical stimulation was about 3.4 ± 0.8 ms and the time constant of decay was 25.7 ± 7.3 ms. We investigated the effect of CSD on dynamic changes of IPSPs. After 10 min of stable IPSPs recordings (9.2 ± 1.7 mV, 262.2 ± 67.4 ms, n = 7; Fig. 1 B1, C1), CSD was induced. IPSPs reemerged within 3-4 min after CSD but the amplitude and duration were significantly reduced within 22.9 ± 3.4 min after CSD induction (4.8 ± 1.8 mV, 172.8 ± 112 ms; p ≤ 0.001; Fig 1 B1, C1). A decrease in the amplitude and duration of IPSPs has been observed after 5 min of CSD initiation. The amplitude and duration of IPSPs continued to decrease before reaching a stable value after about 20 min of CSD induction. First we examined the effect of GBP only on IPSPs. Application of GBP did not affect IPSPs amplitude or duration (Fig. 1 B1, C1). To investigate the effect of GBP on post-CSD IPSPs, GBP (100 µM) was superfused immediately after the depolarization wave of CSD. After 10 min of stable recordings of IPSPs (8.9 ± 3.7 mV, 249.9 ± 77.6 ms, n = 7; Fig. 1 B2, C2), CSD was initiated. After reemergence of IPSPs, post-CSD GBP application induced a significant increase of the amplitude of IPSPs to 11.5 ± 3.5 mV (p ≤ 0.001) after 15.4 ± 3 min. In addition, GBP application inhibited the decrease of the duration of IPSPs after CSD (256.3 ± 129 ms; Fig. 1 B3, C3). 5
Effects of GBP on LTP To investigate the effects of GBP on synaptic plasticity, GBP was applied 30 min prior to tetanic stimulation of thalamocortical projections. LTP induced an increase in fEPSP amplitude in control tissues (n = 6; 143 ± 16 % of baseline value). Application of GBP significantly inhibited LTP induction compared to controls (n = 6; 121 ± 17 % of baseline value; p ≤ 0.001). LTP was significantly enhanced after induction of CSD compared to controls (n = 6; 161 ± 20 % of baseline value; p ≤ 0.001). Administration of GBP after induction of CSD significantly inhibited production of LTP compared to CSD and control groups (n = 6; 120 ± 9 % of baseline value; p ≤ 0.001, Fig. 2 A-B). Importantly, there was no significant difference between the GBP and SDGBP group in terms of LTP induction suggesting that enhanced post-CSD hyperexcitability was fully reduced by GBP. Discussion We here show that GBP modulates synaptic plasticity and post-CSD excitation-to-inhibition balance in rat cortical tissues. GBP augmented the inhibitory tone in the late hyperexcitable phase of CSD. GBP increased the amplitude of IPSPs after CSD suggesting an effect on GABAmediated inhibition. In addition, GBP reduced cortical LTP in response to tetanic stimulation of thalamocortical afferents, suggesting that GBP decreases excitability through modulation of synaptic plasticity. Previous studies have shown that the occurrence of CSD is predictive of brain injury, such as is seen in stroke, and that GBP reduces the stroke volume by inhibiting CSD in tissue at risk (Hoffmann et al., 2011). Disturbance of GABA-mediated inhibition has been shown to contribute to CSD-induced delayed hyperexcitability (Mesgari et al., 2015). Application of GBP did not affect IPSPs. However, we showed that IPSPs after CSD in the presence of GBP were increased suggesting that GBP increases post-CSD GABA-mediated inhibition. Our results provide functional data indicating that plastic alterations in the GABAergic transmission after CSD must be required for GBP to prevent CSD-induced disinhibition in cortical tissues. GABA-mediated inhibition functionally affects CSD-induced hyperexcitability of the human brain (Dreier et al., 2012) and CSD in rat neocortex modulates GABAA binding sites in cortical and subcortical regions (Haghir et al., 2009; Ghaemi et al., 2016). GBP attenuates the neurotransmitter release in brain tissues and this attenuation seems to be more marked when release is evoked by intense 6
stimulation rather than the physiological condition (Dooley et al, 2007). It has been shown that GBP did not inhibit GABA-mediated inhibitory post-synaptic currents in locus coeruleus slices in control mice, whereas reduced these currents via pre-synaptic mechanisms under conditions mimicking neuropathic pain (Takasu et al., 2008). The α2δ-1 subunit of the voltage-dependent calcium channel, the only known binding site to gabapentinoid drugs (Alles and Smith, 2016), was found to correlate with GABAergic neurons in different brain regions, including the neocortex thereby affecting neurotransmitter release (Dolphin, 2013). Using electrophysiological and receptor-binding techniques, pregabalin (a gabapentinoid drug) failed to affect GABAA or GABAB receptors in the rat spinal cord and brain (Li et al., 2011). However several other lines of evidence suggest that GBP has modulatory effects on GABAmediated inhibition. It has been suggested that GBP increases tonic inhibitory conductance of GABAA receptors (Cheng et al., 2006). GBP enhances the activity of glutamic acid decarboxylase, the main enzyme for GABA synthesis, and increases GABA synthesis in human and rat brain (Calabresi et al., 2007). Pervious investigations have shown that GBP is a selective agonist for the GABAB gb1a-gb2 heterodimer subtype, which is coupled to Kir channels and activates inwardly rectifying K+ channels (Ng et al., 2001). It has been suggested that GBP enhances neuronal inhibition during periods of hyperexcitability by increasing cytosolic GABA (Honmou et al., 1995). GBP also enhanced the frequency of spontaneous inhibitory postsynaptic currents in hippocampal pyramidal neurons (Peng et al., 2011) and facilitated GABAergic transmission in the central nucleus of the amygdala (Roberto et al., 2008). Suppression of LTP by administration of gabapentin has been shown before, albeit in a peripheral nerve preparation (Tanabe et al., 2006). In their study Tanabe and colleagues looked at C-fibre evoked field potentials in response to electrical stimulation of the sciatic nerve as a model of pain transmission. We here show that GBP suppresses LTP also at central synapses. Such an effect on central synaptic plasticity likely has an overall effect on the balance of excitation and inhibition in the brain. GBP may suppress LTP via attenuation of the stimulated presynaptic Ca2+ influx (Dooley et al, 2007). In addition, enhancement of inhibitory tone by GBP in the neocortex limits the capacity for induction of plasticity at excitatory synapses (Saez and Friedlander, 2016). GBP has been shown to decreases basal synaptic transmission in the cingulate cortex (Chen et al., 2014) pointing to its pain relieving properties, which are extensively used in a clinical setting. In contrast to our findings, Chen and colleagues were not able to show an effect of GBP on LTP 7
induced by intracortical stimulation in anterior cingulate cortex (Chen et al., 2014). Evoked potentials produced by intracortical stimulations represent a mixture of neuronal and synaptic potentials and this may explain the discrepancy. Whereas Chen and colleagues focused on pain perception pathways, we studied a brain region involved in sensory perception. We here show that GBP suppresses LTP at central synapses. Such an effect on central synaptic plasticity likely has an overall effect on the balance of excitation and inhibition in the brain. CSD trigger factors (such as female hormones; Sachs et al., 2007) facilitate and substances used in inhibition of CSD (such as D2 receptor antagonists; Haarmann et al., 2014) block LTP. Conclusion GBP enhances inhibitory tone in the late excitatory phase of CSD and reduces LTP at excitatory central synapses. These two mechanisms may contribute to GBP´s effect on neurological diseases.
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Acknowledgements This study was supported by Iran National Science Foundation (INSF) to AG. Authors thank Prof. Erwin-Josef Speckmann for his critical reading of the manuscript and Dr. Maryam Jafarian for her histological technical assistance. Conflict of Interest There is no conflict of interest.
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References Alles SR, Smith PA (2016) The Anti-Allodynic Gabapentinoids: Myths, Paradoxes, and Acute Effects. Neuroscientist [Epub ahead of print] Berger M, Speckmann EJ, Pape HC, Gorji A (2008). Spreading depression enhances human neocortical excitability in vitro. Cephalalgia 28(5):558-62. Broicher T, Bidmon HJ, Kamuf B, Coulon P, Gorji A, Pape HC, Speckmann EJ, Budde T (2010) Thalamic afferent activation of supragranular layers in auditory cortex in vitro: a voltage sensitive dye study. Neuroscience 165(2):371-85. Calabresi P, Galletti F, Rossi C, Sarchielli P, Cupini LM (2007) Antiepileptic drugs in migraine: from clinical aspects to cellular mechanisms. Trends Pharmacol Sci 28(4):188-95. Chen T, O'Den G, Song Q, Koga K, Zhang MM, Zhuo M (2014) Adenylyl cyclase subtype 1 is essential for late-phase long term potentiation and spatial propagation of synaptic responses in the anterior cingulate cortex of adult mice. Mol Pain 10;10:65. Cheng VY, Bonin RP, Chiu MW, Newell JG, MacDonald JF, Orser BA (2006) Gabapentin increases a tonic inhibitory conductance in hippocampal pyramidal neurons. Anesthesiology 105(2):325-33. Cruikshank SJ, Rose HJ, Metherate R (2002) Auditory thalamocortical synaptic transmission in vitro. J Neurophysiol 87(1):361-84. Dolphin AC (2013) The α2δ subunits of voltage-gated calcium channels. Biochim Biophys Acta 1828(7):1541-9. Dooley DJ, Taylor CP, Donevan S, Feltner D (2007) Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission. Trends Pharmacol Sci 28(2):75-82. Dreier JP, Major S, Pannek HW, Woitzik J, Scheel M, Wiesenthal D, Martus P, Winkler MK, Hartings JA, Fabricius M, Speckmann EJ, Gorji A; COSBID study group (2012) Spreading convulsions, spreading depolarization and epileptogenesis in human cerebral cortex. Brain 135(Pt 1):259-75. Dreier JP, Reiffurth C (2015) The stroke-migraine depolarization continuum. Neuron 20;86(4):902-22. Eickhoff M, Kovac S, Shahabi P, Ghadiri MK, Dreier JP, Stummer W, Speckmann EJ, Pape HC, Gorji A (2014) Spreading depression triggers ictaform activity in partially disinhibited neuronal tissues. Exp Neurol 253:1-15. Ghaemi A, Sajadian A, Khodaie B, Lotfinia AA, Lotfinia M, Aghabarari A, Khaleghi Ghadiri M, Meuth S, Gorji A (2016) Immunomodulatory Effect of Toll-Like Receptor-3 Ligand Poly I:C on Cortical Spreading Depression. Mol Neurobiol 53(1):143-154. Ghaffarian N, Mesgari M, Cerina M, Göbel K, Budde T, Speckmann EJ, Meuth SG, Gorji A (2016) Thalamocortical-auditory network alterations following cuprizone-induced demyelination. J Neuroinflammation 13(1):160. Haghir H, Kovac S, Speckmann EJ, Zilles K, Gorji A (2009) Patterns of neurotransmitter receptor distributions following cortical spreading depression. Neuroscience 10;163(4):1340-52. Haarmann AM, Jafarian M, Karimzadeh F, Gorji A (2014) Modulatory Effects of Dopamine D2 Receptors on Spreading Depression in Rat Somatosensory Neocortex. Basic Clin Neurosci 5(4):246-52. 10
Hoffmann U, Dileköz E, Kudo C, Ayata C (2010) Gabapentin suppresses cortical spreading depression susceptibility. J Cereb Blood Flow Metab 30(9):1588-92. Hoffmann U, Lee JH, Qin T, Eikermann-Haerter K, Ayata C (2011) Gabapentin reduces infarct volume but does not suppress peri-infarct depolarizations. J Cereb Blood Flow Metab 31(7):1578-82. Honmou O, Kocsis JD, Richerson GB (1995) Gabapentin potentiates the conductance increase induced by nipecotic acid in CA1 pyramidal neurons in vitro. Epilepsy Res 20(3):193-202. Leao AAP (1944) Spreading depression of activity in the cerebral cortex. J Neurophysiol 7(6):359–390. Li Z, Taylor CP, Weber M, Piechan J, Prior F, Bian F, Cui M, Hoffman D, Donevan S (2011) Pregabalin is a potent and selective ligand for α(2)δ-1 and α(2)δ-2 calcium channel subunits. Eur J Pharmacol 667(13):80-90. Marais E, Klugbauer N, Hofmann F (2001) Calcium channel alpha (2) delta subunits-structure and Gabapentin binding. Mol Pharmacol 59(5):1243-8. Mesgari M, Ghaffarian N, Khaleghi Ghadiri M, Sadeghian H, Speckmann EJ, Stummer W, Gorji A (2015) Altered inhibition in the hippocampal neural networks after spreading depression. Neuroscience 304:1907. Ng GY, Bertrand S, Sullivan R, Ethier N, Wang J, Yergey J, Belley M, Trimble L, Bateman K, Alder L, Smith A, McKernan R, Metters K, O'Neill GP, Lacaille JC, Hébert TE (2001) Gamma-aminobutyric acid type B receptors with specific heterodimer composition and postsynaptic actions in hippocampal neurons are targets of anticonvulsant gabapentin action. Mol Pharmacol 59(1):144-52. Peng BW, Justice JA, Zhang K, Li JX, He XH, Sanchez RM (2011) Gabapentin promotes inhibition by enhancing hyperpolarization-activated cation currents and spontaneous firing in hippocampal CA1 interneurons. Neurosci Lett 494(1):19-23. Roberto M, Gilpin NW, O'Dell LE, Cruz MT, Morse AC, Siggins GR, Koob GF (2008) Cellular and behavioral interactions of gabapentin with alcohol dependence. J Neurosci 28(22):5762-71. Sachs M, Pape HC, Speckmann EJ, Gorji A (2007) The effect of estrogen and progesterone on spreading depression in rat neocortical tissues. Neurobiol Dis 25(1):27-34. Saez I, Friedlander MJ (2016) Role of GABAA-Mediated Inhibition and Functional Assortment of Synapses onto Individual Layer 4 Neurons in Regulating Plasticity Expression in Visual Cortex. PLoS One 11(2):e0147642. Tanabe M, Murakami H, Honda M, Ono H (2006) Gabapentin depresses C-fiber-evoked field potentials in rat spinal dorsal horn only after induction of long-term potentiation. Exp Neurol 202(2):280-6. Takasu K, Ono H, Tanabe M (2008) Gabapentin produces PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission in LC neurons following partial nerve injury in mice. J Neurochem 105(3):933-42.
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Legends of figures Figure 1. The effect of gabapentin (GBP) on cortical spreading depression (CSD)-induced inhibitory post-synaptic potentials (IPSPs) in the auditory cortex. A1: Field potential recordings of the auditory neocortex (FP) show negative DC potential waves after injection of KCl (upper panel). Membrane potential (MP) changes are shown during and after CSD in layer IV of the neocortex (lower panel). After application of KCl in layer I–II of the auditory cortex, neurons first hyperpolarized and then depolarized abruptly at very nearly at the same point of time of the beginning of negative extracellular fluctuation. Note the brief burst of high-frequency spikes recorded during an early period of neuronal depolarization. A2: horizontal brain section stained by luxol fast blue followed by a cresyl violet counterstain shows the medial geniculate nucleus (MGN) and auditory cortex. A bipolar stimulation electrode was placed on the thalamocortical projections and intracellular recordings were performed in the layer 4 of the primary auditory cortex. B: Representative traces of IPSPs before and after GPB application (B1), and in CSD group (B2) and CSD and GBP (B3) groups. IPSPs are completely abolished during depolarization phase of CSD and reappeared a few minutes later. C: Box plots summarizing the mean IPSPs amplitude and duration before and after GBP application (C1), CSD induction (C2), and CSD induction and GBP application (C3). Values are represented as mean ± SD. * indicates p < 0.001. Figure 2. The effect of gabapentin (GBP) on long-term potentiation after induction of cortical spreading depression (CSD) in the auditory cortex. A: Long-term potentiation (LTP) of evoked field excitatory postsynaptic potentials (fEPSP) in neocortical tissue. Tetanic stimulation produces a rapid and stable potentiation of fEPSP, calculated as a percentage of baseline mean response amplitude. Changes in the amplitude of fEPSP after application of artificial cerebrospinal fluid (CTRL; black circles), GBP (100 µM; white circles), after induction of CSD (CSD; gray triangle), and after CSD induction in the presence of GBP (CSD-GBP; dark triangle) are shown. Arrow indicates the time of application of tetanic stimulation. Application of GBP significantly decreased LTP induction in CSD and GBP groups compared to the control and CSD rats. B: Representative traces of fEPSP before and after tetanic stimulation in control, GBP, CSD, and CSD-GBP groups. Values are represented as mean ± SD. * indicates p < 0.001.
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Spreading depolarization (SD) plays an important role in migraine and epilepsy. Gabapentin (GBP) is widely used for the treatment of epilepsy and migraine. GBP enhanced evoked inhibitory post synaptic potentials after induction of SD. GBP inhibited long-term potentiation of synaptic transmission after induction of SD GBP modulates synaptic properties and GABAergic function after induction of SD.
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S0306-4522(17)30567-5 http://dx.doi.org/10.1016/j.neuroscience.2017.08.009 NSC 17958
To appear in:
Neuroscience
Received Date: Revised Date: Accepted Date:
28 March 2017 28 July 2017 3 August 2017
Please cite this article as: M. Mesgari, J. Krüger, C.T. Riemer, M.K. Ghadiri, S. Kovac, A. Goji, Gabapentin prevents cortical spreading depolarization-induced disinhibition, Neuroscience (2017), doi: http://dx.doi.org/10.1016/ j.neuroscience.2017.08.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gabapentin prevents cortical spreading depolarization-induced disinhibition
Masoud Mesgari1, Johanna Krüger 1, Christopher Theo Riemer 1, Maryam Khaleghi Ghadiri2, Stjepana Kovac3, Ali Goji1-5* 1
Epilepsy Research Center, Westfälische Wilhelms-Universität Münster, Germany
2
Department of Neurosurgery, Westfälische Wilhelms-Universität Münster, Germany
3
Department of Neurology, Westfälische Wilhelms-Universität Münster, Germany
4
Shefa Neuroscience Research Center, Khatam-Alanbia Hospital, Tehran, Iran
5
Department of Neuroscience, Mashhad University of Medical Sciences, Mashhad, Iran
*Corresponding authors: Prof. Ali Gorji, MD Epilepsy Research Center University of Münster, Robert-Koch-Straße 45 D-48149 Münster, Germany Tel.: +49 251 8355564 Fax: +49 251 8347479 E-mail: [email protected]
1
Abstract Cortical spreading depolarization (CSD) has an important role in brain diseases such as stroke, subarachnoid haemorrhage, migraine with aura, and epilepsy. Several anti-epileptic drugs (AEDs) are used to treat paroxysmal brain diseases and are thus known to suppress CSD. One of these AEDs is gabapentin (GBP) which has been traditionally used for treatment of some CSDrelated neurological diseases. We applied intra- and extracellular recordings to investigate the effect of CSD on inhibitory post synaptic potentials (IPSPs) and synaptic properties of rodent neocortex after application of GBP. Application of GBP after CSD increased the amplitude of IPSPs. In addition, GBP inhibited induction of long-term potentiation after CSD. These data support an effect of GBP on GABA-mediated inhibition in the late hyperexcitable phase of CSD. Modulation of synaptic properties and post-CSD GABAergic function are likely GBP´s mechanisms of action in CSD-related disorders. These mechanisms could be targeted for further drug discovery in CSD-related diseases. Key words: Anticonvulsive, Seizure, Epilepsy, Stroke, Spreading depolarization
2
Introduction
Gabapentin (GBP) is widely used for the treatment of excess hyperexcitability, such as is seen in epilepsy (Calabresi et al., 2007). As mirrored in its name, GBP was initially synthetized in an attempt to enhance GABAergic function, yet more direct evidence for this is lacking. GBP has been shown to bind to the alpha 2 delta subunit of voltage gated calcium channels, a subunit which is closely linked to receptor trafficking, localization, and biophysical properties of the channels (Marais et al., 2001; Calabresi et al., 2007). Cortical spreading depolarization (CSD) has been linked to a plethora of neurological disorders with very robust data confirming a role in stroke, subarachnoid haemorrhage (SAH), migraine with aura and epilepsy (Dreier et al., 2012; Gorji, 2001). Thus, CSD represents the neurophysiological signature of a continuum of diseases ranging from paroxysmal benign diseases, such as migraine with aura, to diseases with a high mortality, such as stroke and subarachnoid haemorrhage (Dreier and Reiffurth, 2015). CSD is associated with a synchronized massive depolarization wave of neurons and glial cells at the velocity of 3-5 mm/min in the cerebral neocortex, which is immediately followed by a long lasting depression of cellular activity (Leao, 1944). More, recent evidence suggests that cellular and synaptic hyperexcitability occurs in the late phase after CSD. This late hyperexcitability seen after CSD can promote seizure activity if GABAergic tone is decreased, i.e. in partially disinhibited brain tissue (Berger et al., 2008; Eickhoff et al., 2014). In humans this late increase in excitability during SD was shown in patients with subarachnoid haemorrhage (Dreier et al., 2012). GBP has been shown to suppress cortical susceptibility to CSD ( Hoffmann et al., 2010), but whether it has an effect on cortical electrophysiological properties once CSD has occurred, is unclear. More importantly, how CSD cellular and synaptic hyperexcitability is influenced by GBP remains unknown. It has been shown that CSD modulated GABA-mediated inhibition and influenced neural network excitability in rodent and human brain (Dreier et al., 2012; Mesgari eta al., 2015). We thus aimed to explore the effects of GBP on (i) CSD induced modulation of GABAergic tone as measured by post-CSD inhibitory post-synaptic potentials (IPSPs) and on (ii) synaptic plasticity as explored by long-term potentiation (LTP). These mechanisms likely explain GBP´s overall inhibitory effect on CSD-induced hyperexcitability. Material and Methods 3
All experimental procedures were in accordance with the guiding principles for the care and use of animals in the University of Münster, Germany (50.0835.2.0/ A-18/2006). Slices were prepared from adult Wistar rats (male, 10–12 week old) in 500-µm thickness. The techniques for auditory thalamocortical brain slice preparation have been described in detail elsewhere (Broicher et al., 2010; Fig. 1). It has been demonstrated that these in vitro preparations contain functionally intact thalamocortical connections from the medial geniculate and the primary auditory cortex (Cruikshank et al., 2002). Briefly, the brain was removed under deep isoflurane anesthesia. Brains were placed into a vibratome and superfused with artificial cerebrospinal fluid (ACSF; 4 °C). The ACSF contained (in mmol/l): NaCl 124, KCl 4, CaCl2 1.0, NaH2PO4 1.24, MgSo4 1.3, NaHCO3 26, and glucose 10. The ACSF was continuously equilibrated with 5 % CO2 in O2, stabilizing the pH at 7.35–7.4. Brain slices were pre-incubated at 28 °C for 60 min and after 30 min CaCl2 was elevated to 2.0 mmol/l. Then, slices were transferred to an interface recording chamber and superperfused with ACSF at 32°C. Intracellular recordings were performed in the fourth layer auditory cortex pyramidal cells. Extracellular DC potential was recorded in the auditory cortex and the hippocampal CA1 region. The technique for intracellular recording is described in detail elsewhere (Ghaffarian et al., 2016). Briefly, intracellular recordings were performed with sharp microelectrodes filled with 2 mol/l potassium methylsulphate (connected to a 2 mol/l KCl solution-bridge through a ceramic junction; 60–100 MΩ). A bipolar extracellular stimulating electrode was placed in the thalamocortical projections in rostral position with respect to the cortex and IPSPs were evoked (Cruikshank et al., 2002). Only the monosynaptic compounds of the IPSPs were analyzed. The traces were digitized by Digidata 1200 (Axon Instruments, CA, USA) and the data were collected and analyzed using Axoscope 10.3 (Axon Instruments, CA, USA). For intracellular recording, the amplitude and duration of IPSPs were measured. For LTP induction, a bipolar platinum stimulus electrode was placed in the thalamocortical projection to apply electrical stimuli. A borosilicate glass microelectrode filled with ACSF and placed in the third layer of the auditory cortex to record field excitatory synaptic potentials (fEPSP). For control experiments, ACSF solution was used whereas, GBP (100 µM; purchased from Fluka, St. Louis/MO, USA) was added to the ACSF 30 min prior to tetanic stimulus in the GBP group. Amplitudes had to be stable (maximum difference of 10%) for at least 45 min before induction of a tetanic stimulus (10 trains of four pulses at 100 Hz, 200 ms apart) was delivered. 4
LTP was inuced 30 min after CSD induction and recordings were continued for 60 min after LTP. CSD was triggered by 3 M KCl application through a glass electrode inserted into the temporal cortex (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). Data are expressed as mean ± SD. One way ANOVA and Mann–Whitney rank sum test were used for comparisons between all data. Significance was established when the probability values were below 0.05. Results Effect of GBP on CSD-induced IPSPs Regular spikes were obtained from the auditory cortex layer IV pyramidal neurons by intracellular recordings (Fig. 1 A, lower panel). Furthermore, to investigate the effect of CSD on IPSPs, thalamocortical projections were stimulated. The synaptic delay of IPSPs evoked by thalamocortical stimulation was about 3.4 ± 0.8 ms and the time constant of decay was 25.7 ± 7.3 ms. We investigated the effect of CSD on dynamic changes of IPSPs. After 10 min of stable IPSPs recordings (9.2 ± 1.7 mV, 262.2 ± 67.4 ms, n = 7; Fig. 1 B1, C1), CSD was induced. IPSPs reemerged within 3-4 min after CSD but the amplitude and duration were significantly reduced within 22.9 ± 3.4 min after CSD induction (4.8 ± 1.8 mV, 172.8 ± 112 ms; p ≤ 0.001; Fig 1 B1, C1). A decrease in the amplitude and duration of IPSPs has been observed after 5 min of CSD initiation. The amplitude and duration of IPSPs continued to decrease before reaching a stable value after about 20 min of CSD induction. First we examined the effect of GBP only on IPSPs. Application of GBP did not affect IPSPs amplitude or duration (Fig. 1 B1, C1). To investigate the effect of GBP on post-CSD IPSPs, GBP (100 µM) was superfused immediately after the depolarization wave of CSD. After 10 min of stable recordings of IPSPs (8.9 ± 3.7 mV, 249.9 ± 77.6 ms, n = 7; Fig. 1 B2, C2), CSD was initiated. After reemergence of IPSPs, post-CSD GBP application induced a significant increase of the amplitude of IPSPs to 11.5 ± 3.5 mV (p ≤ 0.001) after 15.4 ± 3 min. In addition, GBP application inhibited the decrease of the duration of IPSPs after CSD (256.3 ± 129 ms; Fig. 1 B3, C3). 5
Effects of GBP on LTP To investigate the effects of GBP on synaptic plasticity, GBP was applied 30 min prior to tetanic stimulation of thalamocortical projections. LTP induced an increase in fEPSP amplitude in control tissues (n = 6; 143 ± 16 % of baseline value). Application of GBP significantly inhibited LTP induction compared to controls (n = 6; 121 ± 17 % of baseline value; p ≤ 0.001). LTP was significantly enhanced after induction of CSD compared to controls (n = 6; 161 ± 20 % of baseline value; p ≤ 0.001). Administration of GBP after induction of CSD significantly inhibited production of LTP compared to CSD and control groups (n = 6; 120 ± 9 % of baseline value; p ≤ 0.001, Fig. 2 A-B). Importantly, there was no significant difference between the GBP and SDGBP group in terms of LTP induction suggesting that enhanced post-CSD hyperexcitability was fully reduced by GBP. Discussion We here show that GBP modulates synaptic plasticity and post-CSD excitation-to-inhibition balance in rat cortical tissues. GBP augmented the inhibitory tone in the late hyperexcitable phase of CSD. GBP increased the amplitude of IPSPs after CSD suggesting an effect on GABAmediated inhibition. In addition, GBP reduced cortical LTP in response to tetanic stimulation of thalamocortical afferents, suggesting that GBP decreases excitability through modulation of synaptic plasticity. Previous studies have shown that the occurrence of CSD is predictive of brain injury, such as is seen in stroke, and that GBP reduces the stroke volume by inhibiting CSD in tissue at risk (Hoffmann et al., 2011). Disturbance of GABA-mediated inhibition has been shown to contribute to CSD-induced delayed hyperexcitability (Mesgari et al., 2015). Application of GBP did not affect IPSPs. However, we showed that IPSPs after CSD in the presence of GBP were increased suggesting that GBP increases post-CSD GABA-mediated inhibition. Our results provide functional data indicating that plastic alterations in the GABAergic transmission after CSD must be required for GBP to prevent CSD-induced disinhibition in cortical tissues. GABA-mediated inhibition functionally affects CSD-induced hyperexcitability of the human brain (Dreier et al., 2012) and CSD in rat neocortex modulates GABAA binding sites in cortical and subcortical regions (Haghir et al., 2009; Ghaemi et al., 2016). GBP attenuates the neurotransmitter release in brain tissues and this attenuation seems to be more marked when release is evoked by intense 6
stimulation rather than the physiological condition (Dooley et al, 2007). It has been shown that GBP did not inhibit GABA-mediated inhibitory post-synaptic currents in locus coeruleus slices in control mice, whereas reduced these currents via pre-synaptic mechanisms under conditions mimicking neuropathic pain (Takasu et al., 2008). The α2δ-1 subunit of the voltage-dependent calcium channel, the only known binding site to gabapentinoid drugs (Alles and Smith, 2016), was found to correlate with GABAergic neurons in different brain regions, including the neocortex thereby affecting neurotransmitter release (Dolphin, 2013). Using electrophysiological and receptor-binding techniques, pregabalin (a gabapentinoid drug) failed to affect GABAA or GABAB receptors in the rat spinal cord and brain (Li et al., 2011). However several other lines of evidence suggest that GBP has modulatory effects on GABAmediated inhibition. It has been suggested that GBP increases tonic inhibitory conductance of GABAA receptors (Cheng et al., 2006). GBP enhances the activity of glutamic acid decarboxylase, the main enzyme for GABA synthesis, and increases GABA synthesis in human and rat brain (Calabresi et al., 2007). Pervious investigations have shown that GBP is a selective agonist for the GABAB gb1a-gb2 heterodimer subtype, which is coupled to Kir channels and activates inwardly rectifying K+ channels (Ng et al., 2001). It has been suggested that GBP enhances neuronal inhibition during periods of hyperexcitability by increasing cytosolic GABA (Honmou et al., 1995). GBP also enhanced the frequency of spontaneous inhibitory postsynaptic currents in hippocampal pyramidal neurons (Peng et al., 2011) and facilitated GABAergic transmission in the central nucleus of the amygdala (Roberto et al., 2008). Suppression of LTP by administration of gabapentin has been shown before, albeit in a peripheral nerve preparation (Tanabe et al., 2006). In their study Tanabe and colleagues looked at C-fibre evoked field potentials in response to electrical stimulation of the sciatic nerve as a model of pain transmission. We here show that GBP suppresses LTP also at central synapses. Such an effect on central synaptic plasticity likely has an overall effect on the balance of excitation and inhibition in the brain. GBP may suppress LTP via attenuation of the stimulated presynaptic Ca2+ influx (Dooley et al, 2007). In addition, enhancement of inhibitory tone by GBP in the neocortex limits the capacity for induction of plasticity at excitatory synapses (Saez and Friedlander, 2016). GBP has been shown to decreases basal synaptic transmission in the cingulate cortex (Chen et al., 2014) pointing to its pain relieving properties, which are extensively used in a clinical setting. In contrast to our findings, Chen and colleagues were not able to show an effect of GBP on LTP 7
induced by intracortical stimulation in anterior cingulate cortex (Chen et al., 2014). Evoked potentials produced by intracortical stimulations represent a mixture of neuronal and synaptic potentials and this may explain the discrepancy. Whereas Chen and colleagues focused on pain perception pathways, we studied a brain region involved in sensory perception. We here show that GBP suppresses LTP at central synapses. Such an effect on central synaptic plasticity likely has an overall effect on the balance of excitation and inhibition in the brain. CSD trigger factors (such as female hormones; Sachs et al., 2007) facilitate and substances used in inhibition of CSD (such as D2 receptor antagonists; Haarmann et al., 2014) block LTP. Conclusion GBP enhances inhibitory tone in the late excitatory phase of CSD and reduces LTP at excitatory central synapses. These two mechanisms may contribute to GBP´s effect on neurological diseases.
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Acknowledgements This study was supported by Iran National Science Foundation (INSF) to AG. Authors thank Prof. Erwin-Josef Speckmann for his critical reading of the manuscript and Dr. Maryam Jafarian for her histological technical assistance. Conflict of Interest There is no conflict of interest.
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Legends of figures Figure 1. The effect of gabapentin (GBP) on cortical spreading depression (CSD)-induced inhibitory post-synaptic potentials (IPSPs) in the auditory cortex. A1: Field potential recordings of the auditory neocortex (FP) show negative DC potential waves after injection of KCl (upper panel). Membrane potential (MP) changes are shown during and after CSD in layer IV of the neocortex (lower panel). After application of KCl in layer I–II of the auditory cortex, neurons first hyperpolarized and then depolarized abruptly at very nearly at the same point of time of the beginning of negative extracellular fluctuation. Note the brief burst of high-frequency spikes recorded during an early period of neuronal depolarization. A2: horizontal brain section stained by luxol fast blue followed by a cresyl violet counterstain shows the medial geniculate nucleus (MGN) and auditory cortex. A bipolar stimulation electrode was placed on the thalamocortical projections and intracellular recordings were performed in the layer 4 of the primary auditory cortex. B: Representative traces of IPSPs before and after GPB application (B1), and in CSD group (B2) and CSD and GBP (B3) groups. IPSPs are completely abolished during depolarization phase of CSD and reappeared a few minutes later. C: Box plots summarizing the mean IPSPs amplitude and duration before and after GBP application (C1), CSD induction (C2), and CSD induction and GBP application (C3). Values are represented as mean ± SD. * indicates p < 0.001. Figure 2. The effect of gabapentin (GBP) on long-term potentiation after induction of cortical spreading depression (CSD) in the auditory cortex. A: Long-term potentiation (LTP) of evoked field excitatory postsynaptic potentials (fEPSP) in neocortical tissue. Tetanic stimulation produces a rapid and stable potentiation of fEPSP, calculated as a percentage of baseline mean response amplitude. Changes in the amplitude of fEPSP after application of artificial cerebrospinal fluid (CTRL; black circles), GBP (100 µM; white circles), after induction of CSD (CSD; gray triangle), and after CSD induction in the presence of GBP (CSD-GBP; dark triangle) are shown. Arrow indicates the time of application of tetanic stimulation. Application of GBP significantly decreased LTP induction in CSD and GBP groups compared to the control and CSD rats. B: Representative traces of fEPSP before and after tetanic stimulation in control, GBP, CSD, and CSD-GBP groups. Values are represented as mean ± SD. * indicates p < 0.001.
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Spreading depolarization (SD) plays an important role in migraine and epilepsy. Gabapentin (GBP) is widely used for the treatment of epilepsy and migraine. GBP enhanced evoked inhibitory post synaptic potentials after induction of SD. GBP inhibited long-term potentiation of synaptic transmission after induction of SD GBP modulates synaptic properties and GABAergic function after induction of SD.
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