Spreading Depression Enhances Human Neocortical Excitability in vitro

Abstract

Cortical spreading depression (CSD) plays a role in migraine with aura. However, studies of the neuronal effects of CSD in human cortex are scarce. Therefore, in the present study, the effects of CSD on the field excitatory postsynaptic potentials (fEPSP) and the induction of long-term potentiation (LTP) were investigated in human neocortical slices obtained during epilepsy surgery. CSD significantly enhanced the amplitude of fEPSP following a transient suppressive period and increased the induction of LTP in the third layer of neocortical tissues. These results indicate that CSD facilitates synaptic excitability and efficacy in human neocortical tissues, which can be assumed to contribute to hyperexcitability of neocortical tissues in patients suffering from migraine.

Introduction

Cortical spreading depression (CSD) is a pronounced depolarization of neurons and glia that spreads slowly across the cortex. A brief period of excitation heralds CSD, which is immediately followed by prolonged nerve cell depression accompanied by a dramatic failure of brain ion homeostasis and efflux of excitatory amino acids from nerve cells (1). CSD belongs in the domain of the pathophysiology of the brain (2). The hypothesis that aura is the human equivalent of CSD is well established (3). It is believed that changes in neuronal excitability due to CSD propagation play a crucial role in the symptomatology of migraine attacks (4). Therefore, the effect of CSD on neuronal excitability and synaptic function was investigated in human neocortical tissue obtained during epileptic surgery.

Methods

The cortical tissue was obtained from a small portion of that excised for treatment of pharmacoresistant focal epilepsy (n = 13 patients). Temporal lobe tissue used in this study originated from the anterior portion of the inferior temporal gyrus from standard partial temporal lobectomies. Details of patients' data are given in Table 1. The experiments were approved by the local ethics committee (Bezirksregierung Münster, Deutschland; AZ: 50.0835.1.0, G79/2002).

Table 1

Data of patients

Case	Gender	Age (years)	Seizure type	Seizure frequency month	Seizure for n years	AED	MRItext	Path	Tissue
1	F	49	SPS CPS SGS GS	12	39	CBZ VPA	Sclerosis	Gliosis, focal	TLl
2	M	34	SPS CPS SGS GS	3	28	CBZ DPH GPT LTG MSX OCBZ PHB VGB VPA	Sclerosis	dysplasia	TL
3	F	39	SPS CPS SGS GS	3	14	–	–	Gliosis, astrocytosis	TL
4	F	44	SPS CPS SGS GS	300	22	GPT OCBZ VPA	Sclerosis	Gliosis (fibre) astrocytosis atrophy	TL
5	F	47	SPS CPS	2	30	DPH LTG	Sclerosis	Gliosisgliosis (fibre) astrocytosis	TL
6	M	15	SPS GS		1	OCBZ	Tumour	ganglioglioma	TL
7	M	33	SPS CPS SGS GS	4	29	CBZ DPH VPA	Lesion sclerosis	Gliosis, astrocytosis	TL
8	M	18	SPS CPS	2	3	ACTH CBZ OCBZ VPA	Mes atrophy dysplasia lesion sclerosis	Astrocytosis focal dysplasia	TL
9	F	14	SPS CPS	20	12	CBZ ETX LTG OCBZ STM	Lesion sclerosis		TL
10	M	36	CPS	4	30	CBZ LTG OCBZ TGB VPA	Lesion sclerosis	Gliosisastrocytosis focal dysplasia	TL
11	M	50	SPS CPS SGS GS	1	26	BROM CBZ CZ DPH ETX GPT LTG OCBZ PHB PRM STM TGB VGB VPA	–	Focal dysplasia	TL
12	M	46	SPS CPS SGS GS	8	39	CBZ ETX LTG PHB PHT STM VPA	Mes atrophy	Astrocytosis focal dysplasia	TL
13	M	20	SPS CPS SGS GS	7	12	OCBZ VPA	–	Astrocytosis focal dysplasia	TL

ACTH, adrenocorticotropic hormone; BROM, bromide; CBZ, carbamazepine; CLZ, clonazepam; CPS, complex partial seizure; CZ, clobazam; DPH, diphenylhydantoin; ETX, ethosuximide; F, female; FL, frontal lobe; FLB, felbamate; GBT, gabapentine; GS, generalized seizure; LEV, levetiracetam; LTG, lamotrigine; M, male; OCBZ, oxycarbamazepine; PHB, phenobarbital; PHT, phenytoin; PRM, primidone; SGTCS, secondary generalized tonic-clonic seizure; SPS, simple partial seizure; STM, sultiam; TL, temporal lobe; TPM, topiramat; VERA, verapamil; VGB, vigabatrin; VPA, valproic acid.

Slices were prepared from a 1-cm³ tissue block within 5 min of resection. The techniques for slice preparation have been described in detail elsewhere (5). Briefly, neocortical slices 500 µm thick were cut in the frontal plane perpendicular to the pial surface using a vibratome. They were placed in a portable incubation chamber with oxygenated (95% O₂, 5% CO₂) artificial cerebrospinal fluid (ACSF) consisting of (in mm): NaCl, 124; KCl, 4; CaCl₂, 2; NaH₂PO₄, 1.24; MgSO₄, 1.3; NaHCO₃, 26; and glucose, 10 at a temperature of 28°C and pH 7.4. Slices were allowed to recover from transport for a period of 1–2 h before being transferred into a recording interphase-type chamber. In the experimental chamber the temperature was raised to 32°C. During the experiments, the pH, the bath temperature and the flow rate (1.5–2 ml/min) remained constant. Extracellular field potentials were recorded in cortical layers III and V with glass microelectrodes (2 m NaCl; 2–10 Ω) connected to d.c. amplifiers. Field potentials were traced by an ink-writer and recorded by a digital oscilloscope. CSD was elicited by KCl injection (3 m, tip diameter 2 µm; injection pressure 0.5–1.0 bar applied for 200–300 ms, two separate injections, 1–3 nl per pulse). In the controls (i.e. slices not undergoing CSD), ACSF was applied locally in adjacent slices from the same tissue block treated by KCl injection. Control slices were obtained from the same patient as CSD-exposed slices.

Single pulses of electrical stimulation were applied through a bipolar platinum electrode attached to the white matter perpendicular to the recording electrodes. Evoked field excitatory postsynaptic potentials (fEPSP) were recorded in the third layer of neocortical slices. The fEPSP was elicited by adjusting the intensity of stimulation to ∼50% of that at which population spikes after fEPSP began to appear. The amplitude of fEPSP 1 ms after the onset was measured for data analysis. In long-term potentiation (LTP) experiments, the cortex was sequentially stimulated once every minute. Ten trains of four pulses (pulse duration 0.1 ms; interpulse interval 50 ms; intensity 5 V) were repeated at intervals of 10 s. LTP was operationally defined as the mean change in fEPSP amplitude in response to five stimuli given 30 min after tetanic stimulation compared with the mean response to five test pulses applied immediately before the stimulation. Thus, percentage potentiation = post-tetanus amplitude of fEPSP/baseline amplitude of fEPSP. Tetanic stimulation was applied 30 min after induction of CSD. Spreading depression (SD)-like events were evaluated with respect to their repetition, amplitude, duration and velocity rates. SD duration was defined as the interval between the time of half-maximal voltage shift during onset and recovery of the negative d.c. potential deflection. All values are expressed as mean ± s.e.m. The data were statistically processed and compared using the Mann–Whitney rank sum test. Significance was established at P < 0.05.

Results

Local application of KCl in the sixth layer of neocortical tissues induced negative d.c. fluctuations followed by positive waves (amplitude of 15.8 ± 2.5 mV; duration 107 ± 6 s) in all tested slices. SD waves propagated opposite to the direction of the ACSF flow at propagation velocity of 3.3 ± 0.2 mm/min. fEPSP were monitored for at least 30 min before induction of SD. The evoked fEPSP in the third layer of neocortical tissue by stimulation of white substance (with mean amplitude of 0.50 ± 0.02 mV) were temporarily abolished during the negative phase of CSD. They reappeared after 3–5 min and recovered completely after 21 ± 2.4 min. Further recordings showed a significant irreversible increase of the amplitude of the fEPSP without decrement in the next 90 min (0.66 ± 0.04 mV, P < 0.001; n = 18). In the control slices, fEPSP remained unchanged after local injection of ACSF during 90 min of field potential recordings (n = 8, Fig. 1a).

Figure 1

Cortical spreading depression (CSD) enhances field excitatory postsynaptic potentials (fEPSP) and induction of long-term potentiation (LTP) in human neocortical slices. (A1) Representative examples fEPSP recorded from a single slice before, during and after induction of CSD. (A2) A graph of the normalized fEPSP amplitude at time points before, during and after induction of CSD. A representative example of CSD is shown on the left side. (B1) Representative examples of the fEPSP before and after tetanic stimulation in slices affected by CSD or application of artificial cerebrospinal fluid (ACSF) (control). (B2) Tetanic stimulation (10 trains of four pulses, pulse duration 0.1 ms; interpulse interval 50 ms) produces a rapid and stable potentiation in the amplitude of fEPSP, calculated as a percentage of baseline mean response amplitude. Solid hexagons and open circles show the evoked fEPSP after induction of CSD and control, respectively. Arrow shows the time of tetanic stimulation, 45 min after KCl application and ACSF (control). The time points given refer to LTP induction.

A conditioning tetanic stimulation was delivered to the white substance of neocortical slices after induction of CSD or injection of ACSF followed by pulses with stimulation parameters identical to control values. Evoked field potentials were stable for at least 30 min before application of tetanic stimulation (< 10% variation). Tetanic stimulation in slices treated by ACSF injection (control group) produced a rapid, stable and long-lasting enhancement of the amplitude of fEPSP in all tested preparations (n = 6, 130 ± 2.7% control). Induction of CSD 30 min prior to tetanic stimulation significantly increased LTP induction in human neocortical slices (n = 16, 147 ± 4.7% control, P = 0.012; Fig. 1b).

Discussion

The results reveal that induction of CSD initially suppresses fEPSP amplitude, which, followed by irreversible potentiation of human neocortical tissues, evoked responses to stimulation in white substance. Furthermore, the present study has shown that LTP in the third layers of the human neocortical slices is enhanced following induction of CSD. This demonstrates that CSD produces transient suppression of synaptic transmission that is replaced by a sustained increase in the efficacy of synaptic transmission in the affected neocortical tissues. The reduction of fEPSP amplitudes after CSD can be explained by the disturbances in ionic homeostasis of the involved tissues. The depolarization could alter the kinetics of the voltage-sensitive channels, e.g. by inactivating the sodium channels, and suppresses the amplitudes of evoked potentials. Propagation of CSD is accompanied by the release and diffusion of chemical mediators, such as excitatory amino acids, neurokinin, calcitonin gene-related peptide, serotonin and brain-derived neurotrophic factor, into the interstitial space in different neuronal tissues (2). The late facilitatory effect of CSD may relate to the excitatory effects of these mediators on neuronal activity. Enhancement of LTP induction by CSD would not be surprising, as CSD induces prolonged depolarization, activation of N-methyl-D-aspartate channels, and elevated intracellular Ca²⁺ and extracellular K⁺ (1). Transient depression of neuronal activities, which was replaced by hyperexcitability after CSD, has also been observed in rat neocortical (6) and spinal cord tissues (7). A similar pattern of an initial inhibitory effect (during the attacks) and subsequent excitatory effect (1–2 h after the attacks) has been observed in visual evoked potentials of patients suffering from migraine with aura (8). The present experiments were performed on epileptic human brain tissue. It should be noted that it is possible that chronic epilepsy and/or accompanying anticonvulsant therapy could alter the excitability of the tissue in a way that affects the results (5).

CSD-like waves such as observed in animal experiments occurred during the aura phase of migraine attacks in human occipital cortex accompanied by the visual aura (9). Using direct current magnetoencephalography, multiple areas of hyperexcitability in the primary visual cortex and occipital parietal regions have been observed during the aura in migraine attacks (10). Furthermore, electrophysiological studies using multimodal evoked and event-related potentials have found increased cortical excitability also in the headache-free interval in migraineurs (11). It has been suggested that hyperexcitability in widespread regions throughout the occipital cortex may provide the site for triggering potential CSD (12, 13). However, our results indicate that CSD, to some extent, may be responsible for cortical excitability in migraineurs. Reduced occipital cortical inhibition has been demonstrated for migraine with aura and for chronic migraine using an experiment with magnetic suppression of perceptual accuracy (14). Repetitive CSD also causes selective reduction of intracortical inhibition by suppression of GABAergic function (15).

Migraine attacks are characterized by hypersensitivity to visual, auditory and olfactory stimuli (16), and visual and auditory discomfort thresholds fell substantially during a migraine attack (17). An alteration of responsiveness of the sensory system in migraineurs is probably due to dysmodulation of sensor input leading to facilitation of sensory processing. CSD underlies occipital lobe dysfunction during visual aura. Enhancement of synaptic efficacy by CSD can be assumed to contribute to hyperexcitability of neocortical tissues in patients suffering from migraine.