INTRODUCTION

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Cortical spreading depression (CSD) is a transient suppression of neuronal activity, resulting from a temporary disruption of local brain ionic homeostasis that propagates slowly across the cerebral cortex (Lauritzen 1994). More than 50 yr after its discovery by Leão, CSD is attracting renewed attention for three different reasons: 1) it appears to be the primary neurological dysfunction leading to a migraine attack (Cao et al. 1999; Lauritzen 1994); 2) spontaneous peri-infarct CSD promote lesion progression in models of focal ischemia (Mies et al. 1993); and 3) CSD is a reproducible method for the induction of brain preconditioning, i.e., the adaptive cytoprotection that protects against subsequent, potentially lethal insults (Matsushima et al. 1998). Preliminary experiments, carried out in our laboratory as part of a research program involving mice with targeted mutations, suggested that CSD induced by epidural application of KCl to the parietal cortex of mice might not propagate uniformly to frontal and posterior regions. The purpose of this study was to ascertain whether or not CSD spreads symmetrically along the anteroposterior axis of the cortex of mice, and to determine where CSD should be elicited to achieve a uniform exposure of the cortex to this phenomenon (i.e., a requirement for investigations into the molecular changes that underlie CSD-induced preconditioning).

	METHODS

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Animal preparation and CSD induction

Twenty adult, male C57BL/6 mice (weight, 25.6 ± 0.6 g, mean ± SE; Harlan UK, Blackthorn, UK) were used, with food and water available ad libitum. Animal procedures were performed in conformity with the American Physiological Society policy regarding the use and care of animals. Mice were anesthetized throughout with halothane (5% for <1.5 min for induction, 1.5% during surgery, and 1.0% during the rest of the experiment) in O₂:N₂O (1:2). Animals were placed in a stereotaxic frame, and three burr holes (1 mm diam) were drilled in the skull without damaging the dura. They were aligned 1.5 mm to the right of the midline, and positioned +2.5 mm, 0.3 and 3.0 mm from bregma (Fig. 1). These holes were used for CSD induction or the recording of its propagation with glass capillary electrodes. Recurrent CSD was elicited by epidural application of 1 M KCl for 2 h. A steady application of the KCl solution was achieved by using a high precision syringe pump (CMA/100, CMA/Microdialysis, Stockholm) with a 250-µl syringe (Hamilton, Reno, NV) fitted to a fused silica fiber (450 µm OD × 320 µm ID, Polymicro Technologies, Phoenix, AZ) ending right above the dura (Fig. 1). CSD initiation was started by filling rapidly the small hole with KCl, using a relatively fast flow rate (around 1 µl/min), which was then reduced to 0.15 µl/min for the rest of the CSD-induction period. This procedure was found especially appropriate for the reproducible induction of recurrent CSD in mice. The application of KCl remained restricted to the small area of exposed dura throughout the 2-h procedure, with no possibility for lateral diffusion between bone and dura. Removal of the silica fiber and extensive rinsing of the CSD elicitation site with physiological saline stopped the CSD induction.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Location of epidural KCl application and cortical spreading depression (CSD) recording used in series I (top diagram), and representative changes in extracellular DC potential indicative of anterior and posterior CSD propagation (traces). In addition to the obvious, less efficient propagation of CSD in the posterior direction, note the different pattern of the "full" CSD waves (single waves showed over a 3-min time scale) recorded anteriorly and posteriorly. In the DC recordings, the vertical bars indicate 10 mV, and that along the x-axis KCl application for 2 h.

Recording of extracellular DC potential and EEG

Two glass capillary electrodes with 20- to 30-µm diameter tip were inserted 0.5 mm deep into the cerebral cortex. The DC potentials and electroencephalograph (EEG) (monitored to help control the depth of anesthesia) were derived from the potential between each glass capillary electrode and an Ag/AgCl reference placed under the scalp. These signals were first amplified (×10) with a multichannel, high-impedance input preamplifier (NL834, Neurolog System, Digitimer, Welwyn Garden City, UK). With each channel, the AC component in the 1- to 30-Hz window (around 10,000 times overall amplification) provided the EEG, and the DC component (250 times overall amplification) the DC potential. All parameters were continuously digitized, displayed, and stored using a personal computer with A/D converter.

Experimental procedure

CSD induction was started after 30 min of baseline recording. Three series of experiments (n = 4 to 6 for each series) were performed: series I, CSD induction in the middle location, with CSD recording at anterior and posterior sites (Fig. 1); series II, CSD induction in the posterior location, with CSD recording at middle and anterior sites (Fig. 2, top); series III, CSD induction in the anterior location, with CSD recording at middle and posterior sites (Fig. 2, bottom).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Different effectiveness of CSD propagation along the anteroposterior axis of the cerebral cortex in mice, with caudal  rostral propagation (top bar chart) being more effective than in the opposite direction (series II and III). Bar charts represent total integrated areas (TIA) of CSD waves, means ± SE (n = 6); * P < 0.001, paired t-test, comparison between propagating CSD recorded at 2 different sites in each animal;  P < 0.05, unpaired t-test, comparison between series II and III.

Data presentation and analysis

The following variables were measured to assess the efficacy of CSD propagation: average amplitude (A, mV) of all the propagating CSD waves elicited during the 2-h KCl application; number of CSD (N) elicited, with an amplitude >50% of the average amplitude A; and total integrated area of all the SD waves that reached the recording site during the 2-h CSD elicitation period (TIA, mV.min). In series II and III, the rates of CSD propagation were also calculated. In Fig. 1, the polarity of the DC potential was reversed, so that negative shifts of the DC potential (i.e., depolarization) produce an upward deflection. All values in RESULTS are means ± SE. Statistical significance was tested with paired or unpaired t-test for comparisons within or between groups, respectively.

	RESULTS

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Representative CSD propagating from the middle site of elicitation to anterior and posterior regions (series I) are shown in Fig. 1. In this series (n = 4), the number of CSD (N) that reached the recording site with an amplitude >50% of the average amplitude of all CSD was 14.3 ± 2.3 and 9.8 ± 2.4 in the anterior and posterior regions, respectively (P < 0.001, paired t-test), indicating that the propagation of CSD from the middle (parietal) site was not identical along the anteroposterior axis. This was confirmed by the comparison of the TIA, 124.3 ± 8.0 and 61.1 ± 20.4 mV.min in the anterior and posterior regions, respectively (P < 0.05, paired t-test), and the tendency for CSD waves to be broader anteriorly than posteriorly (Fig. 1).

The data from series II and III (Fig. 2) confirmed that CSD propagated much more effectively from posterior to anterior regions than in the opposite direction. The TIAs measured at the middle cortical location were 143 ± 13 and 105 ± 15 mV.min when CSD was elicited posteriorly and anteriorly, respectively (n = 6; no significant difference between these 2 values, P = 0.089). There was only a marked reduction in the TIA measured at the more remote site when CSD was elicited at the anterior site (P < 0.001, paired t-test, comparison between propagating CSD recorded at 2 different sites in each animal). The rate of propagation was also significantly slower from anterior to posterior regions (P < 0.05, unpaired t-test, comparison between series II and III different; Fig. 2). Finally, the fact that, when high KCl was applied to the posterior region, similar or slightly more CSD was recorded at the middle site than when CSD was elicited anteriorly, showed that the asymmetry was due to a different propagation in the two opposite directions, rather than to a reduced susceptibility of the posterior regions to CSD elicitation (Fig. 2).

	DISCUSSION

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Our study demonstrates that CSD propagation in the cerebral cortex of mice is much more effective in the posterior-frontal direction than in the opposite. In contrast, from a study with pentobarbital sodium-anesthetized rats, de Luca and Bure (1977) concluded that there was no difference between the fronto-occipital and occipito-frontal rates of CSD propagation. A number of points may account for this discrepancy. Asymmetric CSD propagation may only occur in mice. Alternatively, several aspects of our experimental procedures might have favored the detection of this asymmetry, including 1) the small size of the mouse cortex, which made that a relatively much larger area of the cortex was explored in our study (5.5 mm along a longitudinal axis of the mouse cortex, vs. a 4-mm region of rat cortex in de Luca and Bure's study); 2) more accurate methods for CSD induction, recording, and analysis; and 3) anesthesia with halothane, which inhibits CSD elicitation (Kitahara et al. 2001), albeit only slightly at the concentration used in our study.

Asymmetric propagation of CSD constitutes a potential pitfall for neurochemical studies of post-CSD changes in mice, as brain tissue samples collected for this purpose are expected to be exposed uniformly to CSD. Occipital sites for CSD induction are clearly optimal for this purpose. If CSD propagation is confirmed to be more effective from occipital to frontal regions in other species, this feature may be relevant to classical migraine because the most frequent aura symptoms (i.e., visual disturbances) originate in the occipital cortex. This predominant occipital origin of auras has been speculatively attributed to K⁺ clearance being less effective in occipital regions where the glia/neurons ratio is the lowest in humans (Lauritzen 1994), but this could also reflect the capacity of CSD originating in the occipital pole to invade a larger area of the cortex. With regards to the cause of asymmetric CSD propagation, it is relevant to mention that gap junctions, a likely contributor to both K⁺ spatial buffering and CSD genesis, are asymmetrical (Zahs 1998), but this explanation would also require a preferential "polar" orientation of these junctions along the anteroposterior axis of the cerebral cortex.