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Optical recording of spreading depression in rat neocortical slices
Brain Research 898 (2001) 288–296 www.elsevier.com / locate / bres
Research report
Optical recording of spreading depression in rat neocortical slices ´ a , Nicole Klapka b , Heiko J. Luhmann b , * Ildiko Vilagi b
a ¨ ¨ Lorand ´ University, Budapest, Hungary Department of Physiology and Neurobiology, Eotvos ¨ ¨ , P.O. Box 101007, D-40001 Dusseldorf , Germany Institute of Neurophysiology, University of Dusseldorf
Accepted 23 January 2001
Abstract A spreading depression (SD) was elicited in adult rat neocortical slices by microdrop application of high potassium and the SD propagation pattern was analyzed by recording simultaneously the extracellular DC potential and the changes in the intrinsic optical signal. The electrical SD with an average peak amplitude of 13.263.4 mV showed a good spatial and temporal correlation with the optical signal. In 79% of the slices, the SD was characterized by an initial increase of light reflectance by 2.361.6%, followed by a reflectance decrease of 0.562.4% and finally a larger and long-lasting increase by 562.4%. In the remaining slices, the SD revealed an initial decrease in light reflectance by 5.861.8% followed by an increase of 1.461.2%. In all slices, the recovery in the DC recording was faster as in the optical signal. The SD preferentially propagated within layers I–IV and could be blocked in most experiments by a vertical incision through upper layers or by local glutamate receptor blockade following microdrop application of kynurenic acid in layers II–III. The SD could be also blocked by bath application of kynurenic acid, MK-801 and octanol, but not by the more specific gap junction blocker carbenoxolone. Our results indicate that the high density of dendritic processes and glutamate receptors in layers II–IV promote the horizontal spread of the SD in these cortical layers and that gap junctions are not required for the propagation of SD in neocortical slices. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Somatosensory cortex; Spreading depression; Propagation; Glutamate antagonist; Gap junction; Cortical layer
1. Introduction The phenomenon of spreading depression (SD), as ˜ [26], is characterized by a initially described by Leao slowly propagating wave of neuronal and glial depolarization that results in a transient depression of synaptic activity (for review see Ref. [31]). Previous studies have demonstrated that SD-like depolarizations may represent the pathophysiological signal promoting the ischemic damage [42,43] and contributing to the gradual enlargement of the infarct area following focal cerebral ischemia [18,33]. During the first 3 h postischemia, each peri-infarct depolarization increases the infarct volume by approximately 20% and glutamate receptor antagonists blocking or *Corresponding author. Tel.: 149-211-811-2616; fax: 149-211-8112706. E-mail address: [email protected] (H.J. Luhmann).
attenuating these depolarizations also reduce the infarct volume (for review see Refs. [8,17,29,39,44]). However, experimental data on the generation and propagation of the SD are controversial. Ionotropic glutamate receptors [18,25,30,41], elevated extracellular potassium concentrations [45], glial cells [23,48], gap junctions [13,24,40] and different cationic inward currents [36] may contribute to the generation and spread of the SD. In order to monitor the SD propagation pattern over a large cortical region, we developed a PC-based optical recording and analyzing system and measured the intrinsic optical signals (IOS) associated with the SD in coronal slices of the adult rat somatosensory cortex in vitro. We were especially interested in the role of the different cortical layers in the process of the SD propagation. Furthermore, the effects of the broad spectrum glutamate antagonist kynurenic acid, the NMDA receptor antagonist MK-801 and the putative gap junction blocking agents
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02196-5
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carbenoxolone and octanol on the initiation and spread of the SD were tested. Parts of these results have been published previously in abstract form [19].
2. Materials and methods Tissue preparation, electrical recording techniques and initiation of SD were similar as described previously [21,22]. In brief, 4–6-week-old male Wistar rats were anesthetized with ether, decapitated and a block of the brain was rapidly removed and stored for 1–2 min in ice-cold, oxygenated artificial cerebrospinal fluid (aCSF). All experiments were conducted in accordance with the national laws for the use of animals in research and approved by the local ethical committee. Coronal slices of 400 mm thickness were cut with a vibratome and preincubated for .90 min in the interface-type recording chamber at 3360.58C and a pH of 7.4 when saturated with 95% O 2 / 5% CO 2 . The chamber was continuously perfused at 0.9 ml / min with aCSF containing 124 mM NaCl, 1.25 mM NaH 2 PO 4 , 1.8 mM MgSO 4 , 1.6 mM CaCl 2 , 3 mM KCl, 26 mM NaHCO 3 and 10 mM glucose. Extracellular recording electrodes were fabricated with a horizontal puller, broken to a tip diameter of 2–3 mm and backfilled with aCSF. An Ag /AgCl metal electrode contacted the aCSF inside the capillaries. Electrode resistances ranged from 2 to 5 MV. Extracellular DC recordings were performed in layers II / III of the primary somatosensory cortex, displayed with a thermo chart recorder (Astro-Med Inc., Rodgau, Germany) and digitized on-line with the TIDA software program (HEKA electronics, Lambrecht, Germany). A single SD episode was elicited by applying a microdrop (|10 nl) of 3 M KCl via a broken pipette on the slice surface either in layers II / III or in layers V/ VI. Following the microdrop application an instantaneous optical signal of 500–800 mm in diameter could be observed. In the same slice, up to three SD waves were elicited at intervals of 20–30 min. The electrical SD was analyzed quantitatively with the TIDA software program by measuring the DC peak amplitude of the second negative component and the duration at half maximal amplitude. For recording of the intrinsic optical signals (IOS), slices were illuminated with a fiber light optical device (Fiber Lite A 3000, Dolan–Jenner Industries Inc., Lawrence, MA) in a 458 angle from above and the reflected light was detected by a 0.5-inch charge-coupled (CCD) color video camera (VC 3031, Euromex, Arnhem, The Netherlands) attached to an upright microscope with variable magnification (Zeiss, Jena, Germany). The changes in light reflectance varied from 213 to 113% during the initial passage of a SD, giving a signal-to-noise ratio of 10:1. The CCD camera was connected to a S-VHS video-tape recorder and video data were captured by a video card (WIN TV, Hauppauge, London, UK) at a rate of
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1 Hz and resolution of 3203240 pixels in RGB color mode. Video data were analyzed by a self-designed program. Each picture was obtained by averaging five images to improve the signal-to-noise ratio. The first picture of a series was regarded as control background and the luminance values were subtracted from all following averaged images and then divided by the source picture. Thus the result shows the relative reflectance changes during the SD. The velocity of SD propagation in different cortical layers was determined by superimposing a protractor on the digitized image of the slice (upper left panel in Fig. 1) and measuring Ddistance /Dtime every 108. For pharmacological experiments, carbenoxolone (100 mM), octanol (2 mM), kynurenic acid (500 mM) or MK-801 (20 mM) were added to the aCSF containing perfusion solution. For local glutamate receptor blockade, a microdrop of 2 mM kynurenic acid (|10 nl) was applied onto the slice surface. All drugs were purchased from Sigma–Aldrich (St. Louis, MO) except for MK-801 (Tocris Cookson Ltd., Bristol, UK). Vertical incisions were performed with a small scalpel after placing the slices in the recording chamber. Values throughout this report are given as mean6S.E.M. The Student’s t-test was used for statistical analyses.
3. Results
3.1. Propagation pattern of spreading depression Experiments were performed on 37 cortical slices prepared from 24 adult male Wistar rats 4–6 weeks of age. The mean amplitude and duration at half maximal amplitude of the KCl-induced SD was 13.263.4 mV and 32.469.3 s (n537), respectively (lower right panel in Fig. 1). The SD could be easily identified in the optical recordings and in most cases the propagation could be observed without any image processing. However, the exact time course and the SD amplitude distribution could be only examined after further digital image processing. Fig. 1 shows a typical example of a SD elicited by KCl drop application to supragranular layers, which propagates uniformly in medial and lateral direction at approximately 3 mm / min. During the first 60 s, the spread in supragranular layers preceded the propagation in infragranular layers. Fig. 1 also illustrates that the onset of the electrical SD coincides with the appearance of the optical signal at the extracellular recording site (open circle in upper row of Fig. 1). An invasion of the SD into the white matter or to subcortical regions could never been observed. As illustrated in Fig. 1, the SD was associated with an initial increase in light reflectance of up to 9% (for details see http: / / www.uni-duesseldorf.de / MedFak / Neurophysiologie /Animation.html). In 34 experiments the change of the IOS during SD was measured close to the electrical recording site in layers II / III. In 27 (79%) of these experiments (Fig. 2), an initial increase of light
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Fig. 1. Digitized pseudocolored images illustrating changes in light reflectance during KCl-induced spreading depression. In this and the following figures the extracellular recording site (s), the KCl application site (d), and the medial (m) and lateral (l) direction is marked in the first pictures. The lower right panel shows the electrical SD following the KCl application at t50 s (↑). Note that the electrical signal starts when the optical SD signal reaches the recording electrode. In the upper left panel a protractor scale for the calculation of the SD propagation velocity was superimposed on the digitized image of the slice.
reflectance of 2.361.6% could be observed 1364.7 s after SD onset. This initial increase was followed by an average decrease of light reflectance by 0.562.4% at 35613.6 s after SD onset. Then the light reflectance increased again to a maximum of 562.4% at 77.9610.2 s after SD onset and gradually decreased to the baseline level within 5–8 min. In the remaining seven experiments (21%), the IOS of the SD showed an initial decrease in light reflectance by
Fig. 2. Simultaneous recording of the extracellular DC potential (thick line) and the intrinsic optical signal (thin line) during the spreading depression. Optical signals were measured every 2 s close to the electrical recording site.
5.861.8% at 24.364.5 s followed by an increase of 1.461.2% at 77.6613.7 s after SD onset. In all 34 experiments, the sudden SD onset could be observed simultaneously in the IOS and the electrical signal (Fig. 2). However, the recovery phase was much faster in the DC signal (2–3 min) as compared to the IOS (.5 min). The layer specificity of the SD spread was further analyzed by applying the KCl microdrop to supra- (n537) or infragranular layers (n517). When KCl was applied to layers II / III, the SD invaded in 62% of the experiments all cortical layers (Fig. 1) and in 38% the SD spread only in supragranular and granular laminae (Fig. 3), indicating that these layers show a propensity for the propagation of the SD. When KCl was applied to layers V/ VI, the SD spread in 47% of the cases initially into upper layers and subsequently propagated horizontally either only in upper layers or in all layers (Fig. 4). In the remaining 53% of the experiments, the SD propagated in horizontal direction simultaneously in infra- and supragranular layers. An exclusive SD propagation in layers V/ VI could never been observed. Besides layer-dependent differences in the SD propagation pattern, we also detected significant differences in the velocity of horizontal spread between upper and lower cortical layers in the same slice on the basis of changes in the IOS. In supragranular layers, the velocity of spread was similar in lateral (3.460.8 mm / min, n535)
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Fig. 3. Example of a spreading depression elicited in layer III (d) and propagating horizontally only in supragranular layers.
and medial (3.661 mm / min, n526) direction. Similar values were obtained in infragranular layers for the propagation in lateral direction (3.762 mm / min, n546), but in medial direction (2.861.3 mm / min, n559) the velocity of spread was significantly (P,0.05) slower as compared to all other values. This slowing in medial direction probably results from the geometry of the neocortical slice preparation. The role of the upper layers in the horizontal propagation of the SD was analyzed further in six slices by producing vertical incisions extending from the pial surface to lower layers. Upon application of a KCl microdrop in supragranular layers medial or lateral to the incision, the SD stopped in nine out of 12 experiments at the site of the incision and did not invade infragranular layers. In these cases the spread towards the intact site was unaffected. In the remaining three experiments the SD circumvented the cut through lower layers, subsequently invaded the supragranular layers and continued to propagate in horizontal
direction (Fig. 5). These experiments confirm the important role of the upper layers in the propagation of the SD.
3.2. Effects of glutamate receptor blockade Since previous studies have demonstrated a participation of ionotropic glutamate receptors in the SD and in periinfarct depolarizations [15,21,25,34,41], we also used the technique of a local glutamate receptor blockade to study the role of the upper layers in the SD spread. In nine slices, which were first tested for showing a prominent SD under control conditions, a microdrop of 2 mM kynurenic acid was applied to the supragranular layers between the extracellular recording site and KCl application site. In five cases, the SD stopped at the kynurenic acid application site and propagated only in the opposite direction (Fig. 6A). This pattern was observed in those slices, which under control conditions showed a SD propagation restricted to
Fig. 4. Example of a spreading depression elicited in layer V and propagating initially into upper layers before spreading in horizontal direction.
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Fig. 5. Vertical incision through layers I–IV (dotted line) delayed SD propagation in the lateral direction until the cut was circumvented through deeper cortical layers. Upper left panel shows photograph of the same slice after Nissl staining.
upper layers as illustrated in Fig. 3. In the remaining four slices the SD circumvented the area of the kynurenic acid application site by invading infragranular layers and subsequently propagated in supra- and infragranular layers in horizontal direction (Fig. 6B). This pattern could be observed in those slices which under control conditions revealed a SD propagating in all cortical layers. Upon
washout of kynurenic acid all nine slices showed a SD as observed under control conditions. In five slices 500 mM kynurenic acid was bath-applied after a SD had been recorded under control conditions in normal aCSF. Kynurenic acid blocked the SD in all cases (Fig. 7) and this effect was reversible after 30 min washout of kynurenic acid. Bath application of the non-competitive
Fig. 6. Effect of glutamate receptor blockade on SD propagation pattern. (A) Local application of 2 mM kynurenic acid to supragranular layers (*) blocks propagation of optical SD and extracellular DC shift. Spread in the untreated part of the slice is not affected. (B) Example of a SD circumventing the kynurenic acid application site. Note delay of electrical SD in lower right panel.
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Fig. 7. Bath application of kynurenic acid blocks propagation of SD. (A) Local KCl application elicits characteristic SD propagating in medial and lateral direction. (B) Same experiment as in A, but after addition of 500 mM kynurenic acid to the bathing solution.
NMDA receptor antagonist MK-801 (20 mM) blocked the SD in four out of four experiments.
3.3. Effects of gap junction blockade Since previous studies have reported a critical role of gap junctions in the propagation of the SD [2,13,24,40], we studied the effects of the putative gap junction blocking agents carbenoxolone (100 mM, n57 slices) and octanol (2 mM, n54) on KCl-induced SDs. After recording a SD under control conditions in aCSF and following a 30-min washing period, we could not detect any obvious effects of carbenoxolone on the properties of the electrically or optically recorded SD (data not shown). In contrast, in all four experiments with octanol, the SD was completely blocked.
4. Discussion
4.1. Optical recording of spreading depression A KCl-induced SD could be easily and reliably monitored in adult rat somatosensory cortical slices by recording of IOS. Illumination and optical recordings from above in an interface-type recording chamber revealed in the majority of the slices an early increase in light reflectance during the initial passage of the SD. This reflectance increase associated with the sudden onset of the SD has been observed previously in rat hippocampal slices [2,37] and rat sensorimotor cortex in vivo [51], and most probably results from an increase in light scattering. The mechanisms underlying this IOS are not completely understood. Since alterations in blood volume and blood flow
[10] can be excluded in brain slice preparations, swelling of neurons and glia accompanied by a shrinkage of the extracellular space volume has been suggested as the process producing changes in light scattering [3,4,9,16,20]. However, Buchheim et al. [6] recently demonstrated that the change in the IOS does not correlate with the extracellular space volume, suggesting that other mechanisms, like alterations in membrane fluidity, intracellular energy status or mitochondrial swelling may contribute to the IOS (for ¨ review see Refs. [1,50]). Muller and Somjen [37] have demonstrated that the increased light scattering is not related to cell swelling and suggested that swelling of mitochondria may be involved. Support for this hypothesis comes from a recent study using R123 fluorescence to monitor mitochondrial membrane potential changes during the SD [5]. Although additional processes such as chromatin clumping, nucleolar condensation, cytoskeletal and microtubule breakdown, etc., may be involved (for discussion see Ref. [37]), mitochondrial responses clearly contribute to the increase in light scattering and reflectance during the initial phase of the SD [5]. In 21% of our experiments the SD was accompanied by an initial decrease in light reflectance, indicating that the direction of change in the IOS may vary under the same experimental conditions from slice to slice. A decrease in light reflectance can be attributed to a reduction in light scattering, which is thought to be linked to cell swelling [16,20,37]. Relative decreases in light reflectance could be also observed in slices showing an initial increase in light reflectance, suggesting that cell swelling is only one of several processes occurring during an SD. Besides intrinsic optical properties of the tissue, artifacts associated with tissue surface scattering also influence the magnitude and the polarity of the signal. Kreisman et al. [20] demon-
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strated that osmotically induced changes in light transmittance are opposite in direction in submerged slices as compared to slices studied in the gas–liquid interface-type chamber. Support for the hypothesis that light scattering artifacts at the tissue surface is the primary cause for these inconsistent observations comes from a recent study by Tao [49], who used a photon-counting fiberoptic system to exclude surface artifacts. In this study, measurements of light scattering within the tissue gave consistent and reproducible results. However, even under this condition small differences in slice thickness may influence the IOS.
4.2. Propagation of the SD and role of glutamate receptors Using the combination of optical and extracellular DC recording techniques, we demonstrate that a KCl-induced SD propagates preferentially in upper cortical layers. A vertical incision or a local glutamate receptor blockade in upper layers prevented the horizontal spread of the SD in the majority of the experiments. Our data are in good agreement with previous observations in the cerebral ˜ and Morison [28] obcortex of different species. Leao served in rabbit cortex that acute transection of the upper layers blocked the SD and concluded that ‘‘The spreading of the phenomenon was found to be independent of . . . at least the three lower layers of the grey matter.’’ In 1951, ˜ [27] reported that the SD developed in the superficial Leao layers first and then propagated into lower layers. Similar results were later obtained in the cat [12] and in the rat [35]. Several factors may contribute to the layer-specific propagation pattern in the cortex. Herreras and Somjen [14,15] have already shown in the hippocampus of anesthetized rats that an SD differs in its properties and velocity of propagation between stratum radiatum and stratum pyramidale and concluded that the layer containing the apical dendrites is more prone to SD as compared to the layer including the pyramidal cell somata. This result is supported by the observation of Andrew et al. [3] that bath-applied NMDA or kainate induced an IOS, particularly in the dendritic region of the hippocampal CA1 area. This signal is mediated by dendritic NMDA and AMPA receptors, which upon massive activation cause sodium influx, followed by chloride and water influx [3]. Therefore the response is predominantly restricted to the dendritic region. Quantitative receptor autoradiography in rat parietal cortex has shown that the binding site density of NMDA and AMPA receptors is highest in layers II–IV (see Fig. 3 in Ref. [52]). Furthermore, the upper neocortical layers also show a high packing density of dendritic processes, since the apical dendrites of pyramidal cells located in infragranular layers as well as the dendrites originating from layers II–IV neurons reach into layers I–III. The high density of apical dendritic processes and of glutamate receptors in upper layers will not only promote
the horizontal propagation of the SD, but will also produce a strong IOS in these layers [3].
4.3. Role of gap junctions Previous studies in the chicken retina and the in vivo and in vitro rat hippocampus have shown that gap junctions are involved in the initiation and propagation of the SD [2,13,24,40]. In contrast to these earlier reports, in our experiments the more specific gap junction blocker carbenoxolone had no effects on properties of the SD. However, octanol consistently blocked the SD. Although the adult rat neocortex expresses an abundance of connexins (Cx 32 and Cx 43), which form gap junctions [38], this would not explain the insensitivity of the neocortical SD to carbenoxolone. We favor the hypothesis that octanol may have some nonspecific effects which will cause a blockade of the cortical SD. Octanol already induces at one hundredth of the concentration used in the present study a decrease in the NMDA channel mean open time and a reduction in the frequency of NMDA channel openings [32]. Since NMDA receptor blockade inhibits neocortical SD [18,21,30,41] (present study), the effect of octanol on the SD may result from its inhibitory action on NMDA receptor function. Another argument also speaks against a major contribution of gap junctions to the SD. Cortical SD is accompanied by a decrease in intracellular pH [7,11] and intracellular acidification closes gap junction channels [46,47]. Therefore it can be assumed that cortical SD will rather be associated with a closure than with a coupling of gap junctions. In conclusion, our experiments indicate that a KClinduced SD (i) preferentially propagates within upper neocortical layers, (ii) depends on NMDA receptor activation and (iii) does not require gap junction coupling.
Acknowledgements This study was supported by DFG Grant SFB 194-B4 to H.J.L. and the Neuroscience Graduate Program at the ¨ Heinrich-Heine-University, Dusseldorf.
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counting fiber optic system, J. Neurosci. Methods 101 (2000) 19–29. [50] A. Villringer, B. Chance, Non-invasive optical spectroscopy and imaging of human brain function, Trends Neurosci. 20 (1997) 435–442. [51] R.S. Yoon, P.W. Tsang, F.A. Lenz, H.C. Kwan, Characterization of cortical spreading depression by imaging of intrinsic optical signals, Neuroreport 7 (1996) 2671–2674. ¨ A. Schleicher, H.J. Luhmann, Characterization of [52] K. Zilles, M. Qu, neuronal migration disorders in neocortical structures: quantitative receptor autoradiography of ionotropic glutamate, GABAA , and GABA B receptors, Eur. J. Neurosci. 10 (1998) 3095–3106.
Research report
Optical recording of spreading depression in rat neocortical slices ´ a , Nicole Klapka b , Heiko J. Luhmann b , * Ildiko Vilagi b
a ¨ ¨ Lorand ´ University, Budapest, Hungary Department of Physiology and Neurobiology, Eotvos ¨ ¨ , P.O. Box 101007, D-40001 Dusseldorf , Germany Institute of Neurophysiology, University of Dusseldorf
Accepted 23 January 2001
Abstract A spreading depression (SD) was elicited in adult rat neocortical slices by microdrop application of high potassium and the SD propagation pattern was analyzed by recording simultaneously the extracellular DC potential and the changes in the intrinsic optical signal. The electrical SD with an average peak amplitude of 13.263.4 mV showed a good spatial and temporal correlation with the optical signal. In 79% of the slices, the SD was characterized by an initial increase of light reflectance by 2.361.6%, followed by a reflectance decrease of 0.562.4% and finally a larger and long-lasting increase by 562.4%. In the remaining slices, the SD revealed an initial decrease in light reflectance by 5.861.8% followed by an increase of 1.461.2%. In all slices, the recovery in the DC recording was faster as in the optical signal. The SD preferentially propagated within layers I–IV and could be blocked in most experiments by a vertical incision through upper layers or by local glutamate receptor blockade following microdrop application of kynurenic acid in layers II–III. The SD could be also blocked by bath application of kynurenic acid, MK-801 and octanol, but not by the more specific gap junction blocker carbenoxolone. Our results indicate that the high density of dendritic processes and glutamate receptors in layers II–IV promote the horizontal spread of the SD in these cortical layers and that gap junctions are not required for the propagation of SD in neocortical slices. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Somatosensory cortex; Spreading depression; Propagation; Glutamate antagonist; Gap junction; Cortical layer
1. Introduction The phenomenon of spreading depression (SD), as ˜ [26], is characterized by a initially described by Leao slowly propagating wave of neuronal and glial depolarization that results in a transient depression of synaptic activity (for review see Ref. [31]). Previous studies have demonstrated that SD-like depolarizations may represent the pathophysiological signal promoting the ischemic damage [42,43] and contributing to the gradual enlargement of the infarct area following focal cerebral ischemia [18,33]. During the first 3 h postischemia, each peri-infarct depolarization increases the infarct volume by approximately 20% and glutamate receptor antagonists blocking or *Corresponding author. Tel.: 149-211-811-2616; fax: 149-211-8112706. E-mail address: [email protected] (H.J. Luhmann).
attenuating these depolarizations also reduce the infarct volume (for review see Refs. [8,17,29,39,44]). However, experimental data on the generation and propagation of the SD are controversial. Ionotropic glutamate receptors [18,25,30,41], elevated extracellular potassium concentrations [45], glial cells [23,48], gap junctions [13,24,40] and different cationic inward currents [36] may contribute to the generation and spread of the SD. In order to monitor the SD propagation pattern over a large cortical region, we developed a PC-based optical recording and analyzing system and measured the intrinsic optical signals (IOS) associated with the SD in coronal slices of the adult rat somatosensory cortex in vitro. We were especially interested in the role of the different cortical layers in the process of the SD propagation. Furthermore, the effects of the broad spectrum glutamate antagonist kynurenic acid, the NMDA receptor antagonist MK-801 and the putative gap junction blocking agents
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02196-5
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carbenoxolone and octanol on the initiation and spread of the SD were tested. Parts of these results have been published previously in abstract form [19].
2. Materials and methods Tissue preparation, electrical recording techniques and initiation of SD were similar as described previously [21,22]. In brief, 4–6-week-old male Wistar rats were anesthetized with ether, decapitated and a block of the brain was rapidly removed and stored for 1–2 min in ice-cold, oxygenated artificial cerebrospinal fluid (aCSF). All experiments were conducted in accordance with the national laws for the use of animals in research and approved by the local ethical committee. Coronal slices of 400 mm thickness were cut with a vibratome and preincubated for .90 min in the interface-type recording chamber at 3360.58C and a pH of 7.4 when saturated with 95% O 2 / 5% CO 2 . The chamber was continuously perfused at 0.9 ml / min with aCSF containing 124 mM NaCl, 1.25 mM NaH 2 PO 4 , 1.8 mM MgSO 4 , 1.6 mM CaCl 2 , 3 mM KCl, 26 mM NaHCO 3 and 10 mM glucose. Extracellular recording electrodes were fabricated with a horizontal puller, broken to a tip diameter of 2–3 mm and backfilled with aCSF. An Ag /AgCl metal electrode contacted the aCSF inside the capillaries. Electrode resistances ranged from 2 to 5 MV. Extracellular DC recordings were performed in layers II / III of the primary somatosensory cortex, displayed with a thermo chart recorder (Astro-Med Inc., Rodgau, Germany) and digitized on-line with the TIDA software program (HEKA electronics, Lambrecht, Germany). A single SD episode was elicited by applying a microdrop (|10 nl) of 3 M KCl via a broken pipette on the slice surface either in layers II / III or in layers V/ VI. Following the microdrop application an instantaneous optical signal of 500–800 mm in diameter could be observed. In the same slice, up to three SD waves were elicited at intervals of 20–30 min. The electrical SD was analyzed quantitatively with the TIDA software program by measuring the DC peak amplitude of the second negative component and the duration at half maximal amplitude. For recording of the intrinsic optical signals (IOS), slices were illuminated with a fiber light optical device (Fiber Lite A 3000, Dolan–Jenner Industries Inc., Lawrence, MA) in a 458 angle from above and the reflected light was detected by a 0.5-inch charge-coupled (CCD) color video camera (VC 3031, Euromex, Arnhem, The Netherlands) attached to an upright microscope with variable magnification (Zeiss, Jena, Germany). The changes in light reflectance varied from 213 to 113% during the initial passage of a SD, giving a signal-to-noise ratio of 10:1. The CCD camera was connected to a S-VHS video-tape recorder and video data were captured by a video card (WIN TV, Hauppauge, London, UK) at a rate of
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1 Hz and resolution of 3203240 pixels in RGB color mode. Video data were analyzed by a self-designed program. Each picture was obtained by averaging five images to improve the signal-to-noise ratio. The first picture of a series was regarded as control background and the luminance values were subtracted from all following averaged images and then divided by the source picture. Thus the result shows the relative reflectance changes during the SD. The velocity of SD propagation in different cortical layers was determined by superimposing a protractor on the digitized image of the slice (upper left panel in Fig. 1) and measuring Ddistance /Dtime every 108. For pharmacological experiments, carbenoxolone (100 mM), octanol (2 mM), kynurenic acid (500 mM) or MK-801 (20 mM) were added to the aCSF containing perfusion solution. For local glutamate receptor blockade, a microdrop of 2 mM kynurenic acid (|10 nl) was applied onto the slice surface. All drugs were purchased from Sigma–Aldrich (St. Louis, MO) except for MK-801 (Tocris Cookson Ltd., Bristol, UK). Vertical incisions were performed with a small scalpel after placing the slices in the recording chamber. Values throughout this report are given as mean6S.E.M. The Student’s t-test was used for statistical analyses.
3. Results
3.1. Propagation pattern of spreading depression Experiments were performed on 37 cortical slices prepared from 24 adult male Wistar rats 4–6 weeks of age. The mean amplitude and duration at half maximal amplitude of the KCl-induced SD was 13.263.4 mV and 32.469.3 s (n537), respectively (lower right panel in Fig. 1). The SD could be easily identified in the optical recordings and in most cases the propagation could be observed without any image processing. However, the exact time course and the SD amplitude distribution could be only examined after further digital image processing. Fig. 1 shows a typical example of a SD elicited by KCl drop application to supragranular layers, which propagates uniformly in medial and lateral direction at approximately 3 mm / min. During the first 60 s, the spread in supragranular layers preceded the propagation in infragranular layers. Fig. 1 also illustrates that the onset of the electrical SD coincides with the appearance of the optical signal at the extracellular recording site (open circle in upper row of Fig. 1). An invasion of the SD into the white matter or to subcortical regions could never been observed. As illustrated in Fig. 1, the SD was associated with an initial increase in light reflectance of up to 9% (for details see http: / / www.uni-duesseldorf.de / MedFak / Neurophysiologie /Animation.html). In 34 experiments the change of the IOS during SD was measured close to the electrical recording site in layers II / III. In 27 (79%) of these experiments (Fig. 2), an initial increase of light
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Fig. 1. Digitized pseudocolored images illustrating changes in light reflectance during KCl-induced spreading depression. In this and the following figures the extracellular recording site (s), the KCl application site (d), and the medial (m) and lateral (l) direction is marked in the first pictures. The lower right panel shows the electrical SD following the KCl application at t50 s (↑). Note that the electrical signal starts when the optical SD signal reaches the recording electrode. In the upper left panel a protractor scale for the calculation of the SD propagation velocity was superimposed on the digitized image of the slice.
reflectance of 2.361.6% could be observed 1364.7 s after SD onset. This initial increase was followed by an average decrease of light reflectance by 0.562.4% at 35613.6 s after SD onset. Then the light reflectance increased again to a maximum of 562.4% at 77.9610.2 s after SD onset and gradually decreased to the baseline level within 5–8 min. In the remaining seven experiments (21%), the IOS of the SD showed an initial decrease in light reflectance by
Fig. 2. Simultaneous recording of the extracellular DC potential (thick line) and the intrinsic optical signal (thin line) during the spreading depression. Optical signals were measured every 2 s close to the electrical recording site.
5.861.8% at 24.364.5 s followed by an increase of 1.461.2% at 77.6613.7 s after SD onset. In all 34 experiments, the sudden SD onset could be observed simultaneously in the IOS and the electrical signal (Fig. 2). However, the recovery phase was much faster in the DC signal (2–3 min) as compared to the IOS (.5 min). The layer specificity of the SD spread was further analyzed by applying the KCl microdrop to supra- (n537) or infragranular layers (n517). When KCl was applied to layers II / III, the SD invaded in 62% of the experiments all cortical layers (Fig. 1) and in 38% the SD spread only in supragranular and granular laminae (Fig. 3), indicating that these layers show a propensity for the propagation of the SD. When KCl was applied to layers V/ VI, the SD spread in 47% of the cases initially into upper layers and subsequently propagated horizontally either only in upper layers or in all layers (Fig. 4). In the remaining 53% of the experiments, the SD propagated in horizontal direction simultaneously in infra- and supragranular layers. An exclusive SD propagation in layers V/ VI could never been observed. Besides layer-dependent differences in the SD propagation pattern, we also detected significant differences in the velocity of horizontal spread between upper and lower cortical layers in the same slice on the basis of changes in the IOS. In supragranular layers, the velocity of spread was similar in lateral (3.460.8 mm / min, n535)
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Fig. 3. Example of a spreading depression elicited in layer III (d) and propagating horizontally only in supragranular layers.
and medial (3.661 mm / min, n526) direction. Similar values were obtained in infragranular layers for the propagation in lateral direction (3.762 mm / min, n546), but in medial direction (2.861.3 mm / min, n559) the velocity of spread was significantly (P,0.05) slower as compared to all other values. This slowing in medial direction probably results from the geometry of the neocortical slice preparation. The role of the upper layers in the horizontal propagation of the SD was analyzed further in six slices by producing vertical incisions extending from the pial surface to lower layers. Upon application of a KCl microdrop in supragranular layers medial or lateral to the incision, the SD stopped in nine out of 12 experiments at the site of the incision and did not invade infragranular layers. In these cases the spread towards the intact site was unaffected. In the remaining three experiments the SD circumvented the cut through lower layers, subsequently invaded the supragranular layers and continued to propagate in horizontal
direction (Fig. 5). These experiments confirm the important role of the upper layers in the propagation of the SD.
3.2. Effects of glutamate receptor blockade Since previous studies have demonstrated a participation of ionotropic glutamate receptors in the SD and in periinfarct depolarizations [15,21,25,34,41], we also used the technique of a local glutamate receptor blockade to study the role of the upper layers in the SD spread. In nine slices, which were first tested for showing a prominent SD under control conditions, a microdrop of 2 mM kynurenic acid was applied to the supragranular layers between the extracellular recording site and KCl application site. In five cases, the SD stopped at the kynurenic acid application site and propagated only in the opposite direction (Fig. 6A). This pattern was observed in those slices, which under control conditions showed a SD propagation restricted to
Fig. 4. Example of a spreading depression elicited in layer V and propagating initially into upper layers before spreading in horizontal direction.
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Fig. 5. Vertical incision through layers I–IV (dotted line) delayed SD propagation in the lateral direction until the cut was circumvented through deeper cortical layers. Upper left panel shows photograph of the same slice after Nissl staining.
upper layers as illustrated in Fig. 3. In the remaining four slices the SD circumvented the area of the kynurenic acid application site by invading infragranular layers and subsequently propagated in supra- and infragranular layers in horizontal direction (Fig. 6B). This pattern could be observed in those slices which under control conditions revealed a SD propagating in all cortical layers. Upon
washout of kynurenic acid all nine slices showed a SD as observed under control conditions. In five slices 500 mM kynurenic acid was bath-applied after a SD had been recorded under control conditions in normal aCSF. Kynurenic acid blocked the SD in all cases (Fig. 7) and this effect was reversible after 30 min washout of kynurenic acid. Bath application of the non-competitive
Fig. 6. Effect of glutamate receptor blockade on SD propagation pattern. (A) Local application of 2 mM kynurenic acid to supragranular layers (*) blocks propagation of optical SD and extracellular DC shift. Spread in the untreated part of the slice is not affected. (B) Example of a SD circumventing the kynurenic acid application site. Note delay of electrical SD in lower right panel.
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Fig. 7. Bath application of kynurenic acid blocks propagation of SD. (A) Local KCl application elicits characteristic SD propagating in medial and lateral direction. (B) Same experiment as in A, but after addition of 500 mM kynurenic acid to the bathing solution.
NMDA receptor antagonist MK-801 (20 mM) blocked the SD in four out of four experiments.
3.3. Effects of gap junction blockade Since previous studies have reported a critical role of gap junctions in the propagation of the SD [2,13,24,40], we studied the effects of the putative gap junction blocking agents carbenoxolone (100 mM, n57 slices) and octanol (2 mM, n54) on KCl-induced SDs. After recording a SD under control conditions in aCSF and following a 30-min washing period, we could not detect any obvious effects of carbenoxolone on the properties of the electrically or optically recorded SD (data not shown). In contrast, in all four experiments with octanol, the SD was completely blocked.
4. Discussion
4.1. Optical recording of spreading depression A KCl-induced SD could be easily and reliably monitored in adult rat somatosensory cortical slices by recording of IOS. Illumination and optical recordings from above in an interface-type recording chamber revealed in the majority of the slices an early increase in light reflectance during the initial passage of the SD. This reflectance increase associated with the sudden onset of the SD has been observed previously in rat hippocampal slices [2,37] and rat sensorimotor cortex in vivo [51], and most probably results from an increase in light scattering. The mechanisms underlying this IOS are not completely understood. Since alterations in blood volume and blood flow
[10] can be excluded in brain slice preparations, swelling of neurons and glia accompanied by a shrinkage of the extracellular space volume has been suggested as the process producing changes in light scattering [3,4,9,16,20]. However, Buchheim et al. [6] recently demonstrated that the change in the IOS does not correlate with the extracellular space volume, suggesting that other mechanisms, like alterations in membrane fluidity, intracellular energy status or mitochondrial swelling may contribute to the IOS (for ¨ review see Refs. [1,50]). Muller and Somjen [37] have demonstrated that the increased light scattering is not related to cell swelling and suggested that swelling of mitochondria may be involved. Support for this hypothesis comes from a recent study using R123 fluorescence to monitor mitochondrial membrane potential changes during the SD [5]. Although additional processes such as chromatin clumping, nucleolar condensation, cytoskeletal and microtubule breakdown, etc., may be involved (for discussion see Ref. [37]), mitochondrial responses clearly contribute to the increase in light scattering and reflectance during the initial phase of the SD [5]. In 21% of our experiments the SD was accompanied by an initial decrease in light reflectance, indicating that the direction of change in the IOS may vary under the same experimental conditions from slice to slice. A decrease in light reflectance can be attributed to a reduction in light scattering, which is thought to be linked to cell swelling [16,20,37]. Relative decreases in light reflectance could be also observed in slices showing an initial increase in light reflectance, suggesting that cell swelling is only one of several processes occurring during an SD. Besides intrinsic optical properties of the tissue, artifacts associated with tissue surface scattering also influence the magnitude and the polarity of the signal. Kreisman et al. [20] demon-
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strated that osmotically induced changes in light transmittance are opposite in direction in submerged slices as compared to slices studied in the gas–liquid interface-type chamber. Support for the hypothesis that light scattering artifacts at the tissue surface is the primary cause for these inconsistent observations comes from a recent study by Tao [49], who used a photon-counting fiberoptic system to exclude surface artifacts. In this study, measurements of light scattering within the tissue gave consistent and reproducible results. However, even under this condition small differences in slice thickness may influence the IOS.
4.2. Propagation of the SD and role of glutamate receptors Using the combination of optical and extracellular DC recording techniques, we demonstrate that a KCl-induced SD propagates preferentially in upper cortical layers. A vertical incision or a local glutamate receptor blockade in upper layers prevented the horizontal spread of the SD in the majority of the experiments. Our data are in good agreement with previous observations in the cerebral ˜ and Morison [28] obcortex of different species. Leao served in rabbit cortex that acute transection of the upper layers blocked the SD and concluded that ‘‘The spreading of the phenomenon was found to be independent of . . . at least the three lower layers of the grey matter.’’ In 1951, ˜ [27] reported that the SD developed in the superficial Leao layers first and then propagated into lower layers. Similar results were later obtained in the cat [12] and in the rat [35]. Several factors may contribute to the layer-specific propagation pattern in the cortex. Herreras and Somjen [14,15] have already shown in the hippocampus of anesthetized rats that an SD differs in its properties and velocity of propagation between stratum radiatum and stratum pyramidale and concluded that the layer containing the apical dendrites is more prone to SD as compared to the layer including the pyramidal cell somata. This result is supported by the observation of Andrew et al. [3] that bath-applied NMDA or kainate induced an IOS, particularly in the dendritic region of the hippocampal CA1 area. This signal is mediated by dendritic NMDA and AMPA receptors, which upon massive activation cause sodium influx, followed by chloride and water influx [3]. Therefore the response is predominantly restricted to the dendritic region. Quantitative receptor autoradiography in rat parietal cortex has shown that the binding site density of NMDA and AMPA receptors is highest in layers II–IV (see Fig. 3 in Ref. [52]). Furthermore, the upper neocortical layers also show a high packing density of dendritic processes, since the apical dendrites of pyramidal cells located in infragranular layers as well as the dendrites originating from layers II–IV neurons reach into layers I–III. The high density of apical dendritic processes and of glutamate receptors in upper layers will not only promote
the horizontal propagation of the SD, but will also produce a strong IOS in these layers [3].
4.3. Role of gap junctions Previous studies in the chicken retina and the in vivo and in vitro rat hippocampus have shown that gap junctions are involved in the initiation and propagation of the SD [2,13,24,40]. In contrast to these earlier reports, in our experiments the more specific gap junction blocker carbenoxolone had no effects on properties of the SD. However, octanol consistently blocked the SD. Although the adult rat neocortex expresses an abundance of connexins (Cx 32 and Cx 43), which form gap junctions [38], this would not explain the insensitivity of the neocortical SD to carbenoxolone. We favor the hypothesis that octanol may have some nonspecific effects which will cause a blockade of the cortical SD. Octanol already induces at one hundredth of the concentration used in the present study a decrease in the NMDA channel mean open time and a reduction in the frequency of NMDA channel openings [32]. Since NMDA receptor blockade inhibits neocortical SD [18,21,30,41] (present study), the effect of octanol on the SD may result from its inhibitory action on NMDA receptor function. Another argument also speaks against a major contribution of gap junctions to the SD. Cortical SD is accompanied by a decrease in intracellular pH [7,11] and intracellular acidification closes gap junction channels [46,47]. Therefore it can be assumed that cortical SD will rather be associated with a closure than with a coupling of gap junctions. In conclusion, our experiments indicate that a KClinduced SD (i) preferentially propagates within upper neocortical layers, (ii) depends on NMDA receptor activation and (iii) does not require gap junction coupling.
Acknowledgements This study was supported by DFG Grant SFB 194-B4 to H.J.L. and the Neuroscience Graduate Program at the ¨ Heinrich-Heine-University, Dusseldorf.
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