The role of extracellular ionic changes in upregulating the mRNA for glial fibrillary acidic protein following spreading depression

BRAIN RESEARCH ELSEVIER

Brain Research 674 (1995) 314-328

Research report

The role of extracellular ionic changes in upregulating the mRNA for glial fibrillary acidic protein following spreading depression D a n i e l J. Bonthius

a,b,l

Eric W. L o t h m a n c, Oswald Steward a,.

a Department of Neuroscience, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA b Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA c Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA Accepted 13 December 1994

Abstract

While spreading depression has been shown to be a powerful stimulus in upregulating glial fibrillary acidic protein (GFAP) mRNA expression, the specific physiological signal underlying the upregulation is unknown. During spreading depression, extracellular ionic concentrations are altered markedly. The present study evaluates the role of these changes in extracellular ionic concentrations as potential signals influencing GFAP mRNA expression. Gel foam pledgets saturated with artificial cerebrospinal fluid (CSF) solutions in which [Na+], [Ca2+], [K +] and [H +] were altered one at a time to match concentrations seen in spreading depression were applied to exposed parietal cortex for one hour. Dot blot and in situ hybridization techniques were used to evaluate GFAP mRNA levels. We found that CSF containing 60 mM KC1 produced a dramatic upregulation of GFAP mRNA levels throughout the cerebral cortex of the ipsilateral hemisphere without causing detectable tissue damage. The pattern and time course of the change were similar to those following application of 3 M KC1. Alteration of other ionic species did not affect GFAP mRNA levels. However, the upregulation of GFAP mRNA was not likely due directly to the increased [K+], but rather to the spreading depression that the elevated [K +] induced. This was demonstrated by the finding that the upregulation in GFAP mRNA induced by the potassium exposure was totally blocked by prior administration of MK-801, an NMDA antagonist that blocks spreading depression. These results demonstrate that an upregulation in GFAP mRNA can occur in the absence of degeneration debris and that the initiating events can be related to physiological changes, but that changes in extracellular ionic concentrations are not the likely molecular signals underlying the upregulation.

Keywords: Glial fibrillary acidic protein; mRNA; Spreading depression; Potassium; MK-801

1. Introduction

Following a variety of injuries to the central nervous system, astrocytes are transformed from a normal to a reactive state [3,33,35,43]. These reactive astrocytes then play important roles in the response of the CNS to the injury, including scar formation [39] and removal of degenerating synaptic terminals [35]. The histological hallmarks of this transformation are hypertrophy and hyperplasia of the involved astrocytes [16], while

* Corresponding author. Department of Neuroscience, University of Virginia Medical Center, Box # 5148, MR4 Annex, Lane Rd, Ext., Charlottesville, VA 22908, USA. Fax: (1) (804) 982 4380. 1 Current Address: Departments of Neurology and Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa 52242 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 0 3 5 - 6

the biochemical hallmark is an increased astrocytic content of glial fibrillary acidic protein (GFAP) [3,46], a cytoskeletal protein unique within the CNS to astrocytes. Until recently, degenerating tissue has been thought to be the primary signal in triggering the astrocytic response [3,33]. It has recently been shown, however, that other types of signals, such as abnormal electrochemical activity, may also influence G F A P gene expression [6,44]. One event that appears to be a powerful stimulus is spreading depression [5,25]. Spreading depression is a cessation of electrical activity which begins as a massive depolarization of cortical neurons and glia, leading to large increases in extracellular potassium concentration, release of neurotransmitters, and a large negative shift in DC potential [27]. Once initiated, spreading depression propogates slowly across

D.J. Bonthius et al./ Brain Research 674 (1995) 314-328

the cortex of the involved hemisphere. The classical and most frequently used method of inducing spreading depression is application of highly concentrated (3 M) KC1 solutions to the cortical surface. Evidence that spreading depression may influence glial cell gene expression was provided by the finding that application of 3 M KC1 to the cortical surface leads to large and transient increases in GFAP mRNA content [5] and GFA protein immunostaining [25] throughout the ipsilateral cortex. The specific molecular signal controlling the upregulation of GFAP mRNA levels following spreading depression is unknown. However, one of the strongest candidates for the molecular signal is interstitial potassium concentration. During spreading depression, potassium is released from depolarizing neurons and glia, and [K+]e rises transiently from its baseline 3 mM to 60 mM [21,24]. Astrocytes play a fundamental role in clearing this excess interstitial potassium by both passive [19,36] and active [50] mechanisms. Furthermore, changes in extracellular potassium concentration can modulate astrocytic proliferation and metabolism under normal and pathologic conditions [38]. Therefore, the rise in [K+]e during spreading depression may play a key role in influencing GFAP gene expression in astrocytes. Interstitial concentrations of other ions also change markedly during spreading depression. For example, [Na+]e fails from 130 mM to 70 mM; [Ca2+]e falls from 1.5 mM to 0.15 mM, and pH falls from 7.4 to 6.9 [21,26]. The principal goal of this study was to test the hypothesis that one of these changes in ionic concentration may serve as a signal regulating GFAP gene expression. We used dot blot and in situ hybridization techniques to evaluate GFAP mRNA levels after application to the parietal cortex of artificial CSF solutions composed of various ionic concentrations, pH and tonicity. Our results revealed that the elevation of [K+]e is the only ionic change that alters GFAP mRNA levels. However, additional experiments indicated that the upregulation of GFAP mRNA is not due directly to the increase in [K+]~, but is rather due to the spreading depression that the potassium treatment induces. This was demonstrated by the finding that the upregulation in GFAP mRNA induced by the potassium exposure is totally blocked by administration of MK-801, an NMDA antagonist that blocks spreading depression. A problem in interpreting the results of previous studies involving spreading depression and GFAP gene expression is that application to the cortex of the highly concentrated (3 M) KCI solutions used to induce spreading depression also induce substantial amounts of tissue injury and necrosis at the site of application and in adjacent tissue [5,22,25]. Focal cortical mechanical injury can alter GFAP gene expression across the ipsilateral cortex and far from the injury site [14,20].

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Thus, it is difficult to dissociate the relative contributions of spreading depression and tissue injury in the response observed following cortical application of highly-concentrated KC1 solutions A second goal of this study was to examine the effect of spreading depression alone - in the absence of detectable tissue injury - on GFAP gene expression. We found that spreading depression, without detectable tissue damage, could be induced by application to the cortex of an artificial CSF containing 60 mM KCI and that spreading depression elicited in this way induces a large transient increase in GFAP mRNA content across the entire ipsilateral neocortex.

2. Materials and methods 2.1. Subjects

A total of 70 adult male Sprague-Dawley rats (225300 g) were used in this study. The animals were anesthetized with pentobarbital (50 m g/ kg i.p.) and were placed in a stereotaxic frame. A 2 cm longitudinal incision was made in the skin exposing the surface of the skull. An oval craniotomy (3 mm x 4 mm) was made through the right parietal bone, and the bone flap was removed. Under a dissecting microscope, the exposed dura was incised and retracted with a hypodermic needle, creating a small (approximately 1 mm diameter) dural window. Great care was exerted not to damage the underlying cortex. The dural opening was positioned over a region devoid of major blood vessels. Thus, its position varied slightly from animal to animal. In general, the dural opening was located 2.5 mm lateral to the sagittal suture and 1-2 mm posterior to Bregma [37]. Gel foam pledgets saturated with artificial CSF of various ionic concentrations were applied to the exposed parietal cortex for 60 min. The baseline composition of the artificial CSF was identical to that described by Westbrook and Lothman [51] and consisted of 127 mM NaCI, 2 mM KC1, 1.5 mM MgSO 4, 1.5 mM CaCla, 25.7 mM NaHCO3, 1.125 mM KH2PO 4 and 10 mM D-glucose, pH 7.4. The pledgets were then removed, the exposed cortex was irrigated with artificial CSF, the skin incision was closed and the animals were allowed to recover. Within two h of administering the anesthesia, the animals displayed normal drinking, feeding and grooming behavior. 2.2. In situ hybridization

To induce spreading depression with minimal tissue damage, the baseline artificial CSF was altered to contain 60 mM KCI. This concentration of KC1 was chosen because it approximates the concentration to which potassium rises during spreading depression

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[21,24] and because concentrations of KCI near 60 m M have been shown to be capable of inducing spreading depression [8]. Animals were killed 1, 2, 4 and 8 days following exposure of the cortex to the artificial CSF. Two animals were included at each timepoint. Four control animals were p r e p a r e d and were killed 4 days after exposure to baseline artifical CSF ([KC1] = 2 mM) (n = 2) or highly concentrated KC1 ([KC1] = 3 M) (n = 2). The animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer while deeply anesthetized with sodium pentobarbital. The brains were removed and stored in fixative overnight. They were then immersed in 30% sucrose in 4% paraformald e h y d e / 0 . 1 M phosphate buffer ( p H 7.4) for 24 h, mounted on the chuck of a cryostat, and frozen by immersion in dry ice. The forebrains were sectioned at 2 0 / x m in the coronal plane using a cryostat. Collection of sections began approximately 1.5 m m anterior to bregma and ended approximately 4 m m posterior to bregma. Every tenth section was thaw-mounted onto a polylysine-coated microscope slide. A total of 25 sections were collected from each animal. The slides with affixed sections were stored frozen at - 8 0 ° C prior to use. Probe preparation. The G F A P probes used in this study were derived from a c D N A for mouse G F A P that has been characterized previously [28]. The original c D N A clone was 2.5 kilobases (kb) in length, which specified over 97% of the G F A P amino acid sequence, and also included a 1.4 kb long portion of the 3' untranslated region. This original 2.5 kb c D N A was cut using the restriction enzyme H i n d I I I , to obtain a 1.26 kb fragment from the 5' (coding) portion of the original c D N A clone. This 1.26 kb fragment was recloned into the H i n d I I I site of a Bluescript ml3-vector by D. Chikaraishi (Tufts University). Samples of the Bluescript plasmid with the G F A P insert were provided to us as a gift. Circular plasmids were isolated using standard procedures and linearized with the restriction enzyme P v u I I . Because there is a P v u I I site within the G F A P cDNA, the restriction enzyme splits the G F A P c D N A into 2 fragments. This allowed the production of an antisense probe of about 1,000 bases in length. 3SSlabeled antisense probes were produced from this vector as described by Maniatis and colleagues [29]. T3 promotor was used to produce antisense probes. The specific activity of the probes ranged from 1.83 x 108 to 8.63 × 108 cpm per /xg of R N A (average = 4.66 x 10s). In situ hybridization was carried out using the procedure described by Rosenthal and colleagues [40]. The slides were allowed to warm to room t e m p e r a t u r e and were then postfixed for 10 min in 4% paraformaldehyde in phosphate buffer. The slides were pre-

treated with proteinase K (1 / x g / m l in R N A s e buffer) for 10-30 min, washed in 0.5 X SSC (saline sodium citrate), dried carefully with a Kimwipe, and placed flat on 3-mm-thick glass bars in a 150-mm Petri dish humidified with a formamide-containing buffer. The sections were covered with 100/zl of hybridization buffer (without the probe) and incubated for 1-3 h at 42°C. A 20/xl aliquot of hybridization buffer containing 1 - 2 / x l of probe (about 1 × 10 6 cpm) and 2 /xg of t R N A was added to the hybridization buffer after heat treatment for 3 min at 95°C. A 22 × 22 m m coverslip was then placed over the hybridization mix. Hybridization was carried out overnight (approximately 18 h) at 55°C. The following day, the coverslips were removed, and the slides were washed in 2 x SSC containing 10 mM beta mercaptoethanol (BME) and 1 m M E D T A . The slides were then incubated for 30 min at room temperature in a solution with 500 mM NaC1, 10 m M Tris p H 8.0, and 2 / x g / m l RNAse. The slides were washed for 2 h at 55°C in a 'stringency' buffer containing 0.1 × SSC, 10 m M BME, and 1 m M E D T A . Finally, the sections were hydrated in graded ethanols in 0.3 M ammonium acetate, and dried. For autoradiography, the slides were exposed for 72 h to Kodak beta-max hyperfilm. The slides were then dipped in Kodak NTB2 emulsion diluted 1:1 with water. The slides were exposed for 1 week and were developed in Kodak D19. The tissue sections were stained with Cresyl violet. To quantitatively evaluate the increases in labelling revealed by in situ hybridization, optical densities were measured using an Image Quant Personal Densitometer. Five autoradiographs were chosen for each case (10 images per timepoint). Optical density was evaluated over neocortex, hippocampus and thalamus both ipsilateral and contralateral to the exposed cortex. For statistical analyses, optical densities over each of the examined brain regions were analyzed with one-way analysis of variance (ANOVA). Analyses were followed by N e w m a n - K e u l s tests for specific between-group differences. 2.3. D o t blot hybridization

In order to verify the increases in G F A P m R N A seen using in situ hybridization, dot blot hybridizations were also performed. Right parietal cortex was exposed for 60 min to artificial CSF containing either 2 m M or 60 m M KC1, and the animals were killed 1, 4, or 8 days post-exposure (2 animals per treatment and per time point). Two naive animals were included as controls. Details of the methods used for R N A isolation, dot blot hybridization with G F A P probe and the controls for nonspecific hybridization have been described [43]. To obtain R N A for the dot blot analyses, animals were deeply anesthetized with sodium pentobarbital and de-

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capitated. Three cubes of parietal cortex (approximately 2 mm per side) were rapidly removed from each animal and were frozen at -70°C. One sample was taken from ipsilateral cortex directly beneath the craniotomy (at the site of the pledget application). The second sample was taken from ipsilateral parietal cortex approximately 4 mm posterior to the site of pledget application. The third sample was taken from the contralateral parietal cortex. Total cellular R N A was isolated by guanidinium isothiocyanate extraction followed by CsC1 gradient centrifugation [1]. The R N A was dissolved in 10 x SSC and 7.5% formaldehyde (pH 7.4). Samples containing 4 /zg of R N A were applied to a Biorad manifold and spotted onto a Nytran membrane. Previous studies have shown that samples containing 4 / z g of R N A are non-saturating [43]. The R N A was fixed to the membrane by cross-linking with ultraviolet light [49]. The membranes were hybridized at 65°C for 18 h with 35S-labelled probe (2.5 n g / m l , 106 c p m / m l in a hybridization buffer consisting of 50% formamide, 5 x standard s a l i n e / p h o s p h a t e / E D T A (SSPE), 5 x Denhardt's solution, 1% SDS, 100/xg of t R N A per ml and 100/zg of poly(A) per ml). The membranes were then washed three times in 1 x S S P E / I % SDS at 65°C, treated with RNAse (10 /~g per ml in 5 X SSC) for 15 min at room temperature, and washed for 30 min at 65°C in a high-stringency buffer (0.1 x S S P E / I % SDS). The membranes were then dried and exposed (for 24 h at - 70°C) to Kodak X-Omat film for autoradiography. Sections of the membrane containing individual spots were cut out, and the total amount of radioactivity in each spot was determined by scintillation counting. As controls for non-specific binding, the extent of hybridization to sections of the nylon membrane with no added R N A (blank) and the extent of hybridization to t R N A were evaluated. There was no binding of the probe to the blank or to the tRNA.

2.4. Electrophysiology Four animals were prepared for electrophysiological recordings. Two of these animals were pre-treated with MK-801 prior to craniotomy, as described below. Two small craniotomies were made through the right parietal bone. The first was a 1 mm diameter craniotomy located approximately 2.5 mm lateral to the sagittal suture and 1 mm posterior to bregma. The second was a 2 mm diameter craniotomy located approximately 2.5 mm lateral to the sagittal suture and 4 mm posterior to bregma. The dura was carefully retracted, exposing the underlying cortex. The animals were grounded through a subcutaneous Ag/AgCI wire in the scapular area. A double barrel glass microelectrode was prepared with one side filled with 1% Fast green in 2 M NaCI and the

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second side silanized and then filled with potassium exchanger [45]. The tip of this electrode was positioned 1 mm below the pial surface of the anterior craniotomy in order to measure extracellular potassium levels. Extracellular field potentials were amplified and displayed in parallel on a digital oscilloscope and paper chart recorder. A gelfoam pledger saturated with artificial CSF containing 60 mM KC1 was applied to the exposed parietal cortex through the posterior craniotomy for one hour. DC potentials, potassium levels and the E E G were recorded continuously. The recording electrode and pledget were then removed, the craniotomies were irrigated with baseline artificial CSF ([KCI] = 2 mM), the skin incision was closed and the animals were allowed to recover. The animals were killed four days later and were prepared for in situ hybridization as described above.

2.5. Effect of MK-801 The non-competitive N M D A antagonist, MK-801, blocks the onset and propogation of spreading depression when administered systemically in sufficient quantities [18,30]. This feature of MK-801 provides a means for directly testing the hypothesis that the upregulation of G F A P m R N A levels following cortical exposure to an artificial CSF containing 60 mM KCI is due to spreading depression. The effect of MK-801 was assessed electrophysiologically and with both in situ hybridization and dot blot hybridization. Two animals were prepared for electrophysiological recordings by receiving two right-sided craniotomies, as described above. The animals received intraperitoneal injections of MK-801 (3 m g / k g ) 30 min prior to topical application of 60 mM KCl-containing artificial CSF to the cortical surface through the posterior craniotomy. Through the anterior craniotomy, DC potentials, potassium levels and the E E G were recorded continuously with a double-barrel potassium-sensitive electrode throughout the hour of exposure. The animals were killed four days later, and the tissue was processed for in situ hybridization. Dot blot hybridization was used to verify and quantify the blocking effect of MK-801 on the 60 mM KCl-induced upregulation of G F A P mRNA. MK-801 was administered systemically (3 m g / k g i.p.) to two animals 30 min prior to topical application to the right cortical surface of the 60 mM KCl-containing artificial CSF. Two animals were treated with the 60 mM KC1containing artificial CSF without prior administration of MK-801, and two additional control animals received MK-801 and were sham-operated. The animals were all killed four days post-exposure. Two naive animals were also included. Samples from ipsilateral, ipsilateral-distal, and contralateral neocortex were har-

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vested, and dot blot hybridizations were performed as described above.

2.6. Effect of changes in extracellular ionic concentrations In order to test the hypothesis that a change in concentration of one of the extracellular ions is responsible for the spreading depression-induced upregulation in G F A P m R N A , right parietal cortex was exposed to one of a variety of artificial CSF solutions. The baseline artificial CSF was altered in one of the

following ways: (a) [NaCI] = 70 mM; (b) [CaC12] = 0.15 mM; (c) [KC1] = 60 mM; (d) p H = 6.9. Gel foam piedgets saturated with one artificial CSF composition were applied for one hour to parietal cortex, as described above. In order to determine whether the upregulation induced by the 60 m M KCl-containing solution was due specifically to the potassium concentration or to the increased tonicity, control animals were exposed to an artificial CSF containing 185 m M NaC1. (The increased tonicity of the control artificial CSF containing 185 m M NaC1 matched the increased tonicity of the experimental artificial CSF containing 60 mM KC1.) All animals were killed one or four days post-exposure (n = 2 for each treatment and timepoint) and were p r e p a r e d for in situ hybridization. In order to verify and quantify the effects seen with in situ hybridization, dot blot hybridizations were also performed. Gelfoam pledgets saturated with baseline artificial CSF altered to contain (a) 70 m M NaC1; (b) 0.15 m M CaCI2; (c) 60 mM KCI; (d) 185 m M NaC1 or (e) p H = 6.9 were positioned on the right parietal cortex for one hour as described above (n = 2 for each treatment). Control animals were naive (n = 2). All animals were killed four days post-exposure, and samples of neocortex from (1) directly beneath the craniotomy; (2) ipsilateral and distal to the site of application and (3) contralateral were harvested and prepared for dot blot hybridization as described above.

3. Results

3.1. Timecourse of changes in GFAP mRNA leuels Application of artificial CSF solutions containing either 60 m M KCI or 3 M KC1 both led to substantial increases in G F A P m R N A levels across the ipsilateral

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Fig. 1. Autoradiographs revealing pattern of labelling of coronally-cut tissue sections following in situ hybridization with 35S-labelled cRNA probes complimentary to GFAP mRNA. In each case, the sections illustrated were taken from directly beneath the site of pledget application on the right cortex. (A) Section from an animal exposed to a gel foam pledget saturated with an artificial CSF containing 60 mM KCI. An upregulation in GFAP m R N A content is evident across the ipsilateral neocortex. (B) Section from an animal exposed to a pledget saturated with 3 M KCI. Again, an increased labelling of GFAP m R N A occurred across the ipsilateral neocortex. The highlyconcentrated KCI solution, however, induced a focal region of tissue necrosis at the site of pledget application (arrows). (C) Section from a control animal exposed to a pledget saturated with artificial CSF containing 2 mM KC1. There is no apparent increase in GFAP m R N A labelling in the ipsilateral neocortex or in any other structure. Throughout the brain, labelling for GFAP m R N A is uniform, except for the fiber tracts and gila limitans at the brain surface. Cx = cortex; H = hippocampus; Th = thalamus. The magnification bar represents 2 mm in A, B and C.

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neocortex (Fig. 1). The upregulation in G F A P m R N A levels appeared to involve all cell layers of and was confined to the ipsilateral neocortex. The only region of ipsilateral neocortex in which G F A P m R N A levels were not upregulated was the cingulate cortex. This sparing of the cingulate cortex occurred consistently in

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animals receiving either the 60 mM or 3 M KCI treatments. The hippocampus, subcortical regions and contralateral neocortex were unaffected. Although, the 3 M KC1 and 60 mM KC1 solutions induced similar responses in G F A P m R N A levels, the two solutions differed markedly in the degree of tissue

Fig. 2. Photomicrographs of cresyl violet-stained cerebral cortex at the site of pledget application. (A) Section of cerebral cortex taken from directly beneath the site of application of a pledget saturated with an artificial CSF containing 60 m M KC1. There is no evidence of tissue injury. T h e surface of the cortex remained intact, and the normal laminar cytoarchitecture of the cortex was preserved. (B) Section of cerebral cortex taken from directly beneath the site of application of a pledget saturated with 3 M KCI. Substantial tissue injury is evident. A wedge of tissue at the cortical surface has disintegrated and left an erosion at the cortical surface (arrows). T h e grey matter beneath the erosion has undergone coagulation necrosis. (C) Section of cerebral cortex taken from directly beneath the site of application of a pledget saturated with baseline (control) CSF containing 2 m M KCl. There is no evidence of tissue injury. (D) Higher power photomicrograph of the grey matter of cerebral cortex taken from the same section as shown in panel A. The image shows normal histological features of parietal cortex. There is no leukocytic infiltration or other evidence of tissue damage. T h e arrows point to cell bodies of cortical neurons. (E) Higher power photomicrograph of the grey matter of cerebral cortex taken from the same section as shown in panel B illustrating an area of coagulation necrosis with few surviving neurons. The curved arrows point to pyknotic nuclei. T h e straight arrows point to clusters of leukocytes. (F) Higher power photomicrograph of the grey matter of cerebral cortex taken from the same section as shown in panel C. There is no evidence of tissue injury. No pyknotic nuclei or leukocytic infiltration is present. The arrows point to cell bodies of cortical neurons. T h e magnification bars represent 0.5 m m in A, B and C and 100/~m in D, E and F.

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injury induced (Fig. 2). At the site of application, the 3 M KC1 solution induced a wedge of coagulation necrosis which extended through the full thickness of the cellular layer of the cortex. The cortical cytoarchitecture was severely disrupted, and pyknotic nuclei and an infiltration of polymorphonuclear leukocytes were evident. In contrast, the 60 m M KC1 solution induced no evident tissue injury. The normal laminar cytoarchitecture of the cortex remained intact. No pyknotic nuclei or leukocytic infiltration were identified. Application of baseline artificial CSF (containing 2 m M KC1) induced no evident tissue damage or changes in G F A P m R N A levels (Figs. 1 and 2). The only exceptions to the uniformly low labelling for G F A P m R N A were the fiber tracts and the glia limitans at the brain surface, where labelling was slightly higher (as is also true in control sections from un-treated animals).

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Fig. 3. Time course of increases in G F A P m R N A following application of an artificial CSF containing 60 m M KCI to the cortical surface. Gel foam pledgets saturated with an artificial CSF containing 60 m M KCI were applied to the parietal cortex for 60 minutes. Animals were killed 1, 2, 4 and 8 days following the pledget application. The forebrains were sectioned coronally and prepared for in situ hybridization with 35S-labelled probes for G F A P m R N A . Optical densities were m e a s u r e d from autoradiographs. T h e graphs illustrate the m e a n optical density ( + S.D.). (A) In the ipsilateral cortex, G F A P m R N A levels rose steadily toward a m a x i m u m at 4 days, then fell toward control levels by 8 days. At the time of m a x i m u m expression, levels of G F A P m R N A were increased greater than 10-fold, relative to control levels. In contrast, labelling in contralateral cortex remained u n c h a n g e d throughout the timecourse. (B) In both the ipsilateral and contralateral hippocampus, the degree of labelling remained near control levels at all time points. (C) In both the ipsilateral and contralateral thalamus, labelling remained low and u n c h a n g e d throughout the time course.

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Naive CSF-1 CSF-4 CSF-8 KCI-1 KCl-4 KCI-8 Fig. 5. Time course of changes in G F A P m R N A levels in cerebral cortex following application of an artificial CSF containing 60 m M KCI to the cortical surface, as measured by dot blot hybridization. Gel foam pledgets saturated with artificial CSF solutions containing either 60 m M KC1 or 2 m M KCI (control) were applied to a focal area of parietal cortex for 60 min. Animals were killed 1, 4 or 8 days later. Naive animals were also included, R N A was obtained from the cerebral cortex directly beneath the site of pledger application (labelled 'ipsilateral'), from the ipsilateral cerebral cortex 4 - 5 m m posterior to the site of pledget application (labelled 'ipsilateral-distal') and from the contralateral cerebral cortex. Duplicate samples containing 4 /xg of R N A were spotted onto nylon m e m b r a n e s and were hybridized with 35S-labelled probes for G F A P m R N A . The graphs illustrate the m e a n counts per minute per dot (_+S.D.). Application of the artificial CSF containing 60 m M KCI resulted in a substantial upregulation of G F A P m R N A levels in ipsilateral cortex both directly beneath the pledger and at the distal ipsilateral site. T h e upregulation was evident on day 1, reached a peak on day 4 and returned toward control levels on day 8. G F A P m R N A levels in contralateral cortex remained unchanged at all time points. Application of the control CSF resulted in no change in G F A P m R N A levels in any of the brain regions examined at any time point,

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exposure (time of peak labelling), the optical density in the ipsilateral cortex of both the 60 mM KCI- and 3 M KCl-exposed groups was significantly higher than controls ( P < 0.01), but the two KCl-exposed groups did not differ significantly from each other.

Optical density analysis of the autoradiographs confirmed the qualitative impressions and revealed a clear temporal pattern. As shown in Fig. 3, G F A P m R N A levels in the cortex ipsilateral to the pledget rose steadily toward a maximum at four days following the 60 mM KCI exposure. At the time of maximum expression, the levels of G F A P m R N A in ipsilateral cortex were greater than 10-fold higher than the hippocampal and thalamic controls and the 2 mM KCl-containing artificial CSF control (Fig. 4). By eight days post-exposure, the level of labelling in ipsilateral cortex had fallen toward, but still slightly exceeded, control levels. In contrast to the large effect seen in the ipsilateral cortex, there were no quantitative changes in labelling in the contralateral cortex. Optical density over the contralateral cortex remained near control levels at each timepoint. Similarly, optical density in the hippocampus and thalamus remained low and unchanged throughout the timecourse. Statistical analysis revealed that optical density was significantly higher in the ipsilateral cortex than in any other region at 1, 2 and 4 days post-exposure ( P < 0.01) though not at 8 days post-exposure. At four days post-

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The finding that artificial CSF containing 60 mM KC1 leads to large and transient increases in G F A P m R N A content across the ipsilateral cortex was confirmed by dot blot hybridization. As shown in Fig. 5, application of a 60 mM KCl-containing solution to the cortex resulted in a substantial upregulation in G F A P m R N A in the ipsilateral cortex. These increases were evident on day 1, reached a peak on day 4, and still exceeded control levels on day 8. The timecourse and magnitude of the increases were similar for samples taken directly beneath the craniotomy (at the site of pledget application) and at the distant ipsilateral cortical site. In contrast, G F A P m R N A levels in the contralateral cortex did not differ significantly from controls at any time point. Application of the 2 mM

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Fig. 6. Electrographic recordings of spreading depression induced by an artificial CSF containing 60 mM KCI. (A) A potassium-sensitive double barrel glass microelectrode was positioned 1 mm below the pial surface of the parietal cortex. A gel foam pledget saturated with an artificial CSF containing 60 mM KCI was applied to a focal area of cortex several millimeters posterior to the electrode. Extracellular potassium concentration (top tracing), tissue DC potential (middle tracing) and EEG (bottom tracing) were recorded simultaneously. Several minutes after application of the pledget to the cortex, recurrent waves of spreading depression occurred. During the waves of spreading depression, extracellular potassium concentration rose markedly, tissue DC potential fell and background EEG activity was suppressed. (B) A continuation in time of the tracings described for panel (A). Throughout the hour of exposure to 60 mM KC1, recurrent waves of spreading depression occurred every 2-3 rain. Background EEG activity remained suppressed both during and between the waves of spreading depression.

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KCl-containing artificial CSF resulted in no change in G F A P m R N A levels in any of the three cortical regions examined at any time point.

across the ipsilateral neocortex (Fig. 7). Again, the only region of ipsilateral neocortex in which an upregulation did not occur was the cingulate cortex.

3.3. Electrophysiology

3.4. Effect of MK-801

Application of pledgets saturated with artificial CSF containing 60 m M KC1 led to recurrent waves of spreading depression (Fig. 6). Electrographic evidence of spreading depression included marked elevations in extracellular potassium concentration accompanied by negative shifts of the D C potential and a suppression of background E E G activity. Both animals in which electrophysiological recordings were made after application of the 60 m M KCl-containing CSF exhibited multiple waves of spreading depression. T h e r e were 22 waves of spreading depression in one case and 24 waves in the other. When brain tissue from these animals was processed for in situ hybridization, both showed a dramatic increase in G F A P m R N A content

Intraperitoneal injection of MK-801 30 min prior to the 60 m M KCI treatment blocked all electrographic evidence of spreading depression. Throughout the hour of pledget application and physiological recording, potassium concentration, D C potential and E E G remained at baseline levels. The MK-801 treatment also blocked the upregulation of G F A P m R N A levels. Fig. 8 shows a series of tissue sections from an animal treated with MK-801 prior to application of artificial CSF containing 60 m M KC1 to the cortex and killed 4 days later. There was no evidence of increased labelling at any point on the ipsilateral or contralateral cortex or at any subcortical site. In the MK-801 treated animals, even the cortex directly beneath the cran-

A

IB

Fig. 7. Effect of cortical spreading depression on the level of GFAP m R N A as revealed by in situ hybridization. Gel foam pledgets saturated with an artificial CSF containing 60 mM KC1 were applied to a focal area of right parietal cortex for 60 min. Extracellular potassium concentration, tissue DC potential and E E G were recorded from ipsilateral cortex and revealed recurrent waves of spreading depression (the electrographic recordings from this specific animal are shown in Fig. 6). The animals were killed 4 days post-exposure. The forebrains were sectioned coronally and were prepared for in situ hybridization with 3sS-labelled cRNA probes complimentary to GFAP mRNA. Panels A - D are autoradiographs taken from a single animal from progressively more caudal locations. There is a dramatic upregulation in labelling across the entire ipsilateral (right) cortex, with the only exception being the cingulate cortex (arrows). Cx = cortex; H = hippocampus; Th = thalamus. The magnification bar represents 2 mm in A, B, C and D.

D.J. Bonthius et al. /Brain Research 674 (1995) 314-328

323

r

Fig. 8. Effect of MK-801 in blocking the upregulation of G F A P m R N A , as revealed by in situ hybridization. Animals were pre-treated with MK-801 prior to application to the parietal cortex of gel foam pledgets saturated with an artificial CSF containing 60 m m KCI. Electrographic recordings revealed that the MK-801 pretreatment blocked the onset of spreading depression. The animals were killed 4 days post-exposure, and the brain tissue was prepared for in situ hybridization with 35S-labelled c R N A probes complimentary to G F A P m R N A . Panels A - D are autoradiographs taken from a single animal from progressively more caudal locations. The upregulation in G F A P m R N A levels which the 60 m M KCI treatment would normally have induced in the ipsilateral cortex has been blocked by the prior administration of MK-801. Cx = cortex; H = hippocampus; Th = thalamus. The magnification bar represents 2 m m in A, B, C, and D.

iotomy site and in direct contact with the 60 mM KCI solution showed no change in G F A P m R N A labelling. Results of the dot blot hybridization confirmed the ability of MK-801 to block the KCl-induced upregulation in G F A P m R N A content. As illustrated in Fig. 9, animals pre-treated with MK-801 showed no detectable changes in G F A P m R N A levels in ipsilateral neocortex - either at the site directly beneath the craniotomy or at the distant ipsilateral site. There were also no changes in G F A P m R N A levels in the animals pre-treated with MK-801 prior to the sham surgery, indicating that MK-801 alone does not alter G F A P m R N A levels.

3.5. Effect of changes in extracellular ionic concentrations When ionic concentrations of the artificial CSF solutions were systematically altered one ion at a time to match the ionic concentrations induced by spreading depression, only the 60 mM KCl-containing solution

led to altered G F A P m R N A levels (Fig. 10). As described above, application of artificial CSF containing 60 mM KCI induced an upregulation in G F A P m R N A content across the ipsilateral neocortex which was evident on day 1 and even more dramatic on day 4. Once again, the only portion of ipsilateral neocortex in which increased labelling of G F A P m R N A did not occur was the cingulate cortex. Alteration in sodium, calcium and hydrogen ion concentrations to levels reflective of spreading depression did not induce any evident changes in G F A P m R N A expression. Even directly beneath the craniotomies, where the pledgers contacted the brain surface, G F A P m R N A levels remained unchanged at 1 day and 4 days post-exposure. Similarly, hypertonic CSF containing 180 mM NaC1 induced no evident changes in G F A P m R N A labelling at 1 or 4 days post-exposure. Results of the dot blot hybridizations confirmed the finding that only alteration of the potassium concentration affected G F A P m R N A levels. As shown in Fig. 11, at four days post-exposure, the CSF containing 60 mM

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2000 t

T[~

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ipsilateral ipsilateral-distal

1000 500

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MK-KCI

MK

Fig. 9. Blockade of GFAP mRNA upregulation by MK-801, as revealed by dot blot hybridization. MK-801 was administered systemically to 4 animals. Thirty minutes later, the animals were treated either with topical application to the right parietal cortex of a pledget saturated with an artificial CSF containing 60 mM KCI (n = 2) or were sham operated (n = 2). Additional animals received the 60 mM KC1 application to the cortex without prior administration of MK-801 (n = 2). Animals were killed 4 days later. Two naive animals were also included. RNA was obtained from the cerebral cortex directly beneath the site of pledget application (labelled 'ipsilateral'), from the ipsilateral cerebral cortex 4-5 mm posterior to the site of pledget application (labelled 'ipsilateral-distal') and from the contralateral cerebral cortex. Duplicate samples containing 4/xg of RNA were spotted onto nylon membranes and were hybridized with 35S-labelled probes complimentary to GFAP mRNA. The graph illustrates the mean counts per minute per dot (+S.D.). Animals exposed to the 60 mM KC1 solution but not pretreated with MK-801 had large increases in levels of GFAP mRNA both directly beneath the site of pledget application and at the distant ipsilateral site. In animals treated with MK-801 prior to the KCI exposure, there were no evident changes in GFAP mRNA levels either directly beneath the pledget or at the distant ipsilateral site. Treatment with MK-801 prior to sham surgery induced no detectable changes in GFAP mRNA levels in any of the regions examined. MK-KCI = animals pre-treated with MK-801 prior to application of artificial CSF containing 60 mM KCI; MK = animals pre-treated with MK-801 prior to sham surgery; KCI = animals treated with artificial CSF containing 60 mM KCI and not pre-treated with MK-801. KC1 i n d u c e d a s u b s t a n t i a l i n c r e a s e in G F A P m R N A c o n t e n t in i p s i l a t e r a l cortex. This i n c r e a s e d G F A P m R N A c o n t e n t o c c u r r e d in s a m p l e s of c o r t e x b o t h d i r e c t l y b e n e a t h t h e c r a n i o t o m y a n d at t h e d i s t a n t i p s i l a t e r a l site. In c o n t r a s t , t h e artificial C S F solutions c o n t a i n i n g 70 m M NaCI, 0.15 m M CaC12, 185 m M NaC1 a n d p H 6.9 i n d u c e d no c h a n g e s in G F A P m R N A c o n t e n t at t h e site of p l e d g e t a p p l i c a t i o n o r at t h e d i s t a n t i p s i l a t e r a l cortical site. I n all cases, G F A P m R N A c o n t e n t in c o n t r a l a t e r a l c o r t e x r e m a i n e d at c o n t r o l levels.

4. D i s c u s s i o n

T h e r e a r e t h r e e p r i n c i p a l findings o f t h e p r e s e n t study. First, a p p l i c a t i o n o f an artificial C S F c o n t a i n i n g 60 m M KCI to t h e cortical s u r f a c e i n d u c e d cortical s p r e a d i n g d e p r e s s i o n w i t h o u t i n d u c i n g e v i d e n t tissue d a m a g e a n d l e d to a l a r g e t r a n s i e n t i n c r e a s e in G F A P

m R N A c o n t e n t across t h e i p s i l a t e r a l neocortex. Second, w h e n ionic c o n c e n t r a t i o n s o f artificial C S F solutions w e r e individually a l t e r e d to m a t c h t h e c o n c e n t r a tions s e e n in s p r e a d i n g d e p r e s s i o n , only t h e c h a n g e in p o t a s s i u m c o n c e n t r a t i o n i n d u c e d an i n c r e a s e in G F A P m R N A c o n t e n t . T h i r d , systemic a d m i n i s t r a t i o n o f M K 801, an N M D A a n a t a g o n i s t , b l o c k e d s p r e a d i n g d e p r e s sion a n d b l o c k e d t h e u p r e g u l a t i o n of G F A P m R N A levels. 4.1. Spreading depression-induced upregulation in G F A P rnRNA T h e results of t h e p r e s e n t study d e m o n s t r a t e t h a t s p r e a d i n g d e p r e s s i o n c a n i n d u c e an u p r e g u l a t i o n in G F A P m R N A levels a n d t h a t this u p r e g u l a t i o n can o c c u r in t h e a b s e n c e of tissue injury. R e s u l t s of previous s t u d i e s have s u g g e s t e d t h a t a b n o r m a l e l e c t r o c h e m ical activity, i n c l u d i n g s p r e a d i n g d e p r e s s i o n [5,25] a n d s e i z u r e activity [6,44,47] c a n a l t e r G F A P g e n e expression. In e a c h of t h e s e p r e v i o u s studies, however, a s u b s t a n t i a l a m o u n t of tissue injury a n d necrosis was i n d u c e d e i t h e r by a p p l i c a t i o n o f highly c o n c e n t r a t e d KCI to the b r a i n surface or by i m p l a n t a t i o n of stimulating e l e c t r o d e s . This is t h e first study to d e m o n s t r a t e an u p r e g u l a t i o n in G F A P m R N A levels in t h e a b s e n c e o f histological e v i d e n c e o f tissue d a m a g e . W h i l e the prese n c e of i n j u r e d a n d n e c r o t i c tissue has long b e e n k n o w n to b e a p o t e n t signal in t r i g g e r i n g t h e glial r e s p o n s e , this study d e m o n s t r a t e s t h a t an u p r e g u l a t i o n in G F A P m R N A can o c c u r in t h e i r a b s e n c e a n d t h a t t h e initiating events a r e r e l a t e d to physiological changes. Several lines o f e v i d e n c e suggest that t h e g e n e r a l m e c h a n i s m by which topical a p p l i c a t i o n of the 60 m M KC1 s o l u t i o n l e a d s to an u p r e g u l a t i o n in G F A P m R N A c o n t e n t is r e l a t e d to s p r e a d i n g d e p r e s s i o n . First, elect r o p h y s i o l o g i c r e c o r d i n g s d e m o n s t r a t e d t h a t t h e 60 m M KC1 t r e a t m e n t did i n d u c e r e c u r r e n t waves o f s p r e a d i n g d e p r e s s i o n . S e c o n d , t h e s p a t i a l d i s t r i b u t i o n of the c h a n g e s in G F A P m R N A levels is c o n s i s t e n t with the spatial d i s t r i b u t i o n e x p e c t e d of s p r e a d i n g d e p r e s s i o n . W h e n s p r e a d i n g d e p r e s s i o n is i n i t i a t e d in o n e hemis p h e r e , it s p r e a d s over t h e i p s i l a t e r a l n e o c o r t e x , b u t typically d o e s not s p r e a d to t h e o p p o s i t e h e m i s p h e r e [32,41] or to subcortical r e g i o n s [8,27]. This e x p e c t e d d i s t r i b u t i o n o f s p r e a d i n g d e p r e s s i o n m a t c h e s t h e obs e r v e d s p a t i a l p a t t e r n of G F A P m R N A u p r e g u l a t i o n . T h i r d , b l o c k a g e of s p r e a d i n g d e p r e s s i o n with MK-801 also b l o c k e d t h e u p r e g u l a t i o n of G F A P m R N A levels. A n i n t e r e s t i n g a n d u n e x p e c t e d finding was t h a t t h e r e s p o n s e o f t h e c i n g u l a t e c o r t e x d i f f e r e d f r o m that of the r e m a i n d e r o f t h e i p s i l a t e r a l n e o c o r t e x . T h e u p r e g u l a t i o n o f G F A P m R N A levels which o c c u r r e d t h r o u g h o u t t h e i p s i l a t e r a l n e o c o r t e x f a i l e d to occur in t h e c i n g u l a t e cortex. This s p a r i n g of the c i n g u l a t e cortex

D.J. Bonthius et al. /Brain Research 674 (1995) 314-328 was o b s e r v e d following b o t h t h e 60 m M a n d 3 M KC1 t r e a t m e n t s . O n e e x p l a n a t i o n is t h a t t h e s p r e a d i n g d e p r e s s i o n i n d u c e d in t h e s e e x p e r i m e n t s m a y n o t have p r o p o g a t e d to t h e c i n g u l a t e c o r t e x [17]. A l t e r n a t i v e l y , the astrocytes of the cingulate cortex may respond d i f f e r e n t l y to s p r e a d i n g d e p r e s s i o n t h a n d o t h e astrocytes o f o t h e r n e o c o r t i c a l regions. I n d e e d , r e g i o n a l d i f f e r e n c e s have b e e n d e m o n s t r a t e d in t h e m o r p h o l o g y a n d g e n e e x p r e s s i o n o f a s t r o c y t e s [15,42]. W h e t h e r the r e g i o n a l d i f f e r e n c e s in G F A P m R N A u p r e g u l a t i o n a r e d u e to factors intrinsic o r extrinsic to t h e a s t r o c y t e s r e m a i n s to b e d e t e r m i n e d . A n i n t e r e s t i n g q u e s t i o n is w h e t h e r t h e effect o f s p r e a d i n g d e p r e s s i o n in u p r e g u l a t i n g G F A P m R N A c o n t e n t is an a l l - o r - n o n e o r a step-wise p h e n o m e n o n . In t h e p r e s e n t e x p e r ! m e n t , all a n i m a l s e x p o s e d to t h e 60 m M KC1 s o l u t i o n w e r e e x p o s e d for an i d e n t i c l e p e r i o d o f t i m e (60 min) a n d likely h a d a similar n u m b e r o f waves o f s p r e a d i n g d e p r e s s i o n [20-25]. T h e r e f o r e , a r e l a t i o n s h i p b e t w e e n n u m b e r o f waves o f s p r e a d i n g d e p r e s s i o n a n d intensity o f G F A P m R N A u p r e g u l a t i o n c a n n o t b e e s t a b l i s h e d r e l i a b l y f r o m t h e p r e s e n t data. H o w e v e r , p r e v i o u s e x p e r i m e n t s in w h i c h e l e c t r o p h y s i o logical c h a n g e s w e r e r e c o r d e d c o n t i n u o u s l y a n d in w h i c h a n i m a l s h a d varying n u m b e r s o f s p r e a d i n g dep r e s s i o n waves s h o w e d t h a t t h e i n t e n s i t y o f G F A P m R N A u p r e g u l a t i o n d e p e n d e d on t h e o c c u r r e n c e b u t

325

n o t on t h e n u m b e r o r d u r a t i o n of waves o f s p r e a d i n g d e p r e s s i o n [6]. T h e s e results s u g g e s t e d t h a t t h e u p r e g u l a t i o n in G F A P m R N A c o n t e n t i n d u c e d by s p r e a d i n g d e p r e s s i o n is an a l l - o r - n o n e , r a t h e r t h a n a step-wise, phenomenon.

4.2. Effect o f altering extracellular ionic concentrations During spreading depression, the concentrations of ions in t h e i n t e r s t i t i u m u n d e r g o p r o f o u n d changes. Specifically, the c o n c e n t r a t i o n s o f s o d i u m a n d c a l c i u m fall p r e c i p i t o u s l y , while h y d r o g e n a n d p o t a s s i u m conc e n t r a t i o n s rise. W e h y p o t h e s i z e d t h a t t h e c h a n g e in e x t r a c e l l u l a r c o n c e n t r a t i o n o f o n e o f t h e s e ions m a y b e t h e p r o x i m a t e signal t r i g g e r i n g the u p r e g u l a t i o n in astrocytic G F A P m R N A c o n t e n t . If so, t h e n cortical a p p l i c a t i o n o f artificial C S F solutions c o n t a i n i n g t h e r e s p o n s i b l e ion at t h e c o n c e n t r a t i o n s e e n in s p r e a d i n g d e p r e s s i o n w o u l d b e e x p e c t e d to i n d u c e a local u p r e g u l a t i o n in G F A P m R N A at t h e site o f p l e d g e t a p p l i c a tion. N o n e o f t h e C S F solutions, with ionic c o n c e n t r a tions a l t e r e d o n e at a time, i n d u c e d such a local response. Manipulation of the sodium, calcium and h y d r o g e n ion c o n c e n t r a t i o n s h a d no effect on G F A P m R N A levels. M a n i p u l a t i o n o f t h e p o t a s s i u m c o n c e n t r a t i o n i n d u c e d an u p r e g u l a t i o n n o t only at t h e site o f a p p l i c a t i o n , b u t across t h e e n t i r e i p s i l a t e r a l n e o c o r t e x .

Fig. 10. Effect of changes in extracellular ionic concentrations on GFAP mRNA expression, as revealed by in situ hybridization. Baseline artificial CSF was altered one ion at a time to match the ionic concentrations typically observed during spreading depression. A gel foam pledget saturated with one of these solutions was applied to a focal area of right parietal cortex for 60 min. The animals were killed 4 days later, and the brain tissue was prepared for in situ hybridization with ass-labelled cRNA probes complimentary to GFAP mRNA. Tissue sections were taken from directly beneath the site of pledget application. The autoradiographs illustrate the pattern of labelling following exposure to artificial CSF containing (A) 70 mM NaCI, (B) 60 mM KCI, (C) 0.15 mM CaCI2, (D) pH 6.9, (E) 185 mM NaCI. Only the alteration in KCI concentration (panel B) induced any upregulation in GFAP mRNA labelling. The upregulation occurred across the entire ipsilateral cortex (except for the cingulate cortex) and was probably due to an induction of spreading depression. None of the other alterations in ionic concentration induced any changes in GFAP mRNA expression. Even directly beneath the craniotomies, where the pledgets directly contacted brain tissue, GFAP mRNA labelling remained unchanged. The magnification bar represents 2 mm in A, B, C, D and E.

D.J. Bonthius et aL / Brain Research 674 (1995) 314-328

326 1000 ¢)

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Fig. 11. Effect of changes in extracellular ionic concentrations on G F A P m R N A expression, as revealed by dot blot hybridization. Baseline artificial CSF was altered one ion at a time to match the ionic concentrations typically observed during spreading depression. A gel foam pledget saturated with one of these solutions was applied to a focal area of right parietal cortex for 60 minutes. The animals were killed 4 days later. R N A was obtained from the cerebral cortex directly beneath the site of pledget application ('ipsilateral'), from the ipsilateral cerebral cortex 4 - 5 m m posterior to the site of pledget application ('ipsilateral-distal') and from the contralateral cerebral cortex. Duplicate samples containing 4 /~g of R N A were spotted onto nylon membranes, and the m e m b r a n e s were hybridized with Sss-labelled probes for G F A P m R N A . T h e graphs illustrate the mean counts per minute per dot (±S.D.). Only the alteration in potassium concentration induced changes in G F A P m R N A levels. The substantial upregulation induced by the altered potassium concentration occurred in cortex directly beneath the site of pledget application and at the distant ipsilateral site (probably reflecting the induction of spreading depression). The artificial CSF solution containing 185 m M NaCI had no effect on G F A P m R N A expression, indicating that the upregulation induced by the 60 m M KC1 CSF solution was not due to its hypertonicity.

As discussed above, this distribution of effect is probably due to the fact that 60 mM potassium not only reflects the concentration seen in spreading depression, but is capable of inducing spreading depression as well. The local and hemispheric upregulation in G F A P m R N A levels following application of 60 mM KC1 to the cortex may, therefore, have been related to spreading depression, but not necessarily to the potassium concentration per se.

4.3. Blockade of GFAP mRNA upregulation by MK-801 Systemic administration of MK-801 prior to application of the 60 mM KCl-containing CSF to the cortex blocked the upregulation of G F A P mRNA. The probable mechanism by which MK-801 blocked the upregulation was by its effect of blocking spreading depression. The endogenous release of excitatory neurotransmitters and their depolarizing actions via the N M D A receptor are believed to play important roles in the initiation and propogation of spreading depression [30,48]. In addition, the role of MK-801 as an anticonvulsant may also have contributed to its effect [13]. It is doubtful that the MK-801 acted directly on the astrocytes to block G F A P m R N A upregulation as astrocytes are not believed to possess N M D A receptors.

The MK-801 treatment prevented the upregulation of G F A P m R N A across the entire ipsilateral neocortex, including the site of pledget application. This pattern of blockade provides several important clues regarding the factors controlling G F A P gene expression. Blockade by MK-801 at the site of pledget application suggests that elevation in extracellular potassium concentration is not the proximate signal influencing G F A P gene expression. Local astrocytes directly beneath the pledget were exposed to 60 mM KC1, irrespective of the presence of MK-801. If elevation in extracellular potassium concentration were the signal controlling the G F A P gene, then application of 60 mM KC1 to the cortex following systemic administration of MK-801 would be expected to induce a local increase in G F A P m R N A content. Because no local increase occurred, it is doubtful 'that potassium is the responsible proximate signal. In a previous study, it was demonstrated that administration of MK-801 prior to application of 3 M KC1 to the cortical surface blocked the upregulation of G F A P m R N A in remote cortical sites, but did not block the upregulation at the site of pledget application, where tissue necrosis occurred [5]. This pattern differs from that of the present study where blockade of upregulation occurred across the entire neocortex, including the site of pledget application. Taken together, these findings suggest that the signals responsible for GFAP m R N A upregulation may be different in injured and intact tissues. The results of this study suggest that change in extracellular concentration of a single ionic species is not the likely signal responsible for the spreading depression-induced upregulation in G F A P mRNA. However, intracellular ionic concentrations, which undergo large changes in spreading depression, and which were not examined in this study, may underlie the upregulation. Of particular potential importance are calcium, the flux of which across cell membranes is large during episodes of spreading depression [21] and intracellular pH, which increases roughly in proportion to the degree of depolarization [10,11] and which often correlates with changes in cellular metabolic activity [9]. Another signal potentially influencing G F A P m R N A levels is tissue DC potential, which undergoes a large negative shift during spreading depression. Astrocytes are sensitive to changes in DC potential as they possess voltage-gated ion channels [2] whose actions can be altered by changes in neuronal activity [31]. Neurotransmitters may also play an important role as they are released in large quantities during spreading depression [48] and can influence glial cells directly via functional receptors on glial cell membranes [7,12,34]. Finally, spreading depression has been shown to upregulate the mRNAs for several growth factors within neurons [23]. The induction of G F A P expression could

D.J. Bonthius et al. / Brain Research 674 (1995) 314-328

be in response to an increased release of such growth factors from neurons. The present study confirms and extends previous observations [5,44] that abnormal electrochemical activity, including spreading depression, can alter GFAP gene expression. An important question which remains unanswered is whether the signals underlying the upregulation in GFAP mRNA during spreading depression are the same as those that trigger astrocytic transformation following tissue injury. How and whether the increases in GFAP expression affect glial cell function are also unknown.

Acknowledgements We thank P. Falk and L. Whitmore for their technical advice, N. Cowan for the GFAP cDNA clone and D. Chikaraishi for recloning the cDNA clone into a riboprobe vector. We thank D. Rempe for production of the glass microelectrodes. Some of these results have been published in abstract form [4]. This work was supported by NIH Grant NS29875 to O.S., by NIH Grants NS21671 and NS25605 to E.W.L., and by a Pediatric Resident Research Grant from the American Academy of Pediatrics with the support of Wyeth Pediatrics to D.J.B.

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