Induction of constitutive heat shock protein 73 mRNA in the dentate gyrus by seizures

Molecular Brain Research, 13 (1992) 19-25

19

(~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00 BRESM 70385

Induction of constitutive heat shock protein 73 m R N A in the dentate gyrus by seizures Ma-Li Wong 1'4, Susan R.B. Weiss 2, Philip W. Gold 1, Sonia Q. Doi 3, Sujit Banerjee 1, Julio Licinio 1'*, R a j n i k a n t Lad 1'**, R o b e r t M. Post 2 and M a r k A. Smith 1 1Clinical Neuroendocrinology Branch, 2Biological Psychiatry Branch NIMH and 3Metabolic Diseases Branch N1DDK, National Institutes of Health, Bethesda, MD 20892 (U.S.A.)

(Accepted 29 August 1991) Key words: Heat shock protein; mRNA; Cocaine; Kindling; Electroconvulsive seizure; Dentate gyrus; Hippocampus; In situ hybridization

We examined the effects of generalized seizures on heat shock protein (hsp) mRNA induction in the rat brain using in situ hybridization. Seizures induced by electroconvulsive shock, electrical or cocaine kindling caused a selective induction of the constitutive hsp 73 gene in the dentate gyrus. In these seizure paradigms, not thought to induce widespread tissue damage, neither the heat-inducible hsp 72 gene nor a member of the hsp 90 family (hsp 84) were induced. Hsp 73 may play a role in the adaptation and/or in the maintainence of dentate granule cell integrity following seizures. INTRODUCTION Heat shock proteins (hsp) or 'stress' proteins comprise a family of evolutionarily conserved genes, some members being expressed constitutively, and others expressed following stressors such as elevated temperature or ischemia 7'2°. Though their function is still largely unknown, at least one class of heat shock proteins, those with molecular weights from 70-73 kDa, may serve a protective role to limit cellular damage or facilitate repair following stress or trauma. For instance competitive inhibition or injection of monoclonal antibodies against the heat-inducible hsp 72 protein impairs the ability of cultured cells to survive high temperatures 17'31. Heat shock proteins may limit cellular damage by preventing inappropriate aggregation of proteins resulting from heat denaturation and by facilitating correct folding of proteins to insure their proper functioning and transport within cells 1. The mammalian central nervous system expresses heat shock proteins in great abundance 4'29, and it has been postulated that hsp 72 may serve as a marker for neuronal injury 14. While elevated temperature causes an increase in hsp 72 which is largely confined to glial cells 3" 6,24, discrete brain lesions induce hsp 72 at the site of tissue injury in both glia and neurons 5. Transient global

ischemia induces hsp 72 gene expression in cortical and hippocampal neurons, particularly in the CA1 pryamidal layer known to be vulnerable to ischemic injury 28'33. Seizures also can cause increases in heat shock proteins in the CNS. Status epilepticus following kainic acid or flurothyl causes an increase in hsp 72 immunoreactivity particularly in the hippocampal pyramidal layer, dentate hilus and cerebral cortex 23'36. However, as the prolonged seizures examined in those studies may have also resulted in hypoxia, the increase in hsp 72 immunoreactivity may have been secondary to hypoxia rather than to the seizures themselves. In the study described here we asked the question whether milder forms of epileptiform activity not known to produce severe damage were sufficient to produce changes in the expression of heat shock protein genes in the CNS. We employed three seizure models: (1) electroconvulsive seizures (ECS) which cause generalized tonic-clonic convulsions, (2) electrical kindling, a model of temporal lobe epilepsy, in which daily electrical stimulation of the amygdala eventually produces a motor seizure, and (3) cocaine kindling in which daily administration of a subconvulsive dose of cocaine eventually induces a generalized seizure. Using in situ hybridization, we examined the effects of seizures on the expression of the constitutive member of the hsp 70 family (hsp

* Present address: Department of Psychiatry, Yale University School of Medicine, VA Medical Center, West Haven, CN, U.S.A. ** Present address: Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA, U.S.A. Correspondence: M.A. Smith, Clinical Neuroendocrinology Branch, NIH/NIMH, Bldg. 36, Room 2D-15, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A. Fax: (1) (301) 402-2200.

20 73) and the heat-inducible form (hsp 72) as well as hsp 84, a constitutively expressed member of the hsp 90 family. MATERIALS AND M E T H O D S

Electroconvulsive seizures Adult male Sprague-Dawley rats (Taconic Farms) were electrically stimulated (80 mA for 0.5 s) via earclips which resulted in tonic-clonic convulsions in all animals. Rats were sacrificed 2 h after their last seizure. Ear clips were attached to control animals which did not receive an electrical stimulation. Epileptiform activity lasted on average about 30 s.

sisting of 3SS-impregnated brain paste of known radioactivity were placed in X-ray cassettes along with 35S-sensitive film (r-max, Amersham) for 3-14 days and developed in D19 (Kodak) for 4 min at 20°C. The relative amount of probe hybridized was determined by measuring regional optical densities of film images with a computerized image analysis system composed of a light box, solid state video camera and a software package called I M A G E developed by Wayne Rasband at the NIMH. Optical densities for hybridization were determined using a threshold function which automatically measured the density above a specified background density. The values were then converted to dprrdmg wet weight using the standard curve generated by the 35S-standards. To determine anatomical localization of probe at the cellular level, sections were dipped in NTB-2 nuclear emulsion (Kodak) as described previously 15, exposed for 7 days, developed in D19 for 2 min at 16°C, and coun-

Amygdala kindling Bipolar platinum-iridium electrodes (Plastic Products no. MS 303/8) were sterotaxically implanted in the amygdala (AP 5.7, LAT 4.5, DV 2.5) 19 under chloral hydrate anesthesia. After a post-operative recovery period of one week, stimulation of the amygdala (800 p A peak to peak intensity, 60 Hz, biphasic square waves for 1 s duration 37 was administered once or twice daily over a period of 2 weeks. Animals had a first major motor seizure (Stages 3-5) 3o after an average of 9 stimulations (range = 3-15 stimulations), and kindled rats had an average of 9.5 major motor seizures prior to sacrifice. The afterdischarge duration averaged approximately 100 s in the kindled animals for the last five stimulations. Rats were sacrificed within 2 h, 24 h, or 3 weeks after the last kindled seizure. Kindled animals were compared to a sham kindled group which was implanted with electrodes and placed in the kindling apparatus but not stimulated.

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Cocaine kindling Rats received 40 mg/kg cocaine-HCl i.p. once daily until they developed a generalized seizure and were then sacrificed 2 h after the first seizure. Results were compared to a control group which received saline injections and another group which received cocaine but never developed a seizure. Cocaine-induced seizures resemble other limbic-type seizures but they tend to occur intermittently for a period of 10-30 min after injection of cocaine. Individual convulsions lasted from 10 to 30 s.

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In situ hybridization We used two synthetic 48-base oligodeoxyribonucleotide probes directed against rat constitutively expressed hsp 73, one directed against bases encoding nucleotides 3586-3633 in the coding region and another against bases 4209-4248 in the 3'-untranslated region 34. Two oligonucleotides for the heat inducible hsp 72 were also used, a 48 mer from the 3'-untranslated region (bp 1264-1303) of the murine gene a2 and a 30 mer from a highly conserved region of the human gene (amino acids 122-129) 16. A 48 mer complementary to the 5'-untranslated region of the murine hsp 84 gene was also used 26. Oligonucleotide probes were endlabelled with [a-3SS]dATP (>1000 Ci/mmol), (Dupont/NEN) using terminal deoxynucleotidyl transferase (25 U//A, Boehringer-Mannheim). In situ hybridization histochemistry was performed as previously described 38. Briefly, cryostat-cut frozen coronal brain sections (15 ~m thick) were fixed in 4% formaldehyde, treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% saline (pH 8.0) to reduce non-specific hybridization of the probe, dehydrated in increasing concentrations of ethanol, delipidated in chloroform, rinsed in ethanol, and air dried. Sections were hybridized overnight at 37°C with 5 x l0 s cpm of labeled probe per section. Then the non-specifically hybridized probe was removed by washing the sections in four 15 min rinses of 2 × SSC (1 × = 0.15 M NaC1/0.015 M sodium citrate, pH 7.2) containing 50% formamide at 40°C and two 30 min rinses of 1 × SSC at room temperature.

Analysis and statistics For analysis of hsp m R N A , the slides along with standards con-

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Fig. 1. Characterization of heat shock protein oligonucleotide probes. A: Northern analysis of 20 pg of poly(A) ÷ RNA extracted from whole rat brain, prepared by standard techniques 32, was used to characterize the 32p-labelled 3'-untranslated hsp 73 and hsp 84 probes. Both probes showed a single band, at 2.1 kb and 2.4 kb, respectively. B: autoradiograph of in situ hybridization using human hsp 72 probe (30 mer) in the right hippocampal region from a rat which had received an injection of saline into the dentate gyrus. C: autoradiograph of in situ hybridization with hsp 72 probe in the right hippocampus from a rat which had received an injection of 14 pg colchicine in the dentate gyrus 3 days previously. Note hybridization to CA1 region. Films in B and C were developed after 14 days exposure. Bar = 0.7 mm.

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Fig. 2. Autoradiograph of hsp 73 hybridization in sham (A) and an animal that had received 3 consecutive electroconvulsive seizures (ECS) (B). Note increase in optical density in the dentate granular layer (DG) in the kindled animal. Bar = 1 mm.

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terstained with Cresyl violet. Statistical significance between the sham and seizure group was determined by analysis of variance (ANOVA) using Apple computer statistical packages. Following ANOVA, the Tukey-Kramer post hoc test was performed to determine differences between seizure groups and sham. Results are expressed as the mean -+ standard deviation (S.D.).

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Fig. 3. Hsp 73 mRNA expression in the dentate gyrus following 1 or 3 electroconvulsive seizures (ECS). Results are presented as mean -+ S.D. and are significantly different from sham at **P < 0.01.

I n situ h y b r i d i z a t i o n r e v e a l e d t h a t t h e c o n s t i t u t i v e l y expressed heat shock mRNAs, distributed throughout

h s p 73 a n d h s p 84, w e r e

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roughly proportional to the neuronal density. Northern

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m Fig. 4. Photomicrographs of hsp 73 mRNA hybridization in neurons of the dentate gyrus granular layer from control (A) and an animal sacrificed 2 h after the last amygdala-kindled seizure (B) and in the CA1 pyramidal layer of the hippocampus from the same control (C) and kindled (D) animals. Note increase in black silver grains over dentate granule cell neurons in the kindled animal (B) indicating increased hsp 73 mRNA. Bar = 15 ~ m .

analysis with the hsp 73 and 84 probes revealed single bands, at 2.1 kb and 2.4 kb, respectively. See Fig. 1A. In contrast, hybridization of the hsp 72 probe was at or below the limits of detection in brains from control animals as determined by Northern analysis or in situ hybridization (Fig. 1B). However, the probe has previously been characterized and shown to be specific for the heat inducible hsp 72 gene 24. Moreover, in our hands this probe was able to detect a signal in the rat hippocampus following damage by an injection of colchicine which is known to completely destroy the dentate granule cells ~2 and do partial damage to the hippocampal pyramidal cells zl. At 3 days after colchicine treatment the signal was most prominent in the CA1 portion of the pyramidal layer which is known to express hsp 72 m R N A for a prolonged period of time after damage 28. See Fig. 1C. Electroconvulsive seizures

Electroconvulsive seizures caused a significant increase in the expression of constitutive hsp 73 m R N A in the granule cell layer of the dentate gyrus (Fig. 2). No con-

sistent changes in hsp 73 m R N A occurred in the pyramidal layer of the hippocampus or cerebral cortex although there were trends towards increases in these areas after a seizure. In contrast to the changes in hsp 73, neither the heat-inducible hsp 72 gene nor hsp 84 in-

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Fig. 5. Hsp 73 mRNA expression in the dentate gyrus following amygdala-kindled seizures. Results are presented as mean ± S . D . and are significantly different from sham at * * P < 0 . 0 1 .

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Fig. 6. Autoradiograph of hsp 73 hybridization in saline injected control (A) and an animal that was sacrficed 2 h after a cocaine kindled seizure (B). Note increase in optical density in the dentate granular layer (DG) in the kindled animal. Same magnification as Fig. 2.

creased significantly in the granule layer of the dentate gyrus following 1 or 3 consecutive ECS (Fig. 3). Only one of the animals experiencing 3 consecutive ECS appeared to express hsp 72 in the brain, and this was confined to the hilar interneurons of the dentate gyrus and in the CA3 and CA4 region of the hippocampal pyramidal layer. No changes in hsp 84 were observed in the hippocampus or cerebral cortex in any of the animals.

Amygdala and cocaine kindling Electrical kindling of the amygdala also induced expression of the constitutive hsp 73 gene in the dentate gyrus (Fig. 4). The increased levels of hsp 73 m R N A were present in animals sacrificed within 2 h of the last seizure but returned to baseline within 24 h and remained at baseline 3 weeks later (Fig. 5). Again no changes in hsp 73 occurred outside the granular layer of the dentate gyrus (see Fig. 4), and no changes in hsp 72

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Fig. 7. Hsp 73 m R N A expression in the dentate gyrus following cocaine-kindled seizures or cocaine alone without seizures. Results are presented as m e a n -+ S.D. and are significantly different from saline at *P < 0.05.

or 84 were observed in any brain region. Cocaine kindled seizures also caused an increase in hsp 73 mRNA in the dentate gyrus (Figs. 6 and 7). Again the increase in hsp 73 was transient and did not occur outside the dentate gyrus. In those animals which were given repeated injections of cocaine but were sacrificed before seizures developed, no increase in hsp 73 expression was observed. Cocaine seizures did not produce any changes in hsp 72 or hsp 84. DISCUSSION

In contrast to previous studies which have demonstrated an increase in the heat-inducible hsp 72 protein following a severe stressor such as status epilepticus 4'23' 36, our results suggest that the member of the hsp 70-73 kDa family most affected by seizure activity of short duration may be the 'constitutive' hsp 73 gene. Previous studies noted that seizures of short duration were not capable of increasing the heat-inducible hsp 72 protein 36, and thus it is not surprising that we too failed to observe a significant increase in expression of hsp 72 mRNA using our seizure models. Another notable difference between our results and those previously reported is that we observed the most consistent increases in heat shock protein gene expression in the granular layer of the dentate gyrus rather than in the hippocampal pyramidal cells. The dentate granular layer is an area relatively resilient to the damaging effects of ischemia or status epilepticus. Though transient anoxia may occur during a tonic-clonic convulsion, there is little evidence for irreversible CNS dam-

age following brief seizures other than to dentate hilus interneurons 8. Hsp 72 immunoreactivity is readily induced in the dentate hilar interneurons as well as in the CA3 portion of the hippocampal pyramidal layer but does not normally appear in the dentate granule layer unless there is more severe tissue damage 2333. Thus our results raise the possibility that the increase in the constitutive hsp 73 mRNA in the granule cells of the dentate gyrus may be a response to the seizures themselves rather than a compensatory response to hypoxia or tissue damage. Though the promoter region of the hsp 73 gene contains a weakly active heat shock element 34, other regulatory elements may be responsible for the induction of hsp 73 mRNA after a seizure. It has been shown that hsp 73 can be induced by some stimuli in cultured cells 34 so this gene may not be strictly 'constitutive'. The increase in hsp 73 in the dentate granule cells resembles the increase in the c-los proto-oncogene that occurs in these same cells following various types of seizures or ischemia w'27'28. C-los is a modulator of transcription, but as the hsp 73 promoter does not contain an AP-1 site, c-fos and hsp 73 are probably not mechanistically linked. The fact that hsp 70 proteins have short half lives 25 may explain why the increases in hsp 73 mRNA had returned to baseline within 24 h of the last seizure. Kindling is strictly dependent on protein synthesis is, and enlargements of postsynaptic densities on dentate granular neurons have been observed following kindled seizures t~. Delivery of newly synthesized proteins to their proper destination would also be an important component of putative changes in synaptic remodelling that might occur during kindling. Transport of proteins to cellular membranes involves clathrin-coated vesicles shuttling back and forth ~3, and it has been previously suggested that the constitutive form of heat shock protein 70 functions as an ATP-dependent uncoating enzyme that releases clathrin triskelia from coated vesicles 9'35. Indeed recent evidence strongly suggests that hsp 73 protein is bound to clathrin during axonal transport 2. This raises the possibility that hsp 73 could be important in transporting correctly folded proteins to plasma membranes following seizures. Increased expression of hsp 73 mRNA may be important to insure an adequate supply of hsp 73 protein during the extraordinary demands made on neurons by seizure activity.

Acknowledgements. The authors thank Allison Lynn for the colchicine-treated animals and appreciate the guidance and support of Miles H e r k e n h a m and the statistical advice of John Bartko.

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