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In vivo regulation of glial cell line-derived neurotrophic factor-inducible transcription factor by kainic acid
GIF induction by kainic acid
Pergamon PII: S0306-4522(99)00302-4
Neuroscience Vol. 94, No. 2, pp. 629–636, 1999 629 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
IN VIVO REGULATION OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR-INDUCIBLE TRANSCRIPTION FACTOR BY KAINIC ACID A. J. EISCH,* C.-H. LAMMERS,†‡ S. YAJIMA,†§ M. M. MOURADIAN† and E. J. NESTLER*k *Laboratory of Molecular Psychiatry, Departments of Psychiatry and Neurobiology, Yale University School of Medicine, Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508, U.S.A. †Genetic Pharmacology Unit, Experimental Therapeutics Branch, National Institute of Neurological Diseases and Stroke, Building 10, Room 5C116, Center Drive, MSC1406 Bethesda, MD 20892-1406, U.S.A.
Abstract—A putative transcription factor induced in vitro by glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-beta was recently cloned and characterized [Yajima S. et al. (1997) J. Neurosci. 17, 8657–8666]. The messenger RNA of this protein, termed murine GDNF-inducible transcription factor (mGIF, hereafter referred to as GIF), is localized within cortical and hippocampal regions of brain, suggesting that GIF might be regulated by perturbations of these brain regions. In an effort to learn more about the role of GIF in vivo, we examined GIF messenger RNA in the brains of rats treated with the glutamatergic agonist kainic acid. This treatment is known to induce seizures and alter the messenger RNA expression of several growth factors, including GDNF, in several brain regions. Rats were given intraperitoneal saline (1 ml/kg) or kainic acid (15 mg/kg) and were killed at various time-points for in situ hybridization of brain sections with a GIF messenger RNA riboprobe. In salinetreated rats, GIF messenger RNA was present at low levels in cerebral cortex, hippocampus and hippocampal remnants such as the taenia tecta. Kainic acid treatment induced robust increases in GIF messenger RNA in several brain regions, including cerebral cortex, hippocampus, caudate–putamen, nucleus accumbens, and several nuclei of the amygdala and hypothalamus. Most brain regions showed the greatest increase in GIF messenger RNA 4–6 h after kainic acid administration and a return towards normal levels at 48 h. The CA3 region of hippocampus, however, showed a more rapid increase in GIF messenger RNA that was also evident 48 h after kainic acid administration. These results demonstrate that GIF messenger RNA can be regulated in vivo, and that this novel factor warrants further study as a central mediator of GDNF and perhaps other neurotrophic factors. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: GDNF, TGF-b, seizure-regulation, hippocampus, accumbens, in situ hybridization.
Initial work with glial cell line-derived neurotrophic factor (GDNF) characterized this distant member of the transforming growth factor-b (TGF-b) superfamily as a survival and morphological differentiation factor for midbrain dopaminergic neurons. 23,42 It is now known that GDNF has trophic, survival, and differentiation effects on a variety of cell types, including noradrenergic neurons and several classes of motor and peripheral neurons. 3,9,34 The neurotrophic and supportive effects of GDNF have encouraged consideration of GDNF’s therapeutic potential in treating neurodegenerative disorders. Indeed, GDNF protects and restores the function of dopaminergic, noradrenergic, motor and hippocampal neurons in animal models of Parkinson’s disease, motor neuron degeneration, limbic seizure and ischemia. 3,13,16,26,44,53,54,56 Additional therapeutic interest in GDNF comes from studies suggesting that GDNF and other neurotrophins may play a role in the selective neuronal vulnerability, axon remodeling and synaptoneogenesis seen after epileptic insults. For example, a seizureinducing dose of kainic acid up-regulates the mRNA for
GDNF and two of its receptor components, c-ret and GFRa-1, in regions of hippocampus that show seizure-induced axonal sprouting. 18,37,49 While several receptor components for GDNF have been identified, 10,20,21,46,48 little is known about the cascade of intracellular events triggered by GDNF receptor activation. Recent work identified a novel mRNA species that was induced in cell culture by GDNF and TGF-b. 55 Designated murine GDNF inducible transcription factor or mGIF (hereafter referred to as GIF), this zinc-finger protein was cloned and characterized in the process of searching for proteins that bind to consensus sequences of the Sp1 DNA response element. The protein sequence of GIF shows a high degree of homology to TGF-b inducible early gene and early growth response gene-a, proteins with Sp1-like zinc finger motifs. 7,43 In vitro, GIF mRNA is induced by GDNF and TGF-b in both a murine cell line and primary cultures from rat cortical neurons. While no regulation studies have been done in vivo, distribution studies of GIF mRNA in mouse brain reveal that it is present at low levels in cerebral cortex and hippocampal regions. Given the distribution of GIF mRNA in brain, and its ability to be induced by GDNF in vitro, we examined whether perturbations of hippocampus and cerebral cortex cause alterations in GIF mRNA expression in vivo. Seizures induced by electroshock, kindling, or systemic or central administration of chemical convulsants produce neuronal damage and axon remodeling in the hippocampus accompanied by alterations in immediate-early genes, growth factors, and receptors for growth factors in several brain areas, including hippocampus. 5,12 Previous work has established the temporal and regional alterations in mRNA for GDNF and its receptor
‡Present address: Zentrum fu¨r Nervenheilkunde, Klink fu¨r Neurologie Station 3B, Rudolf-Bultmann Strasse 8, 35039 Marburg, Germany. §Present address: Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan. kTo whom correspondence should be addressed. Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-isoxazolepropionic acid; CA1 and CA3, Ammon’s horn, areas 1 and 3; DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetate; GDNF, glial cell line-derived neurotrophic factor; GIF, murine glial-derived neurotrophic factor-inducible transcription factor; PBS, phosphate-buffered saline; SSC, standard saline citrate; TGF-b, transforming growth factor-beta; UTP, uridine triphosphate. 629
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described previously. 14 Sections were washed twice in 0.75% glycine in 0.1 M phosphate buffer followed by permeabilization with Proteinase K (1 mg/ml in 0.1 M Tris, pH 8.0, and 50 mM EDTA) for 30 min at 378C. Sections were then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min and rinsed in 2 × standard saline citrate (SSC) until probe application. The 578 base pair murine GIF antisense and sense riboprobes were labeled with 35S-uridine triphosphate (UTP) by in vitro transcription using T7 and SP6, respectively. 55 Resulting riboprobes were purified using a Stratagene NucTrap Column (La Jolla). Tissue sections were incubated overnight at 558C in 1 ml of hybridization buffer [50% formamide, 35 × Denhardt’s, 10% dextran sulfate, 0.14 × SSC, 30 mg/ml denatured salmon sperm DNA, 0.15 mg/ml yeast tRNA, 20 mM dithiothreitol (DTT) and 10 7 c.p.m./ ml 35S-UTP-labeled GIF antisense or sense riboprobe]. The following day, sections were washed with decreasing concentrations of SSC buffer (2–0.5 × ) containing 20 mM DTT at room temperature, two high-stringency washes in 0.1 × SSC with 20 mM DTT at 658C for 30 min each, and final rinses in 0.1 × SSC with 20 mM DTT. Sections were then mounted out of 0.1 M Tris on to Vectabond-treated slides (Vector) and air-dried. Autoradiography Fig. 1. Autoradiographs of rat brain at the level of frontal cortex showing localization of GIF mRNA. Shown is antisense labeling of GIF mRNA 4 h after injection with saline (1 ml/kg, i.p.) or kainic acid (15 mg/kg, i.p.) and 48 h after kainic acid. Sense labeling after injections of either saline (not shown) or kainic acid (4 h time-point shown) was not different from background labeling. AI, agranular insular cortex; d, deep layers of cerebral cortex; Fr, frontal cortex; KA, kainic acid; mPF, medial prefrontal cortex; Pir, piriform cortex; OF, orbitofrontal cortex; s, superficial layers of cerebral cortex; TT, taenia tecta.
components induced by kainic acid. 18,37,49 Therefore, in the present study we examined GIF mRNA at several time-points after kainic acid administration. Our results demonstrate dramatic regulation of GIF mRNA expression in the brain in vivo. EXPERIMENTAL PROCEDURES
Animals, drug treatment and tissue preparation Male Sprague–Dawley rats (250–275 g, Charles River Laboratories, Wilmington, MA) were used for all experiments. Rats were group-housed under conditions of 12 h of light per day in a vivarium approved by the American Association for the Accreditation of Laboratory Animal Care. Food and water were available ad libitum. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 865-23) and were approved by the Yale Animal Care and Use Committee. Rats were given i.p. injections of either 0.9% sodium chloride (saline; 1 ml/kg) or kainic acid (15 mg/kg, Sigma). This dose of kainic acid is known to induce generalized seizures in rats. 5,39,40 Kainic acid was dissolved 5 mg/ml in saline and administered 3 ml/kg. To verify seizure induction by kainic acid, behavioral observations were made every 30 min for 6 h after saline or kainic acid injection. All kainic acid-treated animals, with the exception of those perfused 45 min postinjection, displayed generalized limbic seizure activity within 90 min following kainic acid administration (data not shown). Saline animals (n 4) were perfused 4 h after injection. Kainic acid animals were perfused at 45 min (n 3), 2 h (n 5), 4 h (n 4), 6 h (n 6), 24 h (n 4) or 48 h (n 4) after injection. Prior to perfusion, rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p., Abbott Laboratories) and were exsanguinated with cold 0.1 M phosphate-buffered saline, pH 7.4 (PBS; 75 ml, 15 ml/min) followed by fixation with 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (255 ml, 15 ml/min). Brains were removed and postfixed overnight in 4% paraformaldehyde and then cryoprotected in 30% sucrose in 0.1 M PBS. Parallel series of 30 mm coronal brain sections were cut on a freezing microtome and stored in 4% paraformaldehyde at 48C. In situ hybridization Free-floating sections were processed for in situ hybridization as
The GIF hybridization signal was visualized by both film (B-Max; Amersham) and emulsion (NTB2; Kodak) autoradiography. Sections hybridized to GIF mRNA were exposed for six to 14 days on film and nine weeks on emulsion. Following development of emulsion autoradiograms, sections were counterstained with Cresyl Violet and coverslipped with DPX (Aldrich). Image analysis and statistical methods NIH Image (Bethesda) was used to collect images for qualitative and semi-quantitative examination of GIF mRNA expression. Semi-quantitative measurements were established for six brain regions: frontal cortex, nucleus accumbens core, nucleus accumbens shell, caudate– putamen, and dentate gyrus and CA3 of hippocampus. The NIH Image outline tool was used to circumscribe dentate gyrus (including ventral and dorsal horn), CA3, caudate–putamen and the entire frontal cortex (including claustrum and cingulate, infralimbic, frontal, parietal, agranular insular, lateral orbital and ventral lateral orbital cortices but excluding the forceps minor of the corpus callosum). The square sampling tool was used to measure core and shell of nucleus accumbens. Each measurement was corrected for background by subtracting the value of an unlabeled brain region within the same tissue section (e.g., corpus callosum). Optical density values for an individual animal were calculated by averaging the corrected measurements from six to 12 tissue sections for each brain region. The resulting corrected grey values for each brain region are reported as means and S.E.M. The corrected grey value means for each brain region were analysed using a one-way ANOVA. When the overall analysis showed a main effect of time, individual time-points were compared post hoc using the Fischer’s LSD test. The level of significance for analysis was set as P , 0.05. Subscripts after F-values indicate degrees of freedom for each test. RESULTS
Saline-treated animals The antisense riboprobe for GIF mRNA hybridized to several brain regions of saline-treated rats, while the sense probe did not (Figs 1–3). Regions that displayed a consistent signal for GIF mRNA above background include dentate gyrus and Ammon’s horn (CA1–3) of hippocampus, frontal, anterior cingulate, and piriform cortices, amygdala and taenia tecta. Within cerebral cortical regions, deeper layers of cortex showed slightly greater signal than superficial layers, and medial and lateroventral regions (cingulate, ventral lateral orbital, and lateral orbital cortices) showed slightly greater signal than laterodorsal cortices (anterior parietal cortex; Fig. 1). No significant GIF mRNA signal was observed in other gray matter regions (e.g., caudate–putamen, nucleus accumbens, thalamus or ventral mescencephalon, including
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substantia nigra pars compacta and pars reticulata). This pattern of GIF mRNA in rat brain is similar to that described in the mouse. 55
Kainic acid-treated animals
Fig. 2. Autoradiographs of rat brain at the level of caudate–putamen and nucleus accumbens showing localization of GIF mRNA. Shown is antisense labeling of GIF mRNA 4 h after injection with saline (1 ml/kg, i.p.) or kainic acid (15 mg/kg, i.p.) and 48 h after kainic acid. Sense labeling after injections of either saline (not shown) or kainic acid (4 h time-point shown) was not different from background labeling. AcbSh, nucleus accumbens shell; Cg, cingulate cortex; Cl, claustrum; CPu, caudate–putamen; d, deep layers of cerebral cortex; Fr, frontal cortex; KA, kainic acid; Par, parietal cortex; Pir, piriform cortex; s, superficial layers of cerebral cortex; TT, taenia tecta; TuPy, olfactory tubercle, pyramidal layer.
In contrast to the low basal levels of GIF mRNA seen in the brains of saline-treated rats, kainic acid-treated animals showed robust increases in GIF mRNA in a time- and region-dependent manner (Figs 1–4). Regions that displayed the greatest increase in GIF mRNA signal include frontal cortex, caudate–putamen, core and shell of nucleus accumbens, and dentate gyrus and CA1–3 of hippocampus. For clarity, the time-dependent alterations in GIF mRNA levels in these brain areas are described in separate sections below. Increases in GIF mRNA signal were also observed, but not quantified, in several other brain regions. Consistent increases in GIF mRNA levels were observed in olfactory structures (e.g., pyramidal layer of olfactory tubercle) and frontal– parietal cortices 4–6 h after kainic acid (Figs 1, 2). In more caudal sections, the kainic acid-induced GIF mRNA signal was evident in motor and somatosensory cortices (e.g., forelimb, hindlimb, parietal cortices I and II) yet was distinctly absent in retrosplenial cortices 4–6 h after kainic acid (Figs 2, 3). In addition, the ventromedial hypothalamic nuclei showed induction of GIF mRNA 4–48 h after kainic acid administration (Fig. 3). Finally, the lateral complex of the amygdala (lateral, basolateral, basomedial) and the habenular nuclei showed GIF induction 6–48 h after kainic acid (Fig. 3). No alteration in GIF mRNA signal was observed in regions containing dopaminergic cell bodies, such as the substantia nigra, pars compacta or pars reticulata, or ventral tegmental area.
Frontal cortex
Fig. 3. Autoradiographs of rat brain at the level of hippocampus showing localization of GIF mRNA. Shown is antisense labeling of GIF mRNA 4 h after injection with saline (1 ml/kg, i.p.) or kainic acid (15 mg/kg, i.p.) and 48 h after kainic acid. Sense labeling after injections of either saline (not shown) or kainic acid (4 h time-point shown) was not different from background labeling. AcbSh, nucleus accumbens shell; AI, agranular insular cortex; Am, amygdala; CA1 and CA3, Ammon’s horn, areas 1 and 3; CPu, caudate–putamen; DG, dentate gyrus; Hb, habenula; KA, kainic acid; Par, parietal cortex; Pir, piriform cortex; Rs, retrosplenial cortices; VMH, ventral medial hypothalamic nuclei.
GIF mRNA levels in frontal cortex were dramatically increased by kainic acid administration. As shown in Fig. 1, GIF mRNA signal was increased in frontal cortex by 4 h and began to return to basal levels by 48 h. While all frontal cortical regions showed an increase in GIF mRNA signal 4 h after kainic acid, certain subregions displayed qualitatively more GIF mRNA signal than others. For example, superficial layers of cerebral cortex generally showed a greater increase in GIF mRNA signal after kainic acid than deeper layers (Fig. 1). In addition, the kainic acid inducedincrease in GIF mRNA signal appeared to be greater in limbic-related regions, such as medial prefrontal (cingulate), infralimbic, agranular insular and orbitofrontal (lateral orbital and ventral lateral orbital) cortices, than in motor and sensory-related cortices, such as frontal and anterior parietal cortices (Fig. 1). A one-way ANOVA on the corrected grey values from all time-points showed that the time-dependent increase in GIF mRNA levels in frontal cortex was highly significant (Fig. 4; F6,30 11.05; P , 0.01). Post hoc analysis showed that the kainic acid-induced increase in GIF mRNA signal was significantly greater than saline at 4 h (688% of saline levels), 6 h (800%) and 24 h (544%; all P’s ,0.01). In addition, GIF mRNA signal 48 h after kainic acid was significantly less than the signal at 4 h (P , 0.05) and 6 h (P , 0.01).
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Fig. 4. Systemic administration of kainic acid increases GIF mRNA in a time-dependent manner in several regions of rat brain. Each graph depicts the mean corrected grey value and S.E.M. for saline-treated animals and six time-points after 15 mg/kg i.p. kainic acid (45 min, 2, 4, 6, 24 and 48 h). A one-way ANOVA showed a significant effect of time for all brain regions. Results of post hoc tests are indicated in each graph by asterisks and brackets. Asterisks indicate significant difference from saline-treated value within the same brain region: **P , 0.01; *P , 0.05. Brackets over bars and associated P-values indicate significant difference from either 48 h after kainic acid (frontal cortex, nucleus accumbens core and shell, dentate gyrus and CA 3) or from 45 min or 2 h after kainic acid (caudate–putamen).
Nucleus accumbens The nucleus accumbens also displayed a dramatic kainic acid-induced increase in GIF mRNA levels. While both core and shell showed an increase in GIF mRNA signal 4 h after kainic acid administration, this effect appeared qualitatively greater in shell (Fig. 2). Quantification of the GIF mRNA signal in these regions confirmed this observation (Fig. 4 and below). A one-way ANOVA on the corrected grey values showed that the time-dependent increase in GIF mRNA in nucleus accumbens was highly significant in both core
(F6,30 5.45; P , 0.01) and shell (F6,30 10.43; P , 0.01). For core, post hoc analysis showed that the kainic acidinduced increase in GIF mRNA signal was significantly greater than saline levels at 4 h (1082% of saline levels; P , 0.01) and 6 h (936%; P , 0.05). In addition, GIF mRNA signal in core at 48 h was significantly less than the signal at 4 h (P , 0.01) and 6 h (P , 0.05), indicating a return towards control levels. For shell, post hoc analysis showed that the kainic acid-induced increase in GIF mRNA signal was significantly greater than saline at 4 h (2250% of saline levels; P , 0.01) and 6 h (1090%; P , 0.05). In addition, GIF
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Fig. 5. Emulsion autoradiographs of rat brain at the level of hippocampus showing localization of GIF mRNA. Low-magnification dark-field photomicrograph (left panel) shows dense labeling of GIF mRNA 4 h after kainic acid (15 mg/kg, i.p.) in the dentate gyrus granule cell layer, CA 1–3 and cortex. Adjacent panel (right) shows high magnification of cortical field under bright-field microscopy. Emulsion grains appear over large, pale nuclei but not over small, dark nuclei (neurons, large arrows; glia, small arrows).
mRNA signal in shell at 48 h was significantly less than the signal at 4 h (P , 0.01) and 6 h (P , 0.05). Caudate–putamen In contrast to the large effect of kainic acid in frontal cortex and nucleus accumbens, kainic acid administration produced a small, but notable, increase in GIF mRNA signal in caudate–putamen. The GIF mRNA signal was increased 4 h after kainic acid injection, and returned to basal levels by 48 h. As evident in Fig. 2, the kainic acid-induced GIF mRNA signal in caudate–putamen was greatest in the ventral and medial sectors and least in the dorsal and lateral sectors. A one-way ANOVA of GIF mRNA values in caudate–putamen at all time-points showed a significant effect of time (Fig. 4; F6,30 3.06; P , 0.05). While kainic acid increased the GIF mRNA signal in caudate–putamen relative to saline values at 4 h (688% of saline), 6 h (750%) and 24 h (538%) after injection, post hoc analysis did not show significance for these comparisons (all P . 0.05). Post hoc analysis did show a significant difference between 4 h and 2 h animals (P , 0.05), and between 6 h and both 45 min and 2 h animals (both P’s ,0.05). Hippocampus Kainic acid induced very large increases in GIF mRNA levels in hippocampus (Fig. 3). While both the pyramidal cell layer (CA1–3) and the granule cell layer (dentate gyrus) showed increases in GIF mRNA signal 4 h after kainic acid, the dentate gyrus increase appeared to be much greater than that in CA1–3. By 48 h after kainic acid, the dentate gyrus signal had decreased somewhat but still appeared elevated relative to saline-treated animals. In CA1–3, in contrast, GIF mRNA appeared to increase further at 48 h relative to 4 h after kainic acid. For quantitation of these kainic acid-induced alterations, the entire dentate gyrus and CA3 region of Ammon’s horn were analysed separately to demonstrate kainic acid’s effect on granular and pyramidal cell layers, respectively. A one-way ANOVA on corrected grey values showed that the time-dependent increase in GIF mRNA signal in hippocampus was highly significant in both dentate gyrus (F6,30 27.88; P , 0.01) and CA3 (F6,30 759.29; P , 0.01). As evident from the time-course shown in Fig. 4, however, the pattern of GIF mRNA induction
in each region was distinct. For dentate gyrus, post hoc analysis showed that the increase in GIF mRNA was significantly greater than saline at 4 h (979% of saline levels; P , 0.01), 6 h (744%; P , 0.05), 24 h (447%; P , 0.05) and 48 h (413%; P , 0.05) after kainic acid. In addition, GIF mRNA signal at 48 h was significantly less than the signal at 4 h (P , 0.01), and 6 h (P , 0.01). In contrast, post hoc analysis of CA3 corrected grey values showed that the increase in GIF mRNA was significantly greater than saline at 45 min (377% of saline levels; P , 0.01), 4 h (378%; P , 0.01), 6 h (402%; P , 0.05), 24 h (344%; P , 0.05) and 48 h (559%; P , 0.05) after kainic acid. In addition, GIF mRNA signal at 48 h was significantly greater than the signal at 45 min (P , 0.05), 4 h (P , 0.01), 6 h (P , 0.05), and 24 h (P , 0.01). Cellular localization Kainic acid treatment is known to induce glial hypertrophy and microglial proliferation, 33 and GDNF was initially identified from a glial cell line; therefore, the phenotype of cells expressing GIF mRNA was determined using emulsion autoradiography. In saline-treated animals, grains appeared to be localized over large, pale nuclei, presumably neurons. Grains evident over small, dark nuclei, presumably glia, were not more numerous than background labeling. Kainic acid-treated animals (4–48 h time-point) also showed prefential distribution of emulsion grains over neurons (Fig. 5). For example, superficial layers of parietal cortex showed dense, clustered grains which enabled identification of cellular phenotype (Fig. 5). All grains appeared to be localized over large, pale neuronal nuclei with no grains evident over small, dark glial nuclei. While the high density of grains in the dentate gyrus (Fig. 5) precluded identification of the phenotype of individual cells, the label is presumably neuronal since the dentate gyrus is known to be composed primarily of neurons, not glia. 2,4,8 DISCUSSION
We show here that mRNA levels of the novel, putative transcription factor GIF are dramatically increased in rat brain after systemic kainic acid administration. Most brain regions analysed—frontal cortex, caudate–putamen, nucleus accumbens core and shell, and dentate gyrus—display
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maximum GIF mRNA levels 4–6 h after kainic acid and return towards control values by 48 h. In contrast, the CA3 region of the hippocampus shows a rapid increase in GIF mRNA 45 min after kainic acid that generally persists until 48 h. The pattern of GIF mRNA induction shows some overlap with the distribution of kainic acid receptors in the rat brain. 6,24,31,35,50 However, some brain regions rich in kainic acid binding sites, such as dorsal caudate–putamen, 1 show only a small increase in GIF mRNA following kainic acid, while others, such as the dentate gyrus and nucleus accumbens, show much larger increases in GIF mRNA. Such a pattern suggests that GIF induction is not simply a direct consequence of kainic acid receptor activation. Similarly, while evidence suggests that kainic acid may mediate some of its effects via the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, 30 there is also a lack of correspondence between AMPA, receptor distribution and kainic acid induction of GIF mRNA. 14,30 Rather, the spatiotemporal pattern of GIF induction appears to be a precisely co-ordinated event, resulting not only from direct drug effects at kainate or AMPA receptors, but also from the subsequent neuronal and epileptiform activity. Relationship of murine glial cell line-derived neurotrophic factor-inducible transcription factor messenger RNA induction to neuronal degeneration One major consequence of kainic acid-stimulated neuronal activity is selective neuronal degeneration. 5 However, the changes in metabolic and electrical activity 47 and the wellestablished pattern of neuronal injury 5,39,40 that accompany kainic acid administration show only partial similarities to the specific pattern of GIF induction described here. For example, kainic acid increases GIF mRNA in brain regions known to be resistant to excitotoxic damage (dentate gyrus, caudate–putamen, nucleus accumbens, frontal cortex) as well as in regions vulnerable to this damage (CA3 of hippocampus). This mixed pattern of induction suggests that regulation of GIF is complex, and further work is needed to establish the role that GIF may play in the neurodegenerative effects of kainic acid. Relationship of murine glial cell line-derived neurotrophic factor-inducible transcription factor messenger RNA induction to neurotrophic factor induction An additional consequence of kainic acid is the neuronal remodeling that takes place subsequent to the neuronal injury. A growing body of evidence suggests that seizure-induced neuronal sprouting and synaptoneogenesis observed in hippocampal subregions are linked to seizure-induced alterations in neurotrophins (nerve growth factor, brain-derived neurotrophic factor) 11,52 and, more recently, GDNF and its receptors. 18,37,49 Given recent work showing GIF induction in vitro by GDNF and TGF-b, the region- and time-specific pattern of GIF regulation by kainic acid urges comparison with the pattern of GDNF and related mRNAs after kainic acid. The pattern of GDNF mRNA induction in the dentate gyrus after kainic acid does complement the pattern of GIF mRNA reported here. GDNF mRNA increases in dentate granule cells 3 h after kainic acid, reaches maximal levels at 6 h, and returns to control values by 24 h. 18 While it remains to be determined whether this increase in GDNF mRNA leads to
a corresponding increase in GDNF protein, it is reasonable to consider that increased GDNF in dentate gyrus may underlie the increased GIF expression seen in this region 4–6 h after kainic acid. The fact that receptor components for GDNF are also increased in dentate gyrus after kainic acid is consistent with this idea. 37,49 In contrast to the induction of GDNF mRNA seen in the dentate gyrus, extra-hippocampal induction of GDNF mRNA by kainic acid has not been reported. This raises the question of what signal is responsible for the increase in GIF mRNA seen in other brain regions, such as caudate–putamen, nucleus accumbens and frontal cortex. Two possibilities deserve consideration. First, it is possible that technical barriers have prevented detection of kainic acid-induced increases in GDNF in these regions. Initial studies in the adult rat brain found low or negligible levels of GDNF mRNA in the caudate–putamen, hippocampus and cortex. 38,41,42 More recent examination of GDNF mRNA distribution, however, shows that GDNF mRNA is expressed in several regions of the normal adult rat brain, including caudate–putamen, nucleus accumbens, thalamic nuclei, olfactory regions, hippocampus, cerebellum and cerebral cortex. 36,49 In addition, glutamatergic stimulation of the striatum with kainic acid has been shown to increase GDNF mRNA in this region in vivo. 15 Therefore, it is possible that previously undetected increases in GDNF mRNA are responsible for the kainic acid induced increases in GIF mRNA in the caudate–putamen and other regions. Finally, GDNF has been shown to be a target-derived trophic factor, perhaps eliminating the need for regional correspondence between GIF mRNA and GDNF mRNA. 45 An alternative explanation is that GIF mRNA is regulated in certain brain regions by a TGF-b-related signal other than GDNF. Several isoforms of TGF-b have been identified and localized to the adult CNS. 28,29,51 Indeed, TGF-b2- and TGFb3-immunoreactive neurons are found in the hippocampus, cerebral cortex, hypothalamus and amygdala of the adult rat. While this localization does not completely overlap with the unique pattern of GIF expression described here, these isoforms of TGF-b should be considered for future evaluation of their role in the kainic acid-induced regulation of GIF. A third isoform, TGF-b1, is increased after kainic acid, 32 but the basal signal and the induced signal are generally limited to macrophages within hippocampus. Other candidates that might stimulate GIF mRNA include neurturin and persephin, trophic factors that, with GDNF, comprise the TGF-b-related neurotrophic factor superfamily. 22,27 Initial studies of the regional distribution of neurturin and persephin in the rat brain, however, do not suggest extensive correspondence with the distribution of basal or kainic acid stimulated levels of GIF mRNA. 17,19 It is also feasible that GIF mRNA is regulated by the binding of an undiscovered TGF-b-like factor to one of the many signaling receptors linked to the TGF-b superfamily. For example, TGF-b type I-like receptor, habrec1, shows significant mRNA expression in the striatum, cerebral cortex, and olfactory tubercle, 25 and thus may provide a signaling receptor for regulation of GIF mRNA. Given GIF’s position downstream from GDNF and TGF-b, it is likely that more than one of these trophic factors and/or receptor components is involved in regulation of GIF expression. Further work is needed to establish whether GIF is indeed a site of convergence of various TGF-b-related signaling pathways.
GIF induction by kainic acid CONCLUSIONS
As a transcription factor induced by GDNF and TGF-b, GIF is a primary candidate for mediating intracellular effects of GDNF, related neurotrophic factors and their associated signaling receptors. The identification of paradigms in which GIF mRNA levels are greatly altered in specific brain regions is an essential step to elucidating the physiological role of GIF. The present work establishes kainic acidinduced seizures as the first such paradigm that can regulate GIF mRNA in vivo. The lack of correspondence between the induction of GIF mRNA and GDNF mRNA suggests that GIF expression may be regulated via other TGF-b-related signals,
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such as TGF-b, neurturin or persephin, and/or other receptor components, such as Habrec1. While further work must be done to evaluate the hypothesis that GIF serves as one final common pathway in the signal-transcription coupling events in neurons sensitive to TGF-b-related factors, the present work suggests a multifaceted role for GIF in the injury and plasticity induced by kainic acid.
Acknowledgements—This work was supported by funding from the National Institute of Drug Abuse. We thank Stephen Gold, Chad Messer, Suzanne Numan and Zia Rahman for technical advice, and Amy Duffield for excellent technical assistance.
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Pergamon PII: S0306-4522(99)00302-4
Neuroscience Vol. 94, No. 2, pp. 629–636, 1999 629 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
IN VIVO REGULATION OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR-INDUCIBLE TRANSCRIPTION FACTOR BY KAINIC ACID A. J. EISCH,* C.-H. LAMMERS,†‡ S. YAJIMA,†§ M. M. MOURADIAN† and E. J. NESTLER*k *Laboratory of Molecular Psychiatry, Departments of Psychiatry and Neurobiology, Yale University School of Medicine, Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508, U.S.A. †Genetic Pharmacology Unit, Experimental Therapeutics Branch, National Institute of Neurological Diseases and Stroke, Building 10, Room 5C116, Center Drive, MSC1406 Bethesda, MD 20892-1406, U.S.A.
Abstract—A putative transcription factor induced in vitro by glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-beta was recently cloned and characterized [Yajima S. et al. (1997) J. Neurosci. 17, 8657–8666]. The messenger RNA of this protein, termed murine GDNF-inducible transcription factor (mGIF, hereafter referred to as GIF), is localized within cortical and hippocampal regions of brain, suggesting that GIF might be regulated by perturbations of these brain regions. In an effort to learn more about the role of GIF in vivo, we examined GIF messenger RNA in the brains of rats treated with the glutamatergic agonist kainic acid. This treatment is known to induce seizures and alter the messenger RNA expression of several growth factors, including GDNF, in several brain regions. Rats were given intraperitoneal saline (1 ml/kg) or kainic acid (15 mg/kg) and were killed at various time-points for in situ hybridization of brain sections with a GIF messenger RNA riboprobe. In salinetreated rats, GIF messenger RNA was present at low levels in cerebral cortex, hippocampus and hippocampal remnants such as the taenia tecta. Kainic acid treatment induced robust increases in GIF messenger RNA in several brain regions, including cerebral cortex, hippocampus, caudate–putamen, nucleus accumbens, and several nuclei of the amygdala and hypothalamus. Most brain regions showed the greatest increase in GIF messenger RNA 4–6 h after kainic acid administration and a return towards normal levels at 48 h. The CA3 region of hippocampus, however, showed a more rapid increase in GIF messenger RNA that was also evident 48 h after kainic acid administration. These results demonstrate that GIF messenger RNA can be regulated in vivo, and that this novel factor warrants further study as a central mediator of GDNF and perhaps other neurotrophic factors. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: GDNF, TGF-b, seizure-regulation, hippocampus, accumbens, in situ hybridization.
Initial work with glial cell line-derived neurotrophic factor (GDNF) characterized this distant member of the transforming growth factor-b (TGF-b) superfamily as a survival and morphological differentiation factor for midbrain dopaminergic neurons. 23,42 It is now known that GDNF has trophic, survival, and differentiation effects on a variety of cell types, including noradrenergic neurons and several classes of motor and peripheral neurons. 3,9,34 The neurotrophic and supportive effects of GDNF have encouraged consideration of GDNF’s therapeutic potential in treating neurodegenerative disorders. Indeed, GDNF protects and restores the function of dopaminergic, noradrenergic, motor and hippocampal neurons in animal models of Parkinson’s disease, motor neuron degeneration, limbic seizure and ischemia. 3,13,16,26,44,53,54,56 Additional therapeutic interest in GDNF comes from studies suggesting that GDNF and other neurotrophins may play a role in the selective neuronal vulnerability, axon remodeling and synaptoneogenesis seen after epileptic insults. For example, a seizureinducing dose of kainic acid up-regulates the mRNA for
GDNF and two of its receptor components, c-ret and GFRa-1, in regions of hippocampus that show seizure-induced axonal sprouting. 18,37,49 While several receptor components for GDNF have been identified, 10,20,21,46,48 little is known about the cascade of intracellular events triggered by GDNF receptor activation. Recent work identified a novel mRNA species that was induced in cell culture by GDNF and TGF-b. 55 Designated murine GDNF inducible transcription factor or mGIF (hereafter referred to as GIF), this zinc-finger protein was cloned and characterized in the process of searching for proteins that bind to consensus sequences of the Sp1 DNA response element. The protein sequence of GIF shows a high degree of homology to TGF-b inducible early gene and early growth response gene-a, proteins with Sp1-like zinc finger motifs. 7,43 In vitro, GIF mRNA is induced by GDNF and TGF-b in both a murine cell line and primary cultures from rat cortical neurons. While no regulation studies have been done in vivo, distribution studies of GIF mRNA in mouse brain reveal that it is present at low levels in cerebral cortex and hippocampal regions. Given the distribution of GIF mRNA in brain, and its ability to be induced by GDNF in vitro, we examined whether perturbations of hippocampus and cerebral cortex cause alterations in GIF mRNA expression in vivo. Seizures induced by electroshock, kindling, or systemic or central administration of chemical convulsants produce neuronal damage and axon remodeling in the hippocampus accompanied by alterations in immediate-early genes, growth factors, and receptors for growth factors in several brain areas, including hippocampus. 5,12 Previous work has established the temporal and regional alterations in mRNA for GDNF and its receptor
‡Present address: Zentrum fu¨r Nervenheilkunde, Klink fu¨r Neurologie Station 3B, Rudolf-Bultmann Strasse 8, 35039 Marburg, Germany. §Present address: Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan. kTo whom correspondence should be addressed. Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-isoxazolepropionic acid; CA1 and CA3, Ammon’s horn, areas 1 and 3; DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetate; GDNF, glial cell line-derived neurotrophic factor; GIF, murine glial-derived neurotrophic factor-inducible transcription factor; PBS, phosphate-buffered saline; SSC, standard saline citrate; TGF-b, transforming growth factor-beta; UTP, uridine triphosphate. 629
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described previously. 14 Sections were washed twice in 0.75% glycine in 0.1 M phosphate buffer followed by permeabilization with Proteinase K (1 mg/ml in 0.1 M Tris, pH 8.0, and 50 mM EDTA) for 30 min at 378C. Sections were then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min and rinsed in 2 × standard saline citrate (SSC) until probe application. The 578 base pair murine GIF antisense and sense riboprobes were labeled with 35S-uridine triphosphate (UTP) by in vitro transcription using T7 and SP6, respectively. 55 Resulting riboprobes were purified using a Stratagene NucTrap Column (La Jolla). Tissue sections were incubated overnight at 558C in 1 ml of hybridization buffer [50% formamide, 35 × Denhardt’s, 10% dextran sulfate, 0.14 × SSC, 30 mg/ml denatured salmon sperm DNA, 0.15 mg/ml yeast tRNA, 20 mM dithiothreitol (DTT) and 10 7 c.p.m./ ml 35S-UTP-labeled GIF antisense or sense riboprobe]. The following day, sections were washed with decreasing concentrations of SSC buffer (2–0.5 × ) containing 20 mM DTT at room temperature, two high-stringency washes in 0.1 × SSC with 20 mM DTT at 658C for 30 min each, and final rinses in 0.1 × SSC with 20 mM DTT. Sections were then mounted out of 0.1 M Tris on to Vectabond-treated slides (Vector) and air-dried. Autoradiography Fig. 1. Autoradiographs of rat brain at the level of frontal cortex showing localization of GIF mRNA. Shown is antisense labeling of GIF mRNA 4 h after injection with saline (1 ml/kg, i.p.) or kainic acid (15 mg/kg, i.p.) and 48 h after kainic acid. Sense labeling after injections of either saline (not shown) or kainic acid (4 h time-point shown) was not different from background labeling. AI, agranular insular cortex; d, deep layers of cerebral cortex; Fr, frontal cortex; KA, kainic acid; mPF, medial prefrontal cortex; Pir, piriform cortex; OF, orbitofrontal cortex; s, superficial layers of cerebral cortex; TT, taenia tecta.
components induced by kainic acid. 18,37,49 Therefore, in the present study we examined GIF mRNA at several time-points after kainic acid administration. Our results demonstrate dramatic regulation of GIF mRNA expression in the brain in vivo. EXPERIMENTAL PROCEDURES
Animals, drug treatment and tissue preparation Male Sprague–Dawley rats (250–275 g, Charles River Laboratories, Wilmington, MA) were used for all experiments. Rats were group-housed under conditions of 12 h of light per day in a vivarium approved by the American Association for the Accreditation of Laboratory Animal Care. Food and water were available ad libitum. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 865-23) and were approved by the Yale Animal Care and Use Committee. Rats were given i.p. injections of either 0.9% sodium chloride (saline; 1 ml/kg) or kainic acid (15 mg/kg, Sigma). This dose of kainic acid is known to induce generalized seizures in rats. 5,39,40 Kainic acid was dissolved 5 mg/ml in saline and administered 3 ml/kg. To verify seizure induction by kainic acid, behavioral observations were made every 30 min for 6 h after saline or kainic acid injection. All kainic acid-treated animals, with the exception of those perfused 45 min postinjection, displayed generalized limbic seizure activity within 90 min following kainic acid administration (data not shown). Saline animals (n 4) were perfused 4 h after injection. Kainic acid animals were perfused at 45 min (n 3), 2 h (n 5), 4 h (n 4), 6 h (n 6), 24 h (n 4) or 48 h (n 4) after injection. Prior to perfusion, rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p., Abbott Laboratories) and were exsanguinated with cold 0.1 M phosphate-buffered saline, pH 7.4 (PBS; 75 ml, 15 ml/min) followed by fixation with 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (255 ml, 15 ml/min). Brains were removed and postfixed overnight in 4% paraformaldehyde and then cryoprotected in 30% sucrose in 0.1 M PBS. Parallel series of 30 mm coronal brain sections were cut on a freezing microtome and stored in 4% paraformaldehyde at 48C. In situ hybridization Free-floating sections were processed for in situ hybridization as
The GIF hybridization signal was visualized by both film (B-Max; Amersham) and emulsion (NTB2; Kodak) autoradiography. Sections hybridized to GIF mRNA were exposed for six to 14 days on film and nine weeks on emulsion. Following development of emulsion autoradiograms, sections were counterstained with Cresyl Violet and coverslipped with DPX (Aldrich). Image analysis and statistical methods NIH Image (Bethesda) was used to collect images for qualitative and semi-quantitative examination of GIF mRNA expression. Semi-quantitative measurements were established for six brain regions: frontal cortex, nucleus accumbens core, nucleus accumbens shell, caudate– putamen, and dentate gyrus and CA3 of hippocampus. The NIH Image outline tool was used to circumscribe dentate gyrus (including ventral and dorsal horn), CA3, caudate–putamen and the entire frontal cortex (including claustrum and cingulate, infralimbic, frontal, parietal, agranular insular, lateral orbital and ventral lateral orbital cortices but excluding the forceps minor of the corpus callosum). The square sampling tool was used to measure core and shell of nucleus accumbens. Each measurement was corrected for background by subtracting the value of an unlabeled brain region within the same tissue section (e.g., corpus callosum). Optical density values for an individual animal were calculated by averaging the corrected measurements from six to 12 tissue sections for each brain region. The resulting corrected grey values for each brain region are reported as means and S.E.M. The corrected grey value means for each brain region were analysed using a one-way ANOVA. When the overall analysis showed a main effect of time, individual time-points were compared post hoc using the Fischer’s LSD test. The level of significance for analysis was set as P , 0.05. Subscripts after F-values indicate degrees of freedom for each test. RESULTS
Saline-treated animals The antisense riboprobe for GIF mRNA hybridized to several brain regions of saline-treated rats, while the sense probe did not (Figs 1–3). Regions that displayed a consistent signal for GIF mRNA above background include dentate gyrus and Ammon’s horn (CA1–3) of hippocampus, frontal, anterior cingulate, and piriform cortices, amygdala and taenia tecta. Within cerebral cortical regions, deeper layers of cortex showed slightly greater signal than superficial layers, and medial and lateroventral regions (cingulate, ventral lateral orbital, and lateral orbital cortices) showed slightly greater signal than laterodorsal cortices (anterior parietal cortex; Fig. 1). No significant GIF mRNA signal was observed in other gray matter regions (e.g., caudate–putamen, nucleus accumbens, thalamus or ventral mescencephalon, including
GIF induction by kainic acid
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substantia nigra pars compacta and pars reticulata). This pattern of GIF mRNA in rat brain is similar to that described in the mouse. 55
Kainic acid-treated animals
Fig. 2. Autoradiographs of rat brain at the level of caudate–putamen and nucleus accumbens showing localization of GIF mRNA. Shown is antisense labeling of GIF mRNA 4 h after injection with saline (1 ml/kg, i.p.) or kainic acid (15 mg/kg, i.p.) and 48 h after kainic acid. Sense labeling after injections of either saline (not shown) or kainic acid (4 h time-point shown) was not different from background labeling. AcbSh, nucleus accumbens shell; Cg, cingulate cortex; Cl, claustrum; CPu, caudate–putamen; d, deep layers of cerebral cortex; Fr, frontal cortex; KA, kainic acid; Par, parietal cortex; Pir, piriform cortex; s, superficial layers of cerebral cortex; TT, taenia tecta; TuPy, olfactory tubercle, pyramidal layer.
In contrast to the low basal levels of GIF mRNA seen in the brains of saline-treated rats, kainic acid-treated animals showed robust increases in GIF mRNA in a time- and region-dependent manner (Figs 1–4). Regions that displayed the greatest increase in GIF mRNA signal include frontal cortex, caudate–putamen, core and shell of nucleus accumbens, and dentate gyrus and CA1–3 of hippocampus. For clarity, the time-dependent alterations in GIF mRNA levels in these brain areas are described in separate sections below. Increases in GIF mRNA signal were also observed, but not quantified, in several other brain regions. Consistent increases in GIF mRNA levels were observed in olfactory structures (e.g., pyramidal layer of olfactory tubercle) and frontal– parietal cortices 4–6 h after kainic acid (Figs 1, 2). In more caudal sections, the kainic acid-induced GIF mRNA signal was evident in motor and somatosensory cortices (e.g., forelimb, hindlimb, parietal cortices I and II) yet was distinctly absent in retrosplenial cortices 4–6 h after kainic acid (Figs 2, 3). In addition, the ventromedial hypothalamic nuclei showed induction of GIF mRNA 4–48 h after kainic acid administration (Fig. 3). Finally, the lateral complex of the amygdala (lateral, basolateral, basomedial) and the habenular nuclei showed GIF induction 6–48 h after kainic acid (Fig. 3). No alteration in GIF mRNA signal was observed in regions containing dopaminergic cell bodies, such as the substantia nigra, pars compacta or pars reticulata, or ventral tegmental area.
Frontal cortex
Fig. 3. Autoradiographs of rat brain at the level of hippocampus showing localization of GIF mRNA. Shown is antisense labeling of GIF mRNA 4 h after injection with saline (1 ml/kg, i.p.) or kainic acid (15 mg/kg, i.p.) and 48 h after kainic acid. Sense labeling after injections of either saline (not shown) or kainic acid (4 h time-point shown) was not different from background labeling. AcbSh, nucleus accumbens shell; AI, agranular insular cortex; Am, amygdala; CA1 and CA3, Ammon’s horn, areas 1 and 3; CPu, caudate–putamen; DG, dentate gyrus; Hb, habenula; KA, kainic acid; Par, parietal cortex; Pir, piriform cortex; Rs, retrosplenial cortices; VMH, ventral medial hypothalamic nuclei.
GIF mRNA levels in frontal cortex were dramatically increased by kainic acid administration. As shown in Fig. 1, GIF mRNA signal was increased in frontal cortex by 4 h and began to return to basal levels by 48 h. While all frontal cortical regions showed an increase in GIF mRNA signal 4 h after kainic acid, certain subregions displayed qualitatively more GIF mRNA signal than others. For example, superficial layers of cerebral cortex generally showed a greater increase in GIF mRNA signal after kainic acid than deeper layers (Fig. 1). In addition, the kainic acid inducedincrease in GIF mRNA signal appeared to be greater in limbic-related regions, such as medial prefrontal (cingulate), infralimbic, agranular insular and orbitofrontal (lateral orbital and ventral lateral orbital) cortices, than in motor and sensory-related cortices, such as frontal and anterior parietal cortices (Fig. 1). A one-way ANOVA on the corrected grey values from all time-points showed that the time-dependent increase in GIF mRNA levels in frontal cortex was highly significant (Fig. 4; F6,30 11.05; P , 0.01). Post hoc analysis showed that the kainic acid-induced increase in GIF mRNA signal was significantly greater than saline at 4 h (688% of saline levels), 6 h (800%) and 24 h (544%; all P’s ,0.01). In addition, GIF mRNA signal 48 h after kainic acid was significantly less than the signal at 4 h (P , 0.05) and 6 h (P , 0.01).
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Fig. 4. Systemic administration of kainic acid increases GIF mRNA in a time-dependent manner in several regions of rat brain. Each graph depicts the mean corrected grey value and S.E.M. for saline-treated animals and six time-points after 15 mg/kg i.p. kainic acid (45 min, 2, 4, 6, 24 and 48 h). A one-way ANOVA showed a significant effect of time for all brain regions. Results of post hoc tests are indicated in each graph by asterisks and brackets. Asterisks indicate significant difference from saline-treated value within the same brain region: **P , 0.01; *P , 0.05. Brackets over bars and associated P-values indicate significant difference from either 48 h after kainic acid (frontal cortex, nucleus accumbens core and shell, dentate gyrus and CA 3) or from 45 min or 2 h after kainic acid (caudate–putamen).
Nucleus accumbens The nucleus accumbens also displayed a dramatic kainic acid-induced increase in GIF mRNA levels. While both core and shell showed an increase in GIF mRNA signal 4 h after kainic acid administration, this effect appeared qualitatively greater in shell (Fig. 2). Quantification of the GIF mRNA signal in these regions confirmed this observation (Fig. 4 and below). A one-way ANOVA on the corrected grey values showed that the time-dependent increase in GIF mRNA in nucleus accumbens was highly significant in both core
(F6,30 5.45; P , 0.01) and shell (F6,30 10.43; P , 0.01). For core, post hoc analysis showed that the kainic acidinduced increase in GIF mRNA signal was significantly greater than saline levels at 4 h (1082% of saline levels; P , 0.01) and 6 h (936%; P , 0.05). In addition, GIF mRNA signal in core at 48 h was significantly less than the signal at 4 h (P , 0.01) and 6 h (P , 0.05), indicating a return towards control levels. For shell, post hoc analysis showed that the kainic acid-induced increase in GIF mRNA signal was significantly greater than saline at 4 h (2250% of saline levels; P , 0.01) and 6 h (1090%; P , 0.05). In addition, GIF
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Fig. 5. Emulsion autoradiographs of rat brain at the level of hippocampus showing localization of GIF mRNA. Low-magnification dark-field photomicrograph (left panel) shows dense labeling of GIF mRNA 4 h after kainic acid (15 mg/kg, i.p.) in the dentate gyrus granule cell layer, CA 1–3 and cortex. Adjacent panel (right) shows high magnification of cortical field under bright-field microscopy. Emulsion grains appear over large, pale nuclei but not over small, dark nuclei (neurons, large arrows; glia, small arrows).
mRNA signal in shell at 48 h was significantly less than the signal at 4 h (P , 0.01) and 6 h (P , 0.05). Caudate–putamen In contrast to the large effect of kainic acid in frontal cortex and nucleus accumbens, kainic acid administration produced a small, but notable, increase in GIF mRNA signal in caudate–putamen. The GIF mRNA signal was increased 4 h after kainic acid injection, and returned to basal levels by 48 h. As evident in Fig. 2, the kainic acid-induced GIF mRNA signal in caudate–putamen was greatest in the ventral and medial sectors and least in the dorsal and lateral sectors. A one-way ANOVA of GIF mRNA values in caudate–putamen at all time-points showed a significant effect of time (Fig. 4; F6,30 3.06; P , 0.05). While kainic acid increased the GIF mRNA signal in caudate–putamen relative to saline values at 4 h (688% of saline), 6 h (750%) and 24 h (538%) after injection, post hoc analysis did not show significance for these comparisons (all P . 0.05). Post hoc analysis did show a significant difference between 4 h and 2 h animals (P , 0.05), and between 6 h and both 45 min and 2 h animals (both P’s ,0.05). Hippocampus Kainic acid induced very large increases in GIF mRNA levels in hippocampus (Fig. 3). While both the pyramidal cell layer (CA1–3) and the granule cell layer (dentate gyrus) showed increases in GIF mRNA signal 4 h after kainic acid, the dentate gyrus increase appeared to be much greater than that in CA1–3. By 48 h after kainic acid, the dentate gyrus signal had decreased somewhat but still appeared elevated relative to saline-treated animals. In CA1–3, in contrast, GIF mRNA appeared to increase further at 48 h relative to 4 h after kainic acid. For quantitation of these kainic acid-induced alterations, the entire dentate gyrus and CA3 region of Ammon’s horn were analysed separately to demonstrate kainic acid’s effect on granular and pyramidal cell layers, respectively. A one-way ANOVA on corrected grey values showed that the time-dependent increase in GIF mRNA signal in hippocampus was highly significant in both dentate gyrus (F6,30 27.88; P , 0.01) and CA3 (F6,30 759.29; P , 0.01). As evident from the time-course shown in Fig. 4, however, the pattern of GIF mRNA induction
in each region was distinct. For dentate gyrus, post hoc analysis showed that the increase in GIF mRNA was significantly greater than saline at 4 h (979% of saline levels; P , 0.01), 6 h (744%; P , 0.05), 24 h (447%; P , 0.05) and 48 h (413%; P , 0.05) after kainic acid. In addition, GIF mRNA signal at 48 h was significantly less than the signal at 4 h (P , 0.01), and 6 h (P , 0.01). In contrast, post hoc analysis of CA3 corrected grey values showed that the increase in GIF mRNA was significantly greater than saline at 45 min (377% of saline levels; P , 0.01), 4 h (378%; P , 0.01), 6 h (402%; P , 0.05), 24 h (344%; P , 0.05) and 48 h (559%; P , 0.05) after kainic acid. In addition, GIF mRNA signal at 48 h was significantly greater than the signal at 45 min (P , 0.05), 4 h (P , 0.01), 6 h (P , 0.05), and 24 h (P , 0.01). Cellular localization Kainic acid treatment is known to induce glial hypertrophy and microglial proliferation, 33 and GDNF was initially identified from a glial cell line; therefore, the phenotype of cells expressing GIF mRNA was determined using emulsion autoradiography. In saline-treated animals, grains appeared to be localized over large, pale nuclei, presumably neurons. Grains evident over small, dark nuclei, presumably glia, were not more numerous than background labeling. Kainic acid-treated animals (4–48 h time-point) also showed prefential distribution of emulsion grains over neurons (Fig. 5). For example, superficial layers of parietal cortex showed dense, clustered grains which enabled identification of cellular phenotype (Fig. 5). All grains appeared to be localized over large, pale neuronal nuclei with no grains evident over small, dark glial nuclei. While the high density of grains in the dentate gyrus (Fig. 5) precluded identification of the phenotype of individual cells, the label is presumably neuronal since the dentate gyrus is known to be composed primarily of neurons, not glia. 2,4,8 DISCUSSION
We show here that mRNA levels of the novel, putative transcription factor GIF are dramatically increased in rat brain after systemic kainic acid administration. Most brain regions analysed—frontal cortex, caudate–putamen, nucleus accumbens core and shell, and dentate gyrus—display
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maximum GIF mRNA levels 4–6 h after kainic acid and return towards control values by 48 h. In contrast, the CA3 region of the hippocampus shows a rapid increase in GIF mRNA 45 min after kainic acid that generally persists until 48 h. The pattern of GIF mRNA induction shows some overlap with the distribution of kainic acid receptors in the rat brain. 6,24,31,35,50 However, some brain regions rich in kainic acid binding sites, such as dorsal caudate–putamen, 1 show only a small increase in GIF mRNA following kainic acid, while others, such as the dentate gyrus and nucleus accumbens, show much larger increases in GIF mRNA. Such a pattern suggests that GIF induction is not simply a direct consequence of kainic acid receptor activation. Similarly, while evidence suggests that kainic acid may mediate some of its effects via the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, 30 there is also a lack of correspondence between AMPA, receptor distribution and kainic acid induction of GIF mRNA. 14,30 Rather, the spatiotemporal pattern of GIF induction appears to be a precisely co-ordinated event, resulting not only from direct drug effects at kainate or AMPA receptors, but also from the subsequent neuronal and epileptiform activity. Relationship of murine glial cell line-derived neurotrophic factor-inducible transcription factor messenger RNA induction to neuronal degeneration One major consequence of kainic acid-stimulated neuronal activity is selective neuronal degeneration. 5 However, the changes in metabolic and electrical activity 47 and the wellestablished pattern of neuronal injury 5,39,40 that accompany kainic acid administration show only partial similarities to the specific pattern of GIF induction described here. For example, kainic acid increases GIF mRNA in brain regions known to be resistant to excitotoxic damage (dentate gyrus, caudate–putamen, nucleus accumbens, frontal cortex) as well as in regions vulnerable to this damage (CA3 of hippocampus). This mixed pattern of induction suggests that regulation of GIF is complex, and further work is needed to establish the role that GIF may play in the neurodegenerative effects of kainic acid. Relationship of murine glial cell line-derived neurotrophic factor-inducible transcription factor messenger RNA induction to neurotrophic factor induction An additional consequence of kainic acid is the neuronal remodeling that takes place subsequent to the neuronal injury. A growing body of evidence suggests that seizure-induced neuronal sprouting and synaptoneogenesis observed in hippocampal subregions are linked to seizure-induced alterations in neurotrophins (nerve growth factor, brain-derived neurotrophic factor) 11,52 and, more recently, GDNF and its receptors. 18,37,49 Given recent work showing GIF induction in vitro by GDNF and TGF-b, the region- and time-specific pattern of GIF regulation by kainic acid urges comparison with the pattern of GDNF and related mRNAs after kainic acid. The pattern of GDNF mRNA induction in the dentate gyrus after kainic acid does complement the pattern of GIF mRNA reported here. GDNF mRNA increases in dentate granule cells 3 h after kainic acid, reaches maximal levels at 6 h, and returns to control values by 24 h. 18 While it remains to be determined whether this increase in GDNF mRNA leads to
a corresponding increase in GDNF protein, it is reasonable to consider that increased GDNF in dentate gyrus may underlie the increased GIF expression seen in this region 4–6 h after kainic acid. The fact that receptor components for GDNF are also increased in dentate gyrus after kainic acid is consistent with this idea. 37,49 In contrast to the induction of GDNF mRNA seen in the dentate gyrus, extra-hippocampal induction of GDNF mRNA by kainic acid has not been reported. This raises the question of what signal is responsible for the increase in GIF mRNA seen in other brain regions, such as caudate–putamen, nucleus accumbens and frontal cortex. Two possibilities deserve consideration. First, it is possible that technical barriers have prevented detection of kainic acid-induced increases in GDNF in these regions. Initial studies in the adult rat brain found low or negligible levels of GDNF mRNA in the caudate–putamen, hippocampus and cortex. 38,41,42 More recent examination of GDNF mRNA distribution, however, shows that GDNF mRNA is expressed in several regions of the normal adult rat brain, including caudate–putamen, nucleus accumbens, thalamic nuclei, olfactory regions, hippocampus, cerebellum and cerebral cortex. 36,49 In addition, glutamatergic stimulation of the striatum with kainic acid has been shown to increase GDNF mRNA in this region in vivo. 15 Therefore, it is possible that previously undetected increases in GDNF mRNA are responsible for the kainic acid induced increases in GIF mRNA in the caudate–putamen and other regions. Finally, GDNF has been shown to be a target-derived trophic factor, perhaps eliminating the need for regional correspondence between GIF mRNA and GDNF mRNA. 45 An alternative explanation is that GIF mRNA is regulated in certain brain regions by a TGF-b-related signal other than GDNF. Several isoforms of TGF-b have been identified and localized to the adult CNS. 28,29,51 Indeed, TGF-b2- and TGFb3-immunoreactive neurons are found in the hippocampus, cerebral cortex, hypothalamus and amygdala of the adult rat. While this localization does not completely overlap with the unique pattern of GIF expression described here, these isoforms of TGF-b should be considered for future evaluation of their role in the kainic acid-induced regulation of GIF. A third isoform, TGF-b1, is increased after kainic acid, 32 but the basal signal and the induced signal are generally limited to macrophages within hippocampus. Other candidates that might stimulate GIF mRNA include neurturin and persephin, trophic factors that, with GDNF, comprise the TGF-b-related neurotrophic factor superfamily. 22,27 Initial studies of the regional distribution of neurturin and persephin in the rat brain, however, do not suggest extensive correspondence with the distribution of basal or kainic acid stimulated levels of GIF mRNA. 17,19 It is also feasible that GIF mRNA is regulated by the binding of an undiscovered TGF-b-like factor to one of the many signaling receptors linked to the TGF-b superfamily. For example, TGF-b type I-like receptor, habrec1, shows significant mRNA expression in the striatum, cerebral cortex, and olfactory tubercle, 25 and thus may provide a signaling receptor for regulation of GIF mRNA. Given GIF’s position downstream from GDNF and TGF-b, it is likely that more than one of these trophic factors and/or receptor components is involved in regulation of GIF expression. Further work is needed to establish whether GIF is indeed a site of convergence of various TGF-b-related signaling pathways.
GIF induction by kainic acid CONCLUSIONS
As a transcription factor induced by GDNF and TGF-b, GIF is a primary candidate for mediating intracellular effects of GDNF, related neurotrophic factors and their associated signaling receptors. The identification of paradigms in which GIF mRNA levels are greatly altered in specific brain regions is an essential step to elucidating the physiological role of GIF. The present work establishes kainic acidinduced seizures as the first such paradigm that can regulate GIF mRNA in vivo. The lack of correspondence between the induction of GIF mRNA and GDNF mRNA suggests that GIF expression may be regulated via other TGF-b-related signals,
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such as TGF-b, neurturin or persephin, and/or other receptor components, such as Habrec1. While further work must be done to evaluate the hypothesis that GIF serves as one final common pathway in the signal-transcription coupling events in neurons sensitive to TGF-b-related factors, the present work suggests a multifaceted role for GIF in the injury and plasticity induced by kainic acid.
Acknowledgements—This work was supported by funding from the National Institute of Drug Abuse. We thank Stephen Gold, Chad Messer, Suzanne Numan and Zia Rahman for technical advice, and Amy Duffield for excellent technical assistance.
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