Kainic acid induced seizures cause a marked increase in the expression of neurokinin-3 receptor mRNA in the rat cerebellum

ELSEVIER

Neuroscience Letters 181 (1994) 158-160

NEUROSCIENCE IETTIR$

Kainic acid induced seizures cause a marked increase in the expression of neurokinin-3 receptor m R N A in the rat cerebellum Christine R6der a, Romuald Bellmann b, Kenneth E. McCarson c, James E. Krause c, Giinther Sperk a'* "Department of Pharmacology, University of lnnsbruck, Peter-Mayr-Str. la, 6020 Innsbruck, Austria bDepartment of Internal Medicine, University of Innsbruck, lnnsbruck, Austria 'Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO, USA Received 12 August 1994; Revised version received 21 September 1994; Accepted 23 September 1994

Abstract

Marked changes in the expression of the tachykinin peptide neurokinin B (NKB) have been recently observed in animal models of epilepsy. In this study we investigated mRNA levels encoding the receptor for NKB, the neurokinin-3 receptor (NK-3R), after limbic seizures induced by kainic acid (KA) in the rat. NK-3R mRNA levels were determined by nuclease protection assay at various time intervals after i.p. injection of KA in the rat. Increases of more than 200% were observed in NK-3R mRNA in the cerebellum after 7 and 30 days. In the hippocampus a moderate, reversible increase (of 70%, 1 day after KA) was seen. In the frontal cortex a reduction of NK-3R mRNA (2 days after KA) was found. In the amygdala, levels of the transcript were decreased (by 50% and more) at all intervals investigated. The decreases in mRNA levels in the amygdala are consistent with the severe damage observed in this brain area. The increases in NK-3R mRNA in the cerebellum point to the development of receptor supersensitivity and suggest a functional role of NKB in this animal model of epilepsy.

Key words: Neurokinin B; Tachykinin; Receptor supersensitivity; Epilepsy

Neurokinin B (NKB) belongs to the tachykinin peptide family [6]. NKB is widely distributed within the central nervous system [8] and is thought to exert a role as neuromediator or neurotransmitter through a subclass of the tachykinin receptors, the neurokinin-3 receptors (NK-3R) [4,11]• Limbic seizures induced by the neurotoxin kainic acid (KA) or by pentylenetetrazol kindling cause marked and long-lasting elevations in the expression of N K B [1,7,9,15]. The most pronounced increases in N K B m R N A and peptide levels were seen in the hippocampus, the frontal cortex and the amygdala [15]. Within the hippocampus, increases in N K B immunoreactivity and m R N A concentrations were observed in the granule cell-mossy fiber system and in basket cells of the hilus of the dentate gyrus [9]. This study was undertaken to investigate whether the seizure-

*Corresponding author. Fax: (43) (512) 507-2868. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00745-4

induced adaptation of the N K B system may also involve its corresponding NK-3Rs. Male Sprague-Dawley rats (Forschungsanstalt ffir Versuchstierzucht, Himberg, Austria) were injected intraperitoneally with K A (10 mg/kg). Their behavior was rated [13,14] and animals exhibiting a full limbic seizure syndrome (rating 3 4 ) were selected for the experiment. Animals were decapitated 1, 2, 7 or 30 days later. The brains were rapidly removed and dissected on a cold plate. Samples (100 mg) of the frontal cortex, hippocampus, amygdala and cerebellum were homogenized in 7.6 M guanidium-HC1, 0.1 M potassium acetate buffer pH 5.0 by ultrasonication. The homogenates were immediately frozen on dry-ice and stored at -70°C. Total R N A was extracted according to the method of Cheley and Anderson [3]. Concentrations of total R N A were determined photometrically at 260 nm. The solution hybridization-A/T1 nuclease protection assay was performed according to Berk and Sharp [2] as modified by Krause et al. [5]. We used a uniformly

C. R6der et al./Neuroscience Letters 181 (1994) 158 160

32p-labeled RNA transcript complementary to the bases +1014 to +1359 of the NK-3R cDNA [12] as hybridization probe. It was synthesized from a pBS-rNKBR plasmid as described by McCarson and Krause [10]. The plasmid was linearized with XhoI (Promega, Madison, WI, USA) and the antisense probe was generated using a T3 polymerase (Transprobe T kit, Pharmacia, Upsalla, Sweden) in the presence of 9 mM [32p]-UTP (800 Ci/ mmol; New England Nuclear, Vienna, Austria). The hybridization probe was coprecipitated with 25 #g total RNA extracted from the different brain areas with ethanol/sodium acetate pH 5.0. The precipitates were allowed to dry at room temperature and resuspended in 30 pl hybridization buffer (40 mM PIPES, pH 6.4, 400 mM NaC1, 1 mM EDTA, 80% deionized formamide). The samples were then boiled for 5 min and incubated overnight at 50°C. The non-annealed probe was digested with 80 pg RNase A and 4.2 pg RNase T1 per sample (both Sigma, St. Louis, MI, USA) in 10 mM Tris-HC1, 15 mM NaC1 pH 7.5 at 37°C for 30 min. The reaction was stopped by addition of 10 pg proteinase K/sample (Boehringer, Mannheim, FRG) and incubation at 37°C for 15 min. The mixture was then subjected to an extraction with phenol/chloroform/isoamyl alcohol [3] and the nucleic acids were precipitated with ethanol/sodium acetate. Pellets were washed with ice-cold 70% ethanol and allowed to dry by air before resuspension in a solution containing 98% formamide, 10 mM EDTA pH 8.0, 0.025% xylene cyanol, 0.025% Bromphenol blue, boiling for 5 min and chilling on ice. Electrophoresis was performed on 6% polyacrylamide gels containing 7 M urea for 90 min at 500 V. The gels were fixed, dried and exposed to Curix RP-1 X-ray film (Agfa). Signals were quantified by densitometric scanning. As molecular weight marker we used a HinclI digest of OX174 DNA (USB, Cleveland, Ohio, USA) which was endlabeled 4x

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Fig. 2. Changes in concentrations of neurokinin-3 receptor (NK-3R) m R N A after KA-induzed seizures. NK-3R m R N A was determined at various time intervals (1, 2, 7 and 30 days) after i.p. injection of KA (10 mg/kg) in the frontal cortex (A), hippocampus (B), amygdala (C) and cerebellum (D) using a solution hybridization-nuclease protection assay. Data are expressed as % of controls _+ S.E.M. The small numbers indicate the numbers of animals.

with [32p]-dATP(5000 Ci/mMol; New England Nuclear, Vienna, Austria) using terminal nucleotidyl transferase (Boehringer, Mannheim, FRG). Polyacrylamide gel electrophoresis revealed that more than 95% of the radiolabeled probe migrated in a single band of about 370 base pairs, indicating synthesis of full length probe (Fig. 1). After hybridization with RNA from brain tissue two protected bands of 345 and 300 base pairs, respectively, were found. Both bands in general showed similar changes after KA treatment and likely originate from antisense RNA hybridized to a slightly different extent with NK-3R mRNA. The smaller band could result from partial digestion of A/T rich segments (bases 1034-1050 or 1317-1324) under the stringent hybridization conditions used. The densities of the two bands were therefore summed. Essentially the same% values, however, were obtained when only one band was used for densitometric quantification. In Fig. 1 representative results of a solution hybridization-nuclease protection assay obtained with RNA extracted from the cerebellum of control rats and of rats at various intervals after KA treatment are shown. Values relative to controls are shown in Fig. 2. One day after i.p. injection of KA, a significant increase (by 70%) in NK-3R mRNA concentrations was observed in the dorsal hippocampus. In the frontal cortex a marked but statistically not significant decrease (by 50%) in the receptor message was seen after 2 days. At the same time a slight, not significant decrease in mRNA levels was present in the hippocampus. Seven to 30 days after KA injection NK-3R mRNA levels returned to control

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(7. R6der et al./Neuroscience Letters 181 (1994) 158 160

values in the cortex and were slightly but not significantly increased in the hippocampus. In the amygdala pronounced reductions (50 to 90%) of the message were observed at all time intervals. In the cerebellum, increases in NK-3R mRNA concentrations were found at 2, 7 and 30 days after KA (statistically significant after 7 and 30 days). These increases reached values of more than 270% of control after 30 days. Our data show differential responses of NK-3R mRNA expression in 4 different brain areas induced by intraperitoneal injection of KA in the rat. Only transient and moderate changes were observed in the frontal cortex and the dorsal hippocampus. Although quantitatively quite different, they followed similar patterns in these areas. An early increase in mRNA (1 day after KA) was followed by a decrease (after 2 days) and afterwards a normalization of mRNA levels. NKB has been suggested to be the prefered physiological ligand of NK-3R [4,11]. During acute KA-induced seizures a massive depleation of NKB stores has been observed which lasts for about 2 days [15]. An up-regulation of NK-3R mRNA, could partially substitute for the impaired NKB neurotransmission. The subsequent increase in NKB concentrations in the cortex and hippocampus coincide with a drop and subsequent normalization of NK-3R mRNA levels. The long lasting decrease in NK-3R mRNA concentrations in the amygdala is consistent with the extended brain damage observed in this brain area affecting especially GABA-ergic neurons [13,14]. The most surprising finding, however, was the prominent and persistent increase of NK-3R mRNA in the cerebellum. Although only a minor amount of NKB immunoreactivity was detected in the cerebellum of control rats, this area is rich in mRNA encoding the corresponding receptor [16]. The physiological function of the NK3R in the cerebellum is not known. The marked increase in mRNA may result in the development of NK-3R supersensitivity in this animal model of temporal lobe epilepsy. Provided that the mRNA is translated to functioning NK-3R this can point to a pathophysiological role of the cerebellar NKB/NK-3R system in epilepsy. In conclusion, our results demonstrate heterogeneous responses in NK-3R mRNA expression in various brain areas. These include possible reversible down-regulation as well as loss due to neuronal degeneration of NK-3R mRNA on one side, and up-regulation of NK-3R mRNA on the other side.

This work was supported by the Austrian Science Foundation and by the Dr. Legerlotz Foundation.

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