Introduction role in mediating excitatory amino acids EAA-induced

Brain Research 882 2000 128–138 www.elsevier.com locate bres Research report Crucial role of kainate receptors in mediating striatal kainate injection-induced decrease in acetylcholine M receptor binding 1 in rat forebrain a , a b a Shaoyu Jin , Jian Yang , Wei Ling Lee , Peter T.H. Wong a Department of Pharmacology , Faculty of Medicine, The National University of Singapore, MD2, 18 Medical Drive, Singapore 117597, Singapore b The National Neuroscience Institute of Singapore , Irrawaddy Block, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore Accepted 15 August 2000 Abstract We investigated the roles of kainate-, a-amino-3-hydroxy-5-methylisoxazol-4-propionate AMPA- and N-methyl- D -aspartate NMDA- receptors in mediating striatal kainate injection-induced decrease in the binding of acetylcholine M receptors in rat forebrain. After 1 3 3 3 unilateral intrastriatal injection of kainate 4 nmol, the bindings of [ H]kainate 10 nM, [ H]MK-801 4 nM and [ H]pirenzepine 4 nM to the rat ipsilateral forebrain membranes declined, reaching the lowest on day 2 to 4 and recovering on day 8. Saturation binding studies, performed on day 2 post-injection, showed that kainate 1, 2, 4 nmol dose-dependently decreased B and K of the three max d ligands. 1-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine MK-801, a selective NMDA receptor channel blocker, 3 3 antagonised from a dose of 4 nmol kainate-induced decreases in the bindings of [ H]kainate up to |20, [ H]MK-801 up to |90 3 and [ H]pirenzepine up to |70. In contrast, 6-cyano-7-nitroquinoxaline-2,3-dione CNQX, a selective non-NMDA receptor antagonist, almost completely abolished from a dose of 12 nmol kainate-induced decreases in the bindings of all the three ligands up to |95–98. Cyclothiazide, a selective potentiator that enhances AMPA receptor-mediated responses, significantly enhanced from a dose 3 3 3 of 4 nmol kainate-induced decrease in the binding of [ H]kainate but not that of [ H]pirenzepine or [ H]MK-801. In summary, these results indicate that striatal kainate injection-induced decrease in the binding of acetylcholine M receptors in rat forebrain is dependent 1 on activation of kainate receptors and, to a certain extent, a consequent involvement of NMDA receptors. These and previous studies provide some evidence showing that kainate receptors might play a crucial role in regulating excitatory amino acids EAA-modulated cholinergic neurotransmission in the central nervous system CNS.  2000 Elsevier Science B.V. All rights reserved. Theme : Neurotransmitters, modulators, transporters, and receptors Topic : Excitatory amino acid receptors: physiology, pharmacology and modulation 3 Keywords : Cholinergic neurotransmission; CNQX; Cyclothiazide; Kainate injection; [ H]Pirenzepine binding

1. Introduction role in mediating excitatory amino acids EAA-induced

acetylcholine release in rat striatum in vitro [16], whereas a L -Glutamate, the principal excitatory neurotransmitter in demonstration of kainate receptor-mediated control of the mammalian central nervous system CNS, mediates cholinergic neurotransmission in the CNS under in vivo most of the excitatory neurotransmission through activa- conditions is still lacking. tion of three classes of ionotropic glutamate receptors Local injection of a glutamate analogue, such as kainate, [31,34], named after the agonists N-methyl- D -aspartate is widely used as a method for destroying neurones in vivo NMDA, a-amino-3-hydroxy-5-methylisoxazol-4-prop- [34]. Previous studies have documented that local injection ionate AMPA and kainate [39]. Among the three sub- of kainate in rat brain induced massive neuronal loss and types, kainate receptors have been shown to play a distinct over-expression of amyloid precursor protein APP [7,12,21] as well as corresponding decreases in the expres- sions of choline acetyltransferase ChAT and glutamate Corresponding author. Tel.: 165-874-6061; fax: 165-773-0579. E-mail address : phcjinsynus.edu.sg S. Jin. decarboxylase [12,14] and in the bindings of ionotropic 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 5 7 - 2 S . Jin et al. Brain Research 882 2000 128 –138 129 glutamate- and acetylcholine muscarinic-receptors [15,25]. hypotonic buffer 1 mM EGTA buffered by Tris–HCl to However, the roles of NMDA-, AMPA- and kainate-re- pH 8.0 and left on ice for 30 min. The lysed membranes ceptors in these mechanisms are poorly understood. Gener- were collected by a 30-min centrifugation at 40 0003g, ally, the effect of kainate is thought to be indirect through and then the lysis centrifugation step was repeated one innervations of glutamatergic neurons [10]. It is the release more time. The membranes were then suspended and of glutamate [11,26] that leads to neuronal death through washed twice in a Tris–acetate buffer 100 mM, pH 7.4. activation of postsynaptic NMDA receptors [27,34]. These After each centrifugation, the membrane suspension was studies focusing on rat hippocampus suggested an excitat- sonicated with a tip sonicator at high intensity. The final ory role for presynaptic kainate receptors on glutamatergic pellet was suspended in the assay buffer 100 mM HEPES terminals [27]. On the other hand, conflicting observations containing 50 mM EGTA buffered to pH 7.4 by Tris– regarding the roles of presynaptic kainate- and postsynap- HCl. The membrane suspension was stored in aliquots at tic NMDA-receptors have also been reported [5,6,12]. 2808C. We have recently observed that striatal kainate injection induced a loss in cholinergic neuron and an increase in 2.3. Protein analysis APP expression in rat forebrain. These effects are depen- dent on the activation of kainate receptors with a con- On the day of the binding assay, the frozen membrane sequent involvement of NMDA receptors [42]. In the preparations were thawed on ice and their protein contents present studies, we, therefore, investigated the roles of were determined by Bradford’s method [4]. Bovine serum kainate-, AMPA- and NMDA-receptors in mediating striat- albumin was used for the standard curve. The membrane al kainate injection-induced decreases in the bindings of preparations protein 0.4 mg ml were kept on ice until 3 3 3 ionotropic glutamate- and acetylcholine M -receptors by used in [ H]kainate, [ H]MK-801 and [ H]pirenzepine 1 3 examining the characteristics of the specific [ H]kainate, binding assays. 3 3 [ H]MK-801 and [ H]pirenzepine binding to the mem- branes prepared from the rat ipsilateral forebrain. 2.4. Radioligand binding assays 3 3 3 [ H]Kainate, [ H]MK-801 and [ H]pirenzepine bindings 2. Materials and methods were studied, using the filtration method [8,23,38]. All the