Materials and methods were studied, using the filtration method [8,23,38]. All the
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
binding incubations were performed in disposable sterile 2.1. Animal treatment
96-well plates in a total volume of 300 ml and terminated by rapidly filtering the incubation medium through What-
Male Sprague–Dawley rats 200–250 g were housed man GF B glass fiber filters under suction by means of a
under controlled conditions with a 12-h day-night cycle Skatron cell harvester.
3
and with food and water available ad libitum. Rats were For [ H]kainate binding, aliquots of the membrane
anesthetized with chloral hydrate 400 mg kg, i.p. and suspension in a final concentration of 200 mg ml were
3
mounted in a stereotaxic apparatus Stoelting, Wood Dale, incubated with [ H]kainate 10 nM on ice for 1 h.
IL, USA. One ml of phosphate buffer 4 mM, pH 7.4 Non-specific binding, determined by inclusion of 1 mM
with kainate was slowly injected unilaterally into the
L
-glutamate, amounted to 10–12 of the total binding. For striatum AP510.7 mm, ML563.0 mm, DV526.5 mm
the saturation experiments, the membranes were incubated
3
from bregma, with 5 min allowed for local diffusion with 0.8–400 nM [ H]kainate. Non-specific binding
before removing the microsyringe. The antagonists 1-5- amounted to 10–40 of the total binding. The Skatron cell
Methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-
harvester was set at 111 which meant the filters were imine MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione
washed with three 1-ml aliquots of ice-cold 50 mM Tris– CNQX was injected 15 min before the kainate injection.
HCl buffer, pH 7.4 total filtering and washing time was Cyclothiazide was coinjected with kainate.
3 s.
3
For [ H]MK-801 binding, aliquots of the membrane 2.2. Membrane preparation
suspension in a final concentration of 200 mg ml with 10 mM glutamate and 10 mM glycine were incubated with
3
Rats were decapitated without prior stunning or anaes- [ H]MK-801 4 nM at 258C for 2.5 h. Non-specific
thesia, and the forebrains were rapidly removed on ice. binding, determined by inclusion of 100 mM MK-801,
Membrane preparations were prepared as previously de- amounted to 5–8 of the total binding. For the saturation
scribed [22]. In brief, the ipsilateral forebrains were experiments, the membranes were incubated with 0.5–150
3
homogenized in ten volumes of ice-cold sucrose 300 mM nM [ H]MK-801. Non-specific binding amounted to 5–
containing 1 mM EGTA pH 7.4 in a glass Teflon 30 of the total binding. The Skatron cell harvester was
homogenizer. The homogenate was centrifuged at 10003g set at 333 which meant the filters were washed with three
for 10 min, and then the supernatant was recentrifuged at 3-ml aliquots of ice-cold 50 mM Tris–HCl buffer, pH 7.4
30 0003g for 20 min. The pellet was suspended in total filtering and washing time was 9 s.
130 S
3
For [ H]pirenzepine binding, aliquots of the membrane from Research Biochemicals RBI, Natick, MA, USA. All
suspension in a final concentration of 200 mg ml with 1 other chemicals were of analytical purity and of commer-
mM KCl and 1 mM MgCl were incubated with cial origin.
2 3
[ H]pirenzepine 4 nM at 258C for 3 h. Non-specific binding, determined by inclusion of 1 mM atropine,
amounted to 6–8 of the total binding. For the saturation