Brain Research 880 2000 28–37 www.elsevier.com locate bres
Research report
Changes of GABA receptor binding and subunit mRNA level in rat
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brain by infusion of subtoxic dose of MK-801
a a
b c ,
Hack Seang Kim , Hong Serck Choi , Soo Young Lee , Seikwan Oh
a
College of Pharmacy , Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea
b
Center for Cell Signaling Research , Division of Molecular Life Science, Ewha Womans University, Seoul 120-750, South Korea
c
Department of Neuroscience , Medical Research Center, College of Medicine, Ewha Womans University, Mokdong, Yangchon-ku, Seoul 158-710,
South Korea Accepted 5 July 2000
Abstract
In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA
receptor binding and GABA receptor subunit mRNA level in rat brain. It has been
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reported that NMDA-selective glutamate receptor stimulation alters GABA receptor pharmacology in cerebellar granule neurons in vitro
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by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA receptor
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binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA receptor
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bindings were analyzed by quantitative autoradiography using [ H]muscimol, [ H]flunitrazepam, and [ S]TBPS in rat brain slices. Rats were infused with MK-801 1 pmol 10 ml per h, i.c.v. for 7 days, through pre-implanted cannula by osmotic minipumps Alzet, model
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2ML. The levels of [ H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen,
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thalamus, hippocampus, and cerebellum. However, the [ H]flunitrazepam binding and [ S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly
elevated in parts of thalamus. The levels of b2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of b3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule
layers in MK-801-infused rats. The levels of a6- and d-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by
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MK-801-infusion strongly elevates [ H]muscimol binding throughout the brain, increases regional [ H]flunitrazepam and [ S]TBPS binding, and alters GABA receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on
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various GABA receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor
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with the GABA receptors.
2000 Elsevier Science B.V. All rights reserved.
Theme : Neurotransmitters, modulators, transporters, and receptors
Topic : GABA receptors
Keywords : Autoradiography; In situ hybridization; Osmotic pump; Muscimol; Flunitrazepam
1. Introduction recognition site, but also at allosteric sites that bind
benzodiazepine, barbiturates, and steroids [26,36,44]. Bio- GABA is a major inhibitory transmitter in the central
chemical studies have provided evidence that there are at
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nervous system. The GABA receptor gates a Cl -selec-
least two different conformations of the GABA receptor.
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tive channel in response to the binding of transmitter and The high affinity GABA
sites can be labeled with
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contains multiple sites for pharmacologically distinct [ H]muscimol and are associated with GABA recognition
classes of allosteric modulators. GABA receptor-medi-
sites [28]. Molecular cloning has revealed a multiplicity of
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ated Cl conductance is positively modulated at a GABA
GABA receptor subunits which, based on homology, can
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be divided into subunit classes with multiple members
Corresponding author. Tel.: 182-2-650-5749; fax: 182-2-653-8891.
[26]: a1–6, b1–4, g1–4, and d. Of the 15 GABA
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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 6 8 7 - 1
H .S. Kim et al. Brain Research 880 2000 28 –37
29
receptor subunits known to be present in the adult rodent tional Natick, MA, USA and all other reagents were
brain, the a1, a6, b2, b3, g2, and d subunit mRNAs are purchased from Sigma St. Louis, MO, USA.
expressed at high levels in GABA-responsive cells in the murine cerebellum. When expressed in cell lines, different
2.2. Animals and treatment subunit combinations produce GABA
receptors result in
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different pharmacological responses [42]. [ H]Muscimol Male Sprague–Dawley rats Daehan Laboratory Animal,
labeling revealed that the GABA recognition site is located Eumsung, South Korea weighing 220–240 g were ac-
on the b-subunit [4] or on both the a- and b-subunit [19], climatized for 1 week with free access to rat chow and tap
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while [ H]flunitrazepam labeling indicated that the benzo- water. The temperature 24638C and light 12-h dark of
diazepine recognition site is located mainly on the a- the housing environment were maintained constantly. All
subunit [11] or on both the a- and g-subunit [16]. procedures involving rats were performed using protocols
Glutamate receptors are involved in generating epi- approved by the Animal Care and Use Committee of our
leptiform seizure [5]. The NMDA receptor antagonist, institution. Rats were implanted with guide cannulae for
MK-801, decreases the occurrence and severity of ethanol the drug infusion. Rats were anesthetized with a self
and pentobarbital withdrawal seizures [15,33], and inhibits prepared anesthetic, Equithensin 40 mM sodium pen-
tolerance to ethanol [50]. There is also strong evidence in tobarbital, 250 mM chloral hydrate, 90 mM MgSO ?7H O
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primary cultures of rat cerebellar granule cells that the dissolved in propylene glycol, ethanol, and distilled water,
regulation of mRNA expression of various GABA re-
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3 ml kg i.p., before standard stereotaxic surgery was ceptor subunits is controlled by activation of the NMDA
performed on a Kopf stereotaxic frame. A 21-gauge receptor complex [17,29], and that GABA
receptor
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stainless steel cannula was implanted in the right lateral maturation is reversibly inhibited by MK-801 via modula-
ventricle L, 1.3 mm; A–P, 20.5 mm; and D–V, 24.5 mm tion of receptor subunit expression [48]. Thus, gluta-
of the rat brain with the bregma chosen as the stereotaxic matergic activity may be an important regulator of
reference point [35]. The cannula was held in place with GABAergic activity.
rapid-setting dental acrylic Lang Dental, Wheeling, IL, MK-801 is a potent and selective noncompetitive an-
USA anchored to the skull by an aluminum protective cap tagonist of NMDA receptors, and MK-801 exerts various
and steel screws. Rats were allowed 1 week for recovery behavioral effects such as anxiolytic, anticonvulsant, and
before implantation of osmotic minipumps. The minipump neuroprotective effects [34,40]. MK-801 treatment results
was implanted s.c. as described [20] with minor modi- in an increase in the density of radiolabeled antagonist-
fication. Briefly, under ether anesthesia, a small cut was preferring conformation of the central benzodiazepine-
made behind the ears of the rat and the subcutaneous space binding site [7]. However, MK-801 produces a variety of
was expanded with a hemostatic forceps. Saline vehicle or behavioral effects in rodents ranging from hyperactivity to
MK-801 100 nM in saline was filtered through a 0.2-mm stereotypic motor syndromes such as lateral head weaving,
sterile syringe filter Sterile Acrodisc and was then used to circling, body rolls, and ataxia [21,22,24,46]. We have
fill an osmotic minipump Alzet 2ML 1, Alza, Palo Alto, therefore selected a low dose of MK-801 1 pmol 10 ml
CA, USA. The minipump was implanted and connected per h which does not produce these behavioral effects of
directly to the cannula via 6-cm long PE-60 polyethylene the following studies. The present experiments were
tubing. The infusion rate was 1 pmol MK-801 10 ml per h designed to determine whether prolonged infusion of MK-
for 7 days. The incision on the back was closed with 801 alters GABA receptor binding and whether MK-801
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cyanoacrylate glue, and dental acrylic was layered on top administration alters the expression of GABA
receptor
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of the polyethylene tube. subunit mRNA that is linked to the alteration of GABA
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receptor in adult rats. 2.3. Tissue preparation
Rats infused with MK-801 were decapitated 2 h after the disconnection of osmotic minipumps. After the decapita-
2. Materials and methods
