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
A 3
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
3
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
4 2
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-
A
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
A
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
A
cyanoacrylate glue, and dental acrylic was layered on top administration alters the expression of GABA
receptor
A
of the polyethylene tube. subunit mRNA that is linked to the alteration of GABA
A
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
tion, rat brains were removed immediately and were frozen in liquid nitrogen for 20 s. Horizontal sections, 14-mm
2.1. Chemicals thickness, were cut on an cryostat microtome at 2188C,
3 3
thaw-mounted on gelatin-coated microscope slides and [ H]Muscimol 10.1 Ci mmol, [ H]flunitrazepam 85.8
35 35
stored at 2808C until used. Ci mmol, [ S]TBPS 148.7 Ci mmol, and [ S]dATP
1000 Ci mmol were purchased from New England Nuclear Boston, MA, USA. Nensorb 20 nuclei purifica-
2.4. Autoradiographic procedures tion cartridges and terminal transferase were purchased
3
from New England Nuclear. Muscimol, flunitrazepam and Receptor autoradiography of [ H]muscimol was per-
TBPS were obtained from Research Biochemical Interna- formed according to the method of Titulaer et al. [45] with
30 H
35
modifications. In brief, tissue sections were thawed and perature air before the [ S]TBPS binding. Tissue sections
dried at room temperature, pre-washed in 50 mM Tris– were incubated in 50 mM Tris–citrate buffer containing 3
35
citrate buffer pH 7.1 which contains 150 mM NaCl for nM [ S]TBPS and 200 mM KCl for 3 h at room
30 min at 48C and blown-dry under a stream of cool air temperature, rinsed with cold 50 mM Tris–citrate buffer
3
before the [ H]muscimol binding. Tissue sections were two times for 5 min each, dipped once in distilled water at
incubated in 50 mM Tris–citrate buffer containing 10 nM room temperature, and immediately dried in a stream of
3
[ H]muscimol for 40 min at 48C, rinsed with cold 50 mM cool air. Non-specific binding was determined in the
Tris–citrate buffer three times for 5 min each, dipped once presence of non-radioactive 100 mM picrotoxin.
in ice-cold distilled water, and immediately dried in a Dried tissue sections were placed in X-ray cassettes with
3
stream of cool air. Non-specific binding was determined in a set of tritium standards [ H]Micro-scale RPA 510,
3 3
the presence of non-radioactive 100 mM muscimol. Amersham for [ H]muscimol and [ H]flunitrazepam bind-
3
Receptor autoradiography of [ H]flunitrazepam was ing, and carbon standards Carbon-14 standards, ARC-
35
performed according to the method of Carlson et al. [3] 146C, American Radiolabeled Chemicals for [ S]TBPS
with modifications. In brief, tissue sections were thawed binding, and juxtaposed to Hyperfilm or b-max film
and dried at room temperature. Then, tissue sections were Amersham. Following a certain exposure period 6 weeks
3 3
incubated in 50 mM Tri–citrate buffer containing 150 mM for [ H]muscimol and 2 weeks for [ H]flunitrazepam
3 35
NaCl and 1 nM [ H]flunitrazepam for 90 min at 48C, binding at 48C; 2 days for [ S]TBPS binding at room
rinsed with cold 50 mM Tris–citrate buffer two times for 5 temperature, the film was developed in Kodak D19 at
min each, dipped once in ice-cold distilled water, and room temperature for 3 min and fixed for 5 min. Auto-
immediately dried in a stream of cool air. Non-specific radiograms were analyzed by a digital scanning densitome-
binding was determined in the presence of non-radioactive ter Personal Densitometer, Molecular Dynamics, Sunny-
10 mM flunitrazepam. vale, CA, USA, operating on the image acquisition and
35
Receptor autoradiography of [ S]TBPS was performed analysis program ImageQuant 3.3 Molecular Dynamics.
according to the method of Edgar and Schwartz [8] with Plastic standards exposed simultaneously with the brain
modifications. In brief, tissue sections were thawed and sections were used as reference with the resulting binding
dried at room temperature, pre-washed in 50 mM Tris– values given as radioactivity levels estimated for gray
citrate buffer pH 7.4 containing 200 mM KCl and 1 mM matter areas nCi mg tissue. Non-specific binding was
EDTA to remove endogenous GABA for 10 min at room less than 5 of the total binding and was negligible for
temperature and blown-dry under a stream of room-tem- analyzing the autoradiograms.
3
Fig. 1. Representative autoradiograms of [ H]muscimol binding in horizontal rat brain sections. Tissue sections were incubated in 50 mM Tris–citrate
3
buffer containing 150 mM NaCl and 10 nM [ H]muscimol for 40 min at 48C.
H .S. Kim et al. Brain Research 880 2000 28 –37
31 Table 1
2.5. In situ hybridization
3
Changes in [ H]muscimol binding to brain regions in rats chronically infused with MK-801
In situ hybridization was carried out by our previous
3
Regions [ H]Muscimol binding
described method [32]. Briefly, thaw-mounted brain sec-
nCi mg tissue
tions were rinsed 3 times in phosphate-buffered saline
a
Saline MK-801
PBS for 3 min, rinsed once in 23 SSC for 3 min and rinsed once in deionized water. The probe was designed to
Frontal cortex Layers II, III
1.4160.16 2.4960.21
be complementary to the mRNA of GABA receptor
A
Layers IV, V, VI 0.7960.10
1.5460.11
a6-subunit 59-TGA CTT TGC TAC CGG GGG CTT
Cingulate 1.6760.15
2.7660.19
GGC TGC AGT CTG TGC CTG CCT-39; residues 318–
Caudate-putamen 0.4460.06
0.9460.07
331 [25], b2-subunit 59-TCG TTC CAG GGC GTT GCG GCC AAA ACT ATG CCT AGG CAA CCC AGC-
Thalamic nuclei Anterior ventral
1.4060.14 2.9060.21
39; residues 1292–1336 [51], b3-sbunit 59-CAT GTA
Ventral lateral 1.2860.13
2.7760.21
CCG CCC ATG CCC TTC CTT GGG CAT-39; residues
Lateral geniculate 1.3260.17
2.5960.22
1289–1318 [51], d-subunit 59-GAG GAC AAT GGC
Medial geniculate 1.0560.12
1.9660.11
GTT CCT CAC GTC CAT CTG TGC CCT TGG-39;
Hippocampal formation
residues 1078–1116 [41]. Probe was labeled on its 39-end
CA1 0.5960.07
1.1460.04
35
using terminal transferase and [a- S]dATP. The labeled
Dentate gyrus 1.0160.10
1.9260.16
probe was purified by Nensorb-20 column, and the probe
Cerebellum
5
solution 5310 dpm ml was diluted by 50 times with the
Molecular layer 1.7760.12
2.5160.20
hybridization buffer. Each brain section was hybridized
Granule layer 5.0760.32
8.2860.39
with 30 ml of hybridization buffer under a coverslip and
a
MK-801 was continuously infused flow rate, 1 pmol 10 ml per h into
was incubated at 388C in a high-humidity environment
rat brain i.c.v. by miniosmotic pumps for 7 days. The tissue sections
3
overnight. After hybridization, coverslips were removed in
were incubated with 10 nM [ H]muscimol in the presence of 150 mM NaCl. Values are expressed as mean6S.E. from five to six rats.
13 SSC, and slides were rinsed to remove excess hybridi-
P,0.01 for difference from respective control groups.
zation buffer. Finally, slides were briefly dipped in deion-
3
Fig. 2. Representative autoradiograms of [ H]flunitrazepam binding in horizontal rat brain sections. Tissue sections were incubated in 50 mM Tris–citrate
3
buffer containing 150 mM NaCl and 1 nM [ H]flunitrazepam for 90 min at 48C.
32 H
Table 2
ized water and dried with air. Dried tissue sections were
3
14
Changes in [ H]flunitrazepam binding to brain regions in rats chronically
placed in X-ray cassettes with a set of C-standard ARC-
infused with MK-801
146, St. Louis, MO and juxtaposed to Hyperfilm-bmax
3
Regions [ H]flunitrazepam binding
Amersham. Following a 2-week exposure period at room
nCi mg tissue
temperature, the film was developed and analyzed as
a
Saline MK-801
described in Section 2.4.
Cortex Layers II, III
16.0060.45 17.3360.41
2.6. Quantification and statistics
Frontal layer IV 17.8260.47
18.7960.37 Frontal layers V, VI
13.2360.47 13.4960.35
The density in each region was recorded by marking
Cingulate 19.4360.45
19.9560.33
four to 10 areas over bilateral sides of the brain according
Caudate-putamen 5.7260.26
6.1060.23
to the size and shape of the region. The mean values were
Septum 7.2660.30
8.0160.46
determined from five rats and expressed as the mean6S.E., in nCi mg tissue for autoradiography and nCi g tissue for
Thalamic nuclei
in situ hybridization, respectively. Data were analyzed
Ventral lateral 4.0560.27
4.7060.22 Lateral geniculate
4.8860.15 5.6260.18
using Student’s t-test and analysis of variance followed by
Habenula 5.0660.29
5.4960.27
Newman–Keul’s test.
Hippocampal formation CA1
13.5160.32 14.1860.24
CA3 10.8760.25
11.6760.14
3. Results
