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
Dentate gyrus 17.9460.41
18.8760.42 Brainstem
The distribution of GABA receptor binding e.g.,
A
Inferior colliculus 4.9860.34
4.1560.19
3 3
35
[ H]muscimol, [ H]flunitrazepam, and [ S]TBPS binding
Central gray 13.6460.52
11.9860.24
shows regional discrepancies in the rat brain. Autoradiog-
3
Cerebellum
raphs of [ H]muscimol binding are shown in Fig. 1. When
Molecular layer 13.7860.41
13.5060.33
the bindings were quantitated in 10–12 slices, the level
Granule layer 5.2360.36
5.8360.42
was found to be highest in the granule layer of cerebellum,
a
MK-801 was continuously infused flow rate, 1 pmol 10 ml per h into
moderately high in the cortex and thalamus, and low in the
rat brains i.c.v. by miniosmotic pumps for 7 days. The tissue sections
3
hippocampus and caudate putamen Fig. 1 and Table 1.
were incubated with 1 nM [ H]flunitrazepam in the presence of 150 mM
3
Binding of [ H]muscimol was highly increased 70–
NaCl. Values are expressed as mean6S.E. from five to six rats. P,0.05, P,0.01 for difference from respective control groups.
110 in the cortex, caudate putamen, thalamus and hippocampus by the treatment of MK-801, and also
significantly increased 40–60 in the cerebellum Table mRNA in the rat brain is shown in Fig. 4. When the
1. images were quantitated, levels of a-6 subunit were
3
When the autoradiographs of [ H]flunitrazepam were exclusively localized in cerebellar granule layers, and were
quantitated, binding was found to be highest in the cortex strongly increased 87 by the infusion of MK-801. The
and hippocampus, moderately high in the caudate putamen, levels of b2-subunit were found to be highest in cerebellar
brainstem and molecular layer of the cerebellum, and low granule layers, and significant levels were observed in
in the thalamus and granule layer of the cerebellum Fig. 2 cerebral cortical areas, thalamus, hippocampus, and in-
3
and Table 2. Binding of [ H]flunitrazepam was signifi- ferior colliculus Fig. 4 and Table 4. The levels of
cantly increased 7–15 in part of the frontal cortex b2-subunit were significantly elevated in almost all areas
layers II and III, thalamus lateral geniculate and of brain by the infusion of MK-801; frontal cortex 15,
hippocampus CA3 by the treatment of MK-801, but was thalamus 14, hippocampal area 8–17, inferior
not significantly affected in the cerebellum. However, the colliculus 16, and cerebellar granule layers 19. The
3
level of [ H]flunitrazepam binding was decreased 14 in levels of b3-subunit were found to be highest in hippocam-
parts of the brain stem central gray area Table 2. pal area, and significant levels were observed in frontal
35
When the autoradiographs of [ S]TBPS were quanti- cortex and cerebellar granule layers. In contrast to the
tated, binding was found to be high in the frontal cortex, b2-subunit, the levels of b3-subunit were significantly
thalamus, brainstem and granule layer of the cerebellum, decreased by the infusion of MK-801; frontal cortex
and low in the septum and molecular layer of the cere- 34,
hippocampal area
22–39, and
cerebellar
35
bellum Fig. 3 and Table 3. Binding of [ S]TBPS in granule layers 21 Fig. 4 and Table 4. The levels of
almost all area of the brain was not modulated by the d-subunit were found in frontal cortex, thalamus, and
treatment of MK-801, although the level was increased cerebellar granule layers. The levels of d-subunit were
7–8 in the part of thalamus ventral lateral and lateral significantly decreased 44 in frontal cortex but the
geniculate Table 3. levels were increased 15 in the cerebellar granule
In situ hybridization images of GABA receptor subunit layers by the infusion of MK-801 Fig. 4 and Table 4.
A
H .S. Kim et al. Brain Research 880 2000 28 –37
33
35
Fig. 3. Representative autoradiograms of [ S]TBPS binding in horizontal rat brain sections. Tissue sections were incubated in 50 mM Tris–citrate buffer
35
containing 200 mM and 3 nM [ S]TBPS for 3 h at room temperature. Table 3
35
4. Discussion
