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THE JOURNAL OF COMPARATIVE NEUROLOGY 416:475–495 (2000)
Differential Expression of
␥-Aminobutyric Acid Type B Receptor-1a
and -1b mRNA Variants in GABA and
non-GABAergic Neurons of the Rat Brain
FENGYI LIANG,1* YUMIKO HATANAKA,2 HARUMI SAITO,1 TETSUO YAMAMORI,3
AND TSUTOMU HASHIKAWA1
1Laboratory for Neural Architecture, Brain Science Institute, Riken, Wako,
Saitama 351–0198, Japan
2Laboratory for Neuronal Recognition Molecules, Brain Science Institute, Riken, Wako,
Saitama 351–0198, Japan
3Division of Speciation Mechanisms 1, National Institute for Basic Biology, Okazaki,
Aichi 444–8585, Japan
ABSTRACT
To understand the heterogeneity of ␥-aminobutyric acid type B receptor (GABABR)mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA
and non-GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive
in situ hybridization histochemistry and, in combination with GABA immunocytochemistry,
double labeling. In situ hybridization with a pan probe, which recognizes a common sequence
of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of
GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were
expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only
GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain
structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for
both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed
either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate
putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex
expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did
not display significant levels of either GABABR1a or GABABR1b mRNAs. These data
suggested widespread availability of GABABR-mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain
structures may contribute to the heterogeneity of GABABR-mediated inhibition. Some GABA
neurons possessed, but others might lack the molecular machinery for GABABR-mediated
disinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000. r 2000 Wiley-Liss, Inc.
Indexing terms: GABAB receptor; double labeling; immunocytochemistry; in situ hybridization;
forebrain; cerebellum
As the main inhibitory neurotransmitter in the central
nervous system (CNS), ␥-aminobutyric acid (GABA) plays
important roles in regulating neuronal activity, plasticity,
and pathogenesis. Its action is mediated through distinct
type A, B, or C (GABAA, GABAB, or GABAC) receptors. In
contrast to the ionotropic GABAA and GABAC receptors
that mediate fast inhibitory postsynaptic potentials (IPSP),
the metabotropic GABAB receptor (GABABR) mediates
slow, long-lasting inhibitory neuronal responses that have
been implicated in many important physiological functions and pathologic alterations in the brain (Bowery et al.,
1990; Soltesz and Crunelli, 1992; Bonanno and Raiteri,
1993b; Mott and Lewis, 1994; Misgeld et al., 1995; Bowery,
r 2000 WILEY-LISS, INC.
1997; Deisz, 1997; Bettler et al., 1998). GABABRs have
been demonstrated in both pre- and postsynaptic components of both excitatory and inhibitory neurons in the
Grant sponsor: Riken BSI; Grant number: 57911; Grant sponsor: NIBB
Cooperative Research Program; Grant number: 98–173.
Yumiko Hatanaka’s current address is: Division of Behavior and Neurobiology, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444–
8585, Japan.
*Correspondence to: Dr. Fengyi Liang, Laboratory for Neural Architecture, Brain Science Institute, Riken, 2–1 Hirosawa, Wako, Saitama 351–
0198, Japan. E-mail: [email protected]
Received 26 March 1999; Revised 4 October 1999; Accepted 5 October 1999
476
central nervous system. Their existence in glia cells has
also been reported (Hosli and Hosli, 1990; Hosli et al.,
1990; Fraser et al., 1994). Presynaptic GABABR-mediated
auto-/heteroinhibition represents a classic, well-known
model for regulation of neurotransmitter release and has
been subjected to extensive pharmacologic and electrophysiological studies in a variety of brain structures.
Postsynaptic GABABRs mediate a late IPSP that may
results in long-term changes in neurotransmission and
dynamic response behavior of neurons (Misgeld et al.,
1995; Davies and Collingridge, 1996; Mouginot and
Gahwiler, 1996; Bowery, 1997; Deisz, 1997; Isaacson and
Hille, 1997; Bettler et al., 1998).
Among other findings, previous pharmacologic and electrophysiological studies have suggested the existence of
different subtypes of GABABRs at pre- or postsynaptic
sites and in different cell types and brain structures (Dutar
and Nicoll, 1988; Bonanno and Raiteri, 1993b; Fukuda et
al., 1993; Pitler and Alger, 1994; Bowery, 1997; Deisz et al.,
1997; Bettler et al., 1998). The distribution of GABABR in
the CNS has been demonstrated with receptor binding
autoradiography (Bowery et al., 1987; Chu et al., 1990;
Knott et al., 1993; Turgeon and Albin, 1993). Expression of
GABABR1 mRNA in the rat brain and in the monkey
thalamus has been shown by using radioisotope labeled
riboprobes that recognize both of the two major GABABR1
mRNA variants, GABABR1a and GABABR1b (Kaupmann
et al., 1997; Munoz et al., 1998; Lu et al., 1999). In
addition, GABABR1 immunoreactivity in the rat brain and
retina and GABABR1a and GABABR1b mRNA variant
distribution in the cerebellar cortex and mouse retina have
been reported (Kaupmann et al., 1998b; Koulen et al.,
1998; Zhang et al., 1998; Fritschy et al., 1999; MargetaMitrovic et al., 1999). However, it is still unclear how the
two major GABABR1 mRNA variants are distributed in
different neuronal cell types in the CNS and how the
possible differences in their distribution may contribute to
the heterogeneity of GABABR-mediated late IPSP and
hetero-/autoinhibition of neurotransmitter release in various CNS structures. To address these issues, we mapped,
at cellular level, the expression of GABABR1 gene and its
two major mRNA variants, GABABR1a and GABABR1b, in
the rat brain by using nonradioactive in situ hybridization
histochemistry.
Of particular interests and obvious functional roles are
GABABRs on GABA neurons. GABABRs on somata/
dendrites of GABA neurons may serve as a mechanism to
reduce GABA neuronal activity (disinhibition) by recurrent collaterals or by GABAergic input from other GABA
neurons. GABABRs on GABAergic axon terminals (autoreceptors) play important roles in regulating GABA release
in a frequency-dependent manner. Both processes may act
as gating mechanisms for the generation of long-term
potentiation (LTP) and other types of use-dependent modification of neuronal excitability (Floran et al., 1988; Deisz
and Prince, 1989; Giralt et al., 1990; Davies et al., 1991;
Mott and Lewis, 1991; Metherate and Ashe, 1994; Doze et
al., 1995; Hosford et al., 1995; Morishita and Sastry, 1995;
Brucato et al., 1996; Caddick and Hosford, 1996; Bonanno
et al., 1997). Although GABAB receptors on GABAergic
axon terminals or somata have been demonstrated pharmacologically and electrophysiologically in a variety of brain
structures, further clarification of their distribution and
heterogeneity at molecular level would not only advance
F. LIANG ET AL.
our understanding of the function of this important class of
inhibitory receptor, but also facilitate the search for more
selective GABABR agonists/antagonists (Floran et al.,
1988; Harrison et al., 1988; Giralt et al., 1990; Seabrook et
al., 1991; Davies and Collingridge, 1993; Lambert and
Wilson, 1993; Doze et al., 1995; Ulrich and Huguenard,
1996; Jarolimek and Misgeld, 1997; Kim et al., 1997; Le
Feuvre et al., 1997; Vigot and Batini, 1997; Mouginot et al.,
1998; Koulen et al., 1998). Therefore, the second aim of the
present study was to investigate the expression of
GABABR1a and 1b mRNA variants in different types of
GABA neurons of the rat brain by using immunocytochemistry in situ hybridization histochemistry double-labeling
techniques. Our results indicated that the distribution of
GABABR1a in the CNS markedly differed from that of
GABABR1b in different GABA and non-GABAergic neurons. Preliminary results have been reported in abstract
form (Liang et al., 1998).
MATERIALS AND METHODS
cDNA cloning and in vitro transcription
Rat brain mRNA was isolated from a 12-day-old rat pup
(Wistar strain). First strand cDNA was synthesized by
reverse transcription by using SuperScript II kit (GibcoBRL, Grand Island, NY). GABABR1 cDNA fragments were
amplified by polymerase chain reaction and isolated by
electrophoresis on agarose gel. They were then subcloned
into the EcoRV site of pBluescript II SK vector (Stratagene, La Jolla, CA). JM109 competent cells (Stratagene)
were transformed by the respective recombinant plasmids
and cultured on LB plates containing ampicillin, isopropyl1-thio-␤-D-galactoside (IPTG) and 5-bromo-4-chloro-3indolyl-␤-D-galactoside (Xgal). White colonies were picked
up from the plate and cultured in 2⫻ TY medium containing ampicillin. Recombinant plasmids were purified from
the culture and the inserts were verified by sequence
analysis by using an ABI 310 or ABI 377 DNA sequencer
(Perkin-Elmer, Foster, CA).
Three cDNA fragments were cloned. GABABR1a fragment is specific for GABABR1a variant, has a C⫹G ratio of
61.78% and corresponds to nucleotides 1 to 437 of rat
GABABR1a gene (EMBL accession number Y10369).
GABABR1b is specific for GABABR1b variant, has a C⫹G
ratio of 72.73% and corresponds to nucleotides 21 to 141 of
rat GABABR1b gene (EMBL accession number Y10370).
GABABR1p recognizes both GABABR1a and GABABR1b
variants, has a C⫹G ratio of 57.60% and corresponds to
nucleotides 2408–2841 (2060–2493) of rat GABABR1a
(GABABR1b) gene.
The purified plasmids were linearized and then used as
templates for in vitro transcription of digoxigenin-labeled
antisense (or sense control) RNA probes with T3 (or T7)
RNA polymerase. The nucleoside triphosphate mix for in
vitro transcription consists of 10 mM ATP, 10 mM CTP, 10
mM GTP, 6.5 mM UTP, and 3.5 mM digoxigenin-11-UTP.
According to the supplier (Boehringer Mannheim, Mannheim, Germany) average digoxigenin-11-UTP incorporation into transcribed RNA is about 4–5% of all nucleotides.
In situ hybridization
Sixteen adult rats (Wistar strain) of either sex, ages 10
to 14 weeks and weighing 160–240 grams, were deeply
anesthetized with Nembutal (60 mg/kg, i.p.) and transcardially perfused with physiological saline (about 50 ml)
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
followed by 4% paraformaldehyde in 0.1 M phosphate
buffer (800 ml, pH 7.4). The brain and spinal cord were
removed and postfixed in the same fixative for 4–6 hours at
4°C. They were then cryoprotected in 30% sucrose at 4°C
for 24–48 hours. Thirty-five-micrometer frozen sections
were cut on a freezing microtome. All procedures involving
experimental animals were approved by Riken Animal
Care Committee.
In situ hybridization histochemistry followed a protocol
modified from previous descriptions (Liang and Jones,
1997; Hatanaka and Jones, 1998). Free-floating sections
were rinsed twice in phosphate buffered saline (pH 7.4)
and then treated sequentially with 0.75% glycine (15
minutes ⫻ 2), 0.3% Triton X-100 (10 minutes), 1 µg/ml
proteinase K (15 minutes) and 0.25% acetic anhydride in
0.1 M triethanolamine (15 minutes). After two washes in
2⫻ saline sodium citrate (SSC; 1⫻ SSC ⫽ 0.15 M NaCl,
0.015 M Na3C6H5O7 ⭈ 2H2O) and 1 hour prehybridization
at 60°C in a hybridization buffer (pH 7.0) without probe,
sections were hybridized at 60°C for approximately 14–16
hours in a new hybridization buffer (pH 7.0) that contained
one of the digoxigenin-labeled RNA probes. The hybridization buffer consisted of 2–5⫻ SSC (see below), 2% blocking
reagent (Boehringer Mannheim), 50% deionized formamide, and 0.1% N-lauroylsarcosine. SSC concentration
in the hybridization buffer was varied between 2⫻ and 5⫻
to offset differences in melting temperature derived from
variations in the length and G⫹C content of each probes.
Probe concentrations of approximately 0.07–0.18 µg/ml for
GABABR1a, GABABR1p and 0.25 µg/ml for GABABR1b
were used.
At the end of hybridization, sections were sequentially
treated in the following solutions (except for ribonuclease
A, all containing 0.1% N-lauroylsarcosine): 2⫻ SSC plus
50% deionized formamide, 60°C, twice, 20 minutes each;
ribonuclease A buffer (0.01 M Tris-HCl buffer, pH 8.0, plus
1 mM ethylenediaminetetraacetic acid and 0.5 M NaCl),
room temperature, 10 minutes; ribonuclease A (0.02 mg/ml
in ribonuclease A buffer), 37°C, 30 minutes; 2⫻ SSC, 60°C,
twice, 20 minutes each; 0.2⫻ SSC, room temperature and
then 60°C, 20 minutes each.
To visualize hybridization signals by immunoalkaline
phosphatase histochemistry, sections were rinsed in 0.1 M
Tris-HCl buffered saline (TBS, pH 7.5, 10 minutes), incubated in 1% blocking reagent (in TBS, 60 minutes), and
then in alkaline phosphatase-conjugated sheep antidigoxigenin antibody (Fab fragments, Boehringer Mannheim, 1:1,500 dilution in TBS, pH7.5, plus 1% blocking
reagent, 60 minutes). After three washes in TBS plus 0.1%
Tween 20, alkaline phosphatase (AP) activity was visualized either by nitroblue tetrazolium (NBT) and 5-bromo-4chloro-3-indolyl phosphate (BCIP) or by 2-hydroxy-3naphtoic acid-28-phenylanilide phosphate and fast red TR.
The former reaction gave rise to a blue precipitate in
hybridization-positive cells, whereas the latter produced a
red fluorescent deposit with a maximum absorbance at 550
nm and maximum emission at 562 nm. The NBT-BCIP
reaction lasted for 6–18 hours at room temperature in a
substrate solution consisting of 0.033% NBT, 0.017% BCIP,
0.001 M levamisole, 0.1 M NaCl, and 0.05 M MgCl2 ⭈ 6H2O
in 0.1 M Tris-HCl buffer, pH 9.5. The fluorescent AP
reaction lasted for 0.5–2 hours at room temperature in a
substrate solution consisting of 0.01% 2-hydroxy-3-naphtoic acid-28-phenylanilide phosphate, 0.025% fast red TR,
0.001 M levamisole, 0.1 M NaCl, and 0.01 M MgCl2 in 0.1
477
M Tris-HCl buffer (pH 8.0). Sections were then either
processed further for GABA immunocytochemistry for
double labeling (see below) or mounted on chrome alumgelatin–coated glass slides, air-dried, and cover-slipped by
using an aqueous mounting medium.
Negative control experiments included hybridization
that used sense riboprobes and ribonuclease A treatment
after hybridization in 0.01 M Tris-HCl buffer, pH 8.0, and 1
mM ethylenediaminetetraacetic acid without NaCl. Positive controls consisted of in situ hybridization with antisense riboprobes for glutamic acid decarboxylase-67
(GAD67) and brain-derived neurotrophic factor (BDNF).
For each animal, a series of sections was also stained with
thionin for cytoarchitecture.
GABA immunocytochemistry
For GABA immunocytochemistry or double labeling of
GABA immunoreactivity and GABABR1 mRNA, sections
were rinsed twice in 0.01 M phosphate buffered saline (pH
7.4) before being placed in 5% normal goat serum plus
0.3% Triton X-100 in 0.01 M phosphate buffered saline (pH
7.4) for 1 hour at room temperature. They were then
incubated for 12 hours at room temperature in an affinitypurified, rabbit anti-GABA serum (Sigma, St. Louis, MO;
1:2,000 dilution) and washed in 0.01 M phosphate buffered
saline (pH 7.4). For GABA immunoperoxidase, subsequent
processes followed instructions supplied with the avidin
biotinylated-peroxidase complex (ABC) kit from Vector
Laboratories (Burlingame, CA). Peroxidase activity was
visualized by H2O2 (0.01%) and diaminobenzidine tetrahydrochloride (DAB·4HCl, 0.05%) that gave rise to a brownish deposit at the site of GABA immunoreactivity. For
GABA immunofluorescence, bound antibody was revealed
by goat anti-rabbit IgG secondary antibody conjugated to
fluorescein (Vector Laboratories; 1:100) that yielded green
fluorescence in GABA positive cells. For control reactions
of GABA immunocytochemistry, the primary antibody was
replaced by equal volume of normal rabbit serum.
Double labeling
For studying the distribution of GABABR1 mRNA signal
in GABA neurons, a sequential double-labeling procedure
was carried out on the same histologic sections to visualize
first GABABR1a, GABABR1b or GABABR1p mRNA signals
by in situ hybridization histochemistry and then GABA
immunoreactivity by immunocytochemistry. As with any
double- or multiple-labeling techniques, there exists the
possibility of masking of one reaction by the other(s) and
there were those cells that were marginally (ambiguously)
positive for one marker or the other; therefore, their
identity as single- or double-labeled cells could not be
definitely resolved. These issues were dealt with in the
present study as follows: First, in addition to a nonfluorescent double-labeling technique that used NBT-BCIP for
revelation of GABABR1 mRNAs and DAB/H2O2 for GABA
immunoreactivity, a fluorescent double-labeling procedure
was carried out by using the red fluorescent AP reaction for
visualization of GABABR1 mRNAs and then immunofluorescence for GABA. Preparations from fluorescent doublelabeling experiments were compared with those from
nonfluorescent double labeling to take the advantages of
both methods and to cross-verify the results. Second, in
addition to conventional microscope, Bio-Rad MRC600
laser scanning confocal microscope (Bio-Rad, Hercules,
CA) was used to analyze fluorescent double-labeled materi-
478
F. LIANG ET AL.
als to ascertain the identity of individual cells. Finally,
only those cells with discernible nuclei were considered for
drawing conclusions on the coexistence of GABA and
GABABR1.
Data analysis
Both fluorescent and nonfluorescent histologic preparations were observed under a Nikon Eclipse 800 microscope.
Fluorescent histochemical materials were further analyzed and imaged by a Bio-Rad MRC-600 laser scanning
confocal microscope. Levels of hybridization signals for
GABABR1 mRNAs in various brain structures were estimated by intensity of NBT-BCIP reaction products or
fluorescence. Hybridization signals with antisense probes
in medial habenular nucleus or Purkinje cells (for
GABABR1b and GABABR1p) were denoted as very strong
(⫹⫹⫹⫹, very high levels) and background hybridization
similar to that with sense control probes as negative (-).
Intermediate intensities were designated successively as
strong (⫹⫹⫹, high levels), medium (⫹⫹, medium levels),
weak (⫹, low levels), and very weak or marginal (⫾, very
low levels). Initial ratings of relative signal levels in
various structures were carried out independently by two
investigators and wherever disagreements arose, a third
investigator was called upon to verify the results.
Low-magnification images of nonfluorescent preparations were photomicrographed first and then digitized
with a Nikon LS-4500AF film scanner from negative films.
High magnification photomicrographs were grabbed directly from the microscope to a Macintosh G3 computer by
means of a digital color image acquisition system (Sony
DXC-950 3CCD color video camera, Scion CG-7 color
image grabber and Scion Image 1.62 software). Figures
were prepared from digitized images by using Adobe
Photoshop 5.0 and printed on a full-color digital printer
(Fujix Pictography 3000, Fuji Film, Tokyo, Japan). For
best printouts, the original image resolutions were scaled
to 320 or 400 pixels per inch with Adobe Photoshop
software.
RESULTS
All GABABR1a, GABABR1b, or GABABR1p mRNApositive stainings were located in the cell bodies. Many
cells in a majority of neural structures were GABABR1
mRNA positive, but levels of mRNA signals in different
cells varied to a large extent. Neurons in the medial
habenular nucleus and cerebellar Purkinje cells showed
the strongest hybridization for GABABR1p riboprobe.
Marked differences in the distributions of GABABR1a and
GABABR1b mRNA variants were observed in many brain
structures, with some neuronal cell types expressing neither and others expressing either or both. Different subtypes of GABA neurons were distinguished based on their
possession or lack of GABABR1a and/or GABABR1b mRNA
signals. Table 1 summarizes relative levels of in situ
hybridization signals for GABABR1a, GABABR1b, or
GABABR1p mRNA variant in major brain structures/cell
types. It should be noted that GABABR1b antisense probe
might produce a hybridization signal of lower intensity by
one order of magnitude in comparison with GABABR1a
and GABABR1p because of its short length and extremely
low probability of incorporating digoxigenin-11-UTP (see
Discussion section). Any correlation between staining in-
TABLE 1. Relative Levels of Hybridization Signals for GABABR1 mRNA
Variants in Major CNS Structures1
Brain
regions
Neocortex
Hippocampus
Septum
Habenula
Thalamus
Basal ganglia
Hypothalamus
Cerebellum
Brain stem
Spinal cord
Structure/cells
Layer V pyramidal cells
Other layers
Pyramidal cells
Granule cells
Other neurons
LSD
Others
Medial
Lateral
TRN/VLG
Other nuclei
Caudate
putamen
Subthalamus
SNC
SNR
Supraoptic
nucleus
Others
Basket/stallete
cells
Purkinje cells
Golgi cells
Granule cells
Deep cerebellar
nuclei
Colliculus
Periaquiduct
gray
Pontine nuclei
Cranial nerve
nuclei
Others
Motoneurons
Other laminae
White matter
glia cells
GABABR1a
GABABR1b
GABABR1p
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹⫹)
(⫹⫹⫹)
(⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹⫹)
(⫹⫹⫹)
(⫺)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)
(⫺)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹⫹)
(⫹⫹⫹⫹)
(⫾)–(⫹⫹)
(⫺)–(⫾)
(⫹⫹)–(⫹⫹⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹⫹)
(⫹⫹⫹⫹)
(⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)
(⫹)–(⫹⫹)
(⫺)–(⫾)
(⫺)–(⫾)
(⫹)
(⫺)
(⫹)–(⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫺)–(⫾)
(⫺)–(⫾)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹)
(⫺)–(⫾)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫺)
(⫹⫹⫹⫹)
(⫺)
(⫺)
(⫾)
(⫹⫹⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹)–(⫹⫹)
(⫹)–(⫹⫹)
(⫺)
(⫺)–(⫾)
(⫹)–(⫹⫹)
(⫹)–(⫹⫹)
(⫹)–(⫹⫹⫹)
(⫹⫹)
(⫺)–(⫾)
(⫺)–(⫾)
(⫹)–(⫹⫹⫹)
(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫺)–(⫹)
(⫺)–(⫾)
(⫹)
(⫺)–(⫾)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹⫹)–(⫹⫹⫹)
(⫹)–(⫹⫹)
(⫹)
(⫺)–(⫾)
(⫹)
1Some
but not necessarily all neurons in specified structures were positive for the
mRNA. Hybridization signals: undetected (⫺), marginal/very weak (⫾), weak (⫹),
medium (⫹⫹), strong (⫹⫹⫹), very strong (⫹⫹⫹⫹). Ratings were not for direct
comparison between GABABR1a/1p and GABABR1b signals for reasons stated in the
Discussion section. LSD, dorsal lateral septal nucleus; SNC, substantia nigra pars
compacta; SNR, substantia nigra pars reticulata; TRN, thalamic reticular nucleus;
VLG, ventral lateral geniculate nucleus.
tensity and relative mRNA levels for GABABR1a/1p was
not directly applicable to that for GABABR1b. Also for this
reason, the levels of GABABR1b mRNA expression in the
CNS as presented in the present study may represent an
underestimation. Although intensity of hybridization signals in sections from different animals slightly varied, its
relative ratings among different brain structures in individual animals kept consistent.
GABABR1b sense control probe yielded a very weak
hybridization signal in the pyramidal cell layer of hippocampal CA1–3 and granule cell layer of the dentate gyrus
(Fig. 1B). Other CNS structures did not show any detectable hybridization other than a very faint background
staining. Hybridization with sense control probes for
GABABR1a and GABABR1p did not give rise to positive
signal above background in any CNS structures (Fig. 1A).
Posthybridization treatment of sections with ribonuclease
A at low salt concentration also resulted in loss of positive
hybridization signals.
Neocortex
Many neurons across all neocortical layers were GABABR1 mRNA positive. Figure 2 compares the expression
pattern of GABABR1a (Fig. 2A) and GABABR1p (Fig. 2B)
in the primary somatosensory cortex. Left panels in Figure
3 illustrates GABABR1b hybridization in different layers
of the motor cortex.
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
479
Fig. 1. Photomicrographs showing hybridization by ␥-aminobutyric acid (GABA)BR1a and GABABR1b sense control probes in the
hippocampus. A: Hybridization with GABABR1a sense probe produced
no hybridization signal above background. Gr, granule cell layer; Or,
stratum oriens; Py, stratum pyramidalis; Rad, stratum radiatum.
B: Hybridization with GABABR1b sense probe gave rise to a very weak
hybridization signal in pyramidal cells of CA1-CA3 and in granule
cells of the dentate gyrus. Scale bar ⫽ 200 µm in B (applies to A,B).
In layer I, scattered neurons showed medium levels of
GABABR1a/1p mRNA, but only a few neurons in this layer
were weakly positive for GABABR1b mRNA. In layers II
and III, hybridization signals for GABABR1a/1p variant
were medium to high in many neurons of variable sizes,
but those for GABABR1b were low to marginal. Among
different neocortical areas, layers II-III neurons of the
temporal, occipital, and the barrel cortex showed more
pronounced hybridization than other areas. Hybridization
signals in layer IV neurons were generally weak for
GABABR1a/1p and very weak to unobservable for
GABABR1b mRNA. In the barrel cortex, neurons inside
the barrels were visibly weaker in their hybridization
signal for GABABR1a and GABABR1p (Fig. 2). Layer V
pyramidal neurons, especially those of the motor cortex,
showed the strongest hybridization of all neocortical neu-
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Fig. 2. Photomicrographs showing variable expression levels of ␥-aminobutyric acid (GABA)BR1a (A)
or GABABR1p (B) mRNA in different layers of the primary somatosensory cortex of the rat brain. Cortical
layers are indicated in A. Scale bar ⫽ 200 µm in B (applies to A,B).
rons for GABABR1a, 1b, or 1p probe, but hybridization
signals in neurons of smaller sizes in layer V were generally medium to low for GABABR1a/1p and low to below
detection for GABABR1b mRNA. mRNA levels in most
layer VI neurons were low for GABABR1a/1p and low to
marginal for GABABR1b (Figs. 2A,B, 3A,C,E).
As revealed by fluorescent double labeling and illustrated in Figure 4, a majority of GABA neurons in the
neocortex expressed GABABR1a/1p. Many neocortical
GABA neurons, especially those in deep layers, also displayed low to medium levels of GABABR1b mRNA (Fig.
3A–F). In layer I, virtually all GABABR1a/1p-positive
neurons were GABAergic. Some GABA neurons in layer I
also showed low levels of GABABR1b mRNA expression.
GABA neurons in layers II–VI were scattered among
non-GABAergic GABABR1 mRNA positive neurons. Those
in layers II to IV expressed variable levels of GABABR1a/1p
and very low to indiscernible GABABR1b mRNA. Most
GABA neurons in layers V and VI had weak to medium
GABABR1a, 1b, or 1p mRNA expression (Figs. 3A–F,
4A–D). Same results were obtained from nonfluorescent
double-labeling experiments (data not shown).
Hippocampus and septum
Pyramidal neurons of CA1–3 expressed strong
GABABR1a and medium GABABR1b mRNA with relatively higher levels of expression for the latter variant in
CA1 than in CA2–3. GABABR1p expression represented
the summation of GABABR1a and GABABR1b mRNA
variants in that pyramidal neurons in CA1 showed more
prominent hybridization signals than in CA2–3. Granule
cells of the dentate gyrus expressed medium levels of
GABABR1a/1p and low levels of GABABR1b mRNA. Scattered neurons in strata oriens, radiatum, and lacunosum
moleculare of CA1–3 and molecular, polymorphic layers of
the dentate gyrus exhibited medium levels of GABABR1a/1p
mRNA. Most of them were also weakly positive for
GABABR1b mRNA (Fig. 5A–C).
GABA immunocytochemistry revealed a distinct band of
dense GABA axon terminals in stratum radiatum at the
proximal dendritic segment of CA3 pyramidal cells (Fig.
6A,B). On double-labeling preparations, most GABA neurons stained positive for GABABR1a/1p, but a few of them
in strata oriens and pyramidalis displayed little
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
481
Fig. 3. Laser scanning confocal photomicrographs illustrating
double labeling of ␥-aminobutyric acid (GABA)BR1b mRNA (left
panels) and GABA immunoreactivity (right panels) on the same
histologic section of the rat motor cortex. Expression of GABABR1b
mRNA in GABA and non-GABAergic neurons in layers I–III (A,B) was
very weak or below detection. Many cells in layers V (C,D) and VI
(E,F) were moderately positive for GABABR1b. Note the relatively
strong hybridization in layer V pyramidal neurons. Arrows in the right
panels point to examples of GABA neurons and corresponding arrows
at the same locations in the left panels indicate the GABABR1b
expression status of the same neurons. Scale bar ⫽ 50 µm in F (applies
to A–F).
GABABR1a/1p hybridization signal. Both GABA and
GABABR1a/1p mRNA levels varied to a certain extent in
hippocampal GABA neurons (Fig. 6A–D). GABABR1b signals were also observed in a large proportion of hippocampal GABA neurons (Fig. 7A–D).
GABABR1a and GABABR1b coexpressed in the septum.
Although there existed considerable variation of GABABR1 mRNA staining intensity among individual neuronal
cells, neurons in the dorsal lateral septal nucleus showed
stronger GABABR1a, GABABR1b as well as GABABR1p
mRNA staining than those in ventral or medial septal
nuclei (Fig. 8A). Panels B to G in Figure 8 show fluorescent
double labeling of GABABR1 mRNA variants and GABA
immunoreactivity in the septum. All GABA neurons in the
septum appeared positive for GABABR1a, GABABR1b, and
GABABR1p mRNA.
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F. LIANG ET AL.
Fig. 4. Laser scanning confocal photomicrographs showing fluorescent double labeling of ␥-aminobutyric acid (GABA)BR1a (A,C) and
GABA (B,D) on the same histologic section of the rat motor cortex. All
GABA neurons in layers I, II (A,B), and a majority of GABA neurons in
layers V and VI (C,D) were GABABR1a mRNA positive. Arrows in the
right panels point to examples of GABA neurons, and corresponding
arrows at the same locations in the left panels indicate the GABABR1a
expression status of the same neurons. Cortical layers are indicated on
the right in A and C. Scale bar ⫽ 100 µm in D (applies to A–D).
Thalamus and caudate putamen
neurons in this structure seemed GABAergic and expressed low to medium levels of GABABR1a/1p mRNA
without detectable GABABR1b (Figs. 9A–C, 10A,B). In the
dorsal lateral geniculate nucleus, GABA neurons were
smaller in size than the surrounding non-GABAergic
neurons, showed strong GABA immunoreactivity, but gave
only marginal GABABR1a and no GABABR1b or
GABABR1p mRNA signal (Fig. 11A–F). GABA neurons in
ventral lateral geniculate nucleus had medium levels of
hybridization signals for GABABR1a and GABABR1p (data
not shown). Most other thalamic nuclei lacked significant
number of GABA neurons.
In the caudate putamen and globus pallidus, many
neurons expressed low to medium levels of GABABR1a and
GABABR1p mRNA, but GABABR1b hybridization signal
was mostly not observed. Scattered GABA neurons in the
caudate putamen and globus pallidus contained weak to
moderate levels of GABABR1a/1p mRNA but expressed no
apparent GABABR1b mRNA (Fig. 10C,D).
Neurons in a majority of dorsal thalamic nuclei displayed medium to high levels of hybridization signals for
both GABABR1a and GABABR1b mRNA. Thalamic reticular nucleus and ventral lateral geniculate nucleus, however, lacked significant hybridization signal for GABABR1b
under the present experimental condition. As a result,
GABABR1p mRNA levels in these nuclei were markedly
lower than those in other thalamic nuclei, although levels
of GABABR1a hybridization signals were comparable to
those in the latter. Among different nuclei in the dorsal
thalamus, mRNA levels varied in a similar pattern for
GABABR1a, 1b, and GABABR1p. Hybridization signals for
all three cRNA probes were visibly stronger in neurons of
anterodorsal, mediodorsal, laterodorsal, medial geniculate, and dorsal lateral geniculate nuclei than in other
thalamic subdivisions. Ventrolateral nucleus exhibited
slightly higher signal intensities than the neighboring
ventral posteromedial and ventral posterolateral nuclei.
Neurons in intralaminar nuclei displayed weaker hybridization signals (Fig. 9A–F, results from anterodorsal and
medial geniculate nuclei not illustrated).
Upper panels in Figure 10 show fluorescent double
labeling of GABABR1p mRNA hybridization (A) and GABA
immunoreactivity (B) in thalamic reticular nucleus. All
Cerebellum
Different cell types in the cerebellar cortex had their
unique patterns of expression of GABABR1 mRNA variants. Stellate/basket cells in the molecular layer showed
weak GABA immunoreactivity, weak GABABR1a, very
Fig. 5. Photomicrographs showing ␥-aminobutyric acid
(GABA)BR1a (A), GABABR1b (B), or GABABR1p (C) mRNA expression
in the hippocampus. Note the stronger hybridization signal in CA1
pyramidal neurons than in CA2–3 for GABABR1b and 1p. Gr, granule
cell layer; Or, stratum oriens; Py, stratum pyramidalis; Rad, stratum
radiatum. Scale bar ⫽ 200 µm in C (applies to A–C).
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F. LIANG ET AL.
Fig. 6. Laser scanning confocal photomicrographs showing double
labeling of ␥-aminobutyric acid (GABA)BR1 mRNA and GABA immunoreactivity in the hippocampus. A,B: GABABR1p mRNA and GABA
immunoreactivity in CA2–3. Note a band of GABAergic axon termi-
nals in stratum radiatum in CA3. C,D: GABABR1a mRNA expression
in pyramidal neurons and GABAergic interneurons in CA2–3. Scale
bars ⫽ 200 µm in B (applies to A,B); 100 µm in D (applies to C,D).
weak GABABR1p and undetectable GABABR1b mRNA
expression. Purkinje cells were weakly GABA immunoreactive, had strong signals for GABABR1b and GABABR1p,
but very weak signals for GABABR1a mRNA. Both Golgi
and granule cells showed no signals for GABABR1b mRNA.
Golgi cells had medium levels of GABA immunoreactivity
and GABABR1a/1p mRNAs. The granule cells were negative for GABA, but weakly positive for GABABR1a/1p
mRNA (Fig. 12A–F).
Although Purkinje cells showed very weak hybridization
signal for GABABR1a at probe concentrations used in the
present study, they did show medium to strong hybridization for GABABR1a at higher probe concentrations (⬎0.4
µg/ml) as demonstrated in a series of pilot experiments
designed to test the suitable probe concentration and to
optimize the specificity and sensitivity of the technique.
This positivity for GABABR1a at higher probe concentrations was arbitrarily defined as ‘‘false positive.’’ However,
together with the observation that hybridization signal for
GABABR1a was stronger than that for GABABR1p in the
stellate/basket cells, these phenomena might have resulted from the possible existence of truncated forms of
GABABR1a or other unidentified GABABR1 mRNA variants/subunits in the cerebellar cortex.
In the deep cerebellar nuclei, two populations of neurons
could be distinguished according to their size, GABA
immunoreactivity, and GABABR1 mRNA signals. The relatively large neurons, which lacked GABA immunoreactivity, were moderately GABABR1a and GABABR1p mRNA
positive. The small GABAergic neurons were weakly
GABABR1a and GABABR1p mRNA positive. No GABABR1b
mRNA-positive neurons were detected in the deep cerebellar nuclei (data not illustrated).
Other CNS structures
Low to medium levels of GABABR1a/1p hybridization
signals were observed in various hypothalamic nuclei,
with relatively strong signal in the supraoptic nucleus
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
485
Fig. 7. Laser scanning confocal photomicrographs showing double labeling of ␥-aminobutyric acid
(GABA)BR1b mRNA (A,C) and GABA immunoreactivity (B,D) in CA1 strata oriens, pyramidalis, and
radiatum (upper panels) or in stratum radiatum and lacunosum moleculare (lower panels) of the
hippocampus. Scale bar ⫽ 50 µm in D (applies to A–D).
(Fig. 13A,B). GABABR1b hybridization in most cases could
not be ascertained in the hypothalamus because of low
signal. GABA neurons in the hypothalamus showed low to
medium levels of GABABR1a/1p mRNA.
Neurons in both the pars compacta and reticulata of
substantia nigra hybridized at variable signal intensities
for GABABR1a and GABABR1p (Fig. 13C). Weak expression of GABABR1b mRNA was only detected in substantia
nigra pars compacta. Many neurons in various brainstem
structures and all laminae of the spinal cord gray matter
showed low to medium levels of GABABR1a/GABABR1p
mRNA. Some cells in the spinal cord white matter also
displayed weak GABABR1a/1p hybridization signal. Figure 13D shows the superior colliculus hybridized for
GABABR1a mRNA. Although detected at low levels in the
motoneurons of various cranial nerve nuclei and of lamina
IX of spinal ventral horn, GABABR1b mRNA expression in
most other structures of the brainstem and spinal cord
could not be definitely established due to poor hybridization signal.
Low to medium levels of GABABR1a/1p hybridization
signal were detected in many GABA neurons of the brain
stem and spinal cord structures including the superior and
inferior colliculi, nucleus of the solitary tract, the superficial laminae of the spinal trigeminal nucleus and laminae
II-III of the spinal cord dorsal horn. Figure 13E and F
illustrate such an example of GABABR1p expression in
GABA neurons in laminae II-III of the spinal cord dorsal
horn.
DISCUSSION
The present study demonstrated widespread expression
of GABABR1 receptor gene but differential distributions of
the 1a and 1b mRNA variants in a variety of brain
structures and neuronal cell types. GABA neurons of
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Fig. 8. A: Photomicrograph showing ␥-aminobutyric acid
(GABA)BR1p mRNA expression in the septum. B–G: Laser scanning
confocal photomicrographs showing double labeling of GABA (C, E, G)
and GABABR1a (B), GABABR1b (D), or GABABR1p (F) mRNA in
lateral septal nucleus. Middle column depicts GABABR1 mRNA
hybridization and right column illustrates GABA immunoreactivity in
the same fields. Arrows in the right panels point to some of the GABA
neurons and corresponding arrows at the same locations in the middle
panels indicate the expression status of GABABR1 mRNA variants in
the same neurons. Scale bars ⫽ 100 µm in A,G (apply to A and B–G,
respectively).
different areal origins and cell types expressed very differently the GABABR1a and GABABR1b variants.
fects have been described in many neural structures and
cell types in spinal dorsal horn, cerebellum, substantia
nigra, thalamus, hypothalamus, hippocampus, and neocortex, in line with the present findings of widespread expression of GABABR1 mRNA variants in the CNS (Bowery et
al., 1980; Newberry and Nicoll, 1984, 1985; Andrade et al.,
1986; Howe et al., 1987; Connors et al., 1988; Soltesz et al.,
1989a; Hausser and Yung, 1994; Misgeld et al., 1995;
Davies and Collingridge, 1996; Mouginot and Gahwiler,
1996; Deisz, 1997; Isaacson and Hille, 1997; Bettler et al.,
1998). However, it has been shown that in heterologous
mammalian expression systems GABABR1 subunit alone
could not fully function as a mature receptor. A GABABR2
receptor subunit has been cloned recently, and heterodimerization of the GABABR1 and GABABR2 subunits appears
to be necessary for the assembly of a fully functional
GABAB receptor (Jones et al., 1998; Kaupmann et al.,
1998a; White et al., 1998; Kuner et al., 1999). Low-level
expression of GABABR1 mRNA has also been detected in
peripheral organs (Castelli et al., 1999). It remains to be
seen whether expression products of the GABABR1 variants serve functions other than mediating late IPSP and
auto-/heteroinhibition of transmitter release.
Widespread availability of GABABR-mediated
inhibition?
GABABR1 mRNAs were expressed by surprisingly numerous neuronal cell types throughout the CNS. Although
their levels varied considerably in different cell types and
CNS structures, these results may still suggest that the
GABAB receptor-mediated mechanisms are widely disposable to a variety of neuronal cell types and CNS structures.
This corroborated previous data from molecular biological,
electrophysiological, pharmacologic, receptor binding autoradiographic as well as recent immunocytochemical works
on GABABRs in a variety of brain structures and cell types.
The present results of strong GABAB1 receptor gene
expression in the medial habenular nucleus, dorsal thalamus, and cerebellar Purkinje cells conform to previous
results by receptor binding, in situ hybridization, and
immunocytochemistry (Bowery et al., 1987; Chu et al.,
1990; Knott et al., 1993; Turgeon and Albin, 1993; Kaupmann et al., 1997, 1998b; Munoz et al., 1998; MargetaMitrovic et al., 1999; Fritschy et al., 1999; Lu et al., 1999).
GABABR-mediated physiological and pharmacologic ef-
Fig. 9. Photomicrographs showing expression of ␥-aminobutyric
acid (GABA)BR1 mRNA variants in the thalamus. A–C: Expression of
GABABR1a (A), GABABR1b (B), or GABABR1p mRNA (C), in different
nuclei of the thalamus. D–F: Differential expression of GABABR1a (D),
GABABR1b (E), or GABABR1p (F) mRNA in the dorsal and ventral
lateral geniculate nuclei (DLG and VLG). CL, centrolateral thalamic
nucleus; LD, laterodorsal thalamic nucleus; MD, mediodorsal thalamic nucleus; R, thalamic reticular nucleus; VL, ventrolateral nucleus;
VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bars ⫽ 400 µm in C (applies to A–C);
200 µm in F (applies to D–F).
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F. LIANG ET AL.
Fig. 10. Laser scanning confocal photomicrographs showing expression of ␥-aminobutyric acid (GABA)BR1p mRNA in GABA and nonGABAergic neurons in thalamic reticular nucleus and ventral posterolateral nucleus (A,B) and in the caudate putamen (C,D). Left column
illustrates expression of GABABR1p mRNA and right column, GABA
immunoreactivity. Arrows in the right panels point to some of the
GABA neurons, and corresponding arrows at the same locations in the
left panels indicate the GABABR1p expression status of the same
neurons. For abbreviations, see Figure 9. Scale bar ⫽ 100 µm in D
(applies to A–D).
In general, the present results on the distribution of
GABABR1 mRNAs agree well with that of GABABR1
immunoreactivities (Koulen et al., 1998; Malitschek et al.,
1998; Fritschy et al., 1999; Margeta-Mitrovic et al., 1999).
The lack of detectable GABABR1b mRNA expression in the
caudate putamen, thalamic reticular nucleus, and most
brain stem structures, as shown in the present study, is
reflected by a negative immunostaining thereof (Fritschy
et al., 1999). However, in contrast to the present results of
stronger GABABR1a hybridization signals in most CNS
structures, Western blotting results suggest GABABR1b as
a dominant variant in the adult rat brain (Malitschek et
al., 1998; Fritschy et al., 1999; Margeta-Mitrovic et al.,
1999). This discrepancy may arise from the fact that
mRNA levels do not always reflect the synthesis of protein
or the functional relevance of the gene (Tecott et al., 1994;
see Lu et al., 1999 for further discussions). More importantly, it should be noted that sequence information specific for GABABR1b is only 140 bp nucleotides. The relative
low GABABR1b signal levels were likely to be attributed to
the short length and very low U content of GABABR1b
antisense probe (U contents of GABABR1a, 1p, and 1b
antisense probes were 76, 98, and 8, respectively). Thus, if
one digoxigenin-11-UTP is incorporated out of every five
probe uracil bases, each copy of GABABR1b antisense
probe had on average 1.6 digoxigenin molecules, whereas
each copy of GABABR1a and GABABR1p antisense probe
might include 15.2 and 19.6 digoxigenin molecules, respectively. Considering the amplifying effect by anti-digoxigenin antibody, the difference could be even bigger in
number of bound alkaline phosphatase molecules per
mRNA copy between GABABR1a/1p and GABABR1b. Further complicated by a nonlinear correlation between intensity of AP reaction deposits and the number of bound
enzyme molecules or the duration of AP reaction, it may be
misleading to directly compare the hybridization signal
intensity for GABABR1b variant with those for the
GABABR1a or 1p probe. Also for the same reason, low-level
expression of GABABR1b mRNA in certain structures
might have escaped detection.
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
489
Fig. 11. Laser scanning confocal photomicrographs comparing
␥-aminobutyric acid (GABA) immunoreactivity (right column) and
GABABR1 mRNA expression (left column) in dorsal lateral geniculate
nucleus. Many non-GABAergic neurons stained positive for GABABR1a
(A), GABABR1b (C) or GABABR1p (E) mRNA. However, GABA neurons in the same structure, as revealed by simultaneous staining of
GABA immunoreactivity on the same histologic sections and illus-
trated in B, D, and F, respectively, were only marginally positive for
GABABR1a and mostly devoid of GABABR1b or GABABR1p mRNA
expression. Arrows in the right panels point to some of the GABA
neurons, and corresponding arrows at the same locations in the left
panels indicate the expression status of GABABR1 mRNA variants in
the same neurons. Scale bar ⫽ 50 µm in F (applies to A–F).
In addition to neurons, glia-like cells in the spinal white
matter near the pia mater showed weak GABABR1a/1p
mRNA expression. This result supports previous findings
of GABABR-mediated effects in glial cells of the CNS (Hosli
and Hosli, 1990; Hosli et al., 1990; Fraser et al., 1994).
Expression of GABABR1 mRNA in glial cells, however,
490
Fig. 12. Photomicrographs showing expression of ␥-aminobutyric
acid (GABA)BR1a (A,B), GABABR1b (C), and GABABR1p (E,F) mRNA
expression in the cerebellar cortex. A, C, and E depict in situ
hybridization results. B and F show non-fluorescent double labeling of
GABABR1a or GABABR1p mRNA variants (blue) and GABA immuno-
F. LIANG ET AL.
reactivity (brown) on the same histologic sections. D: Distribution of
GABA-immunoreactive cells in the cerebellar cortex. G, Golgi cell; gr,
granule cell layer; P, Purkinje cell; s/b, stellate/basket cell. Scale bar ⫽
100 µm in F (applies to A–F).
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
Fig. 13. Photomicrographs showing expression of ␥-aminobutyric
acid (GABA)BR1p mRNA in the hypothalamus (A,B), GABABR1a
mRNA in the substantia nigra (C) and superior colliculus (D), and
GABABR1p mRNA in spinal cord dorsal horn (E). F: GABAimmunoreactive neurons in the same field as E. GABA neurons were
GABABR1p mRNA positive in laminae II-III of the gray matter of the
491
spinal dorsal horn. InG, intermediate gray layer of superior colliculus;
LA, lateral anterior hypothalamic nucleus; LH, lateral hypothalamic
area; SCh, suprachiasmatic nucleus; SNC, pars compacta of substantia nigra; SNR, pars reticulata of substantia nigra; SO, supraoptic
nucleus; SuG, superficial gray layer of superior colliculus. Scale bars ⫽
200 µm in D (applies to A–D), 100 µm in F (applies to E,F).
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Fig. 14. Schematic diagrams showing the four expression profiles
of ␥-aminobutyric acid (GABA)BR1a and/or GABABR1b mRNA variants in different GABA neurons of the rat brain. The possibly
differential targeting of GABABR1a and GABABR1b variants into preand postsynaptic GABABR receptors is according to a recent hypothesis, which remains unconfirmed (Bettler et al., 1998; Kaupmann et
al., 1998b; Zhang et al., 1998). I: Most GABA neurons in the neocortex,
hippocampus, and septum expressed both GABABR1a and GABABR1b.
II: GABA neurons in thalamic reticular nucleus, caudate putamen,
and in stellate/basket cells of the cerebellum showed mainly GABABR1a
mRNA signal. III: As exemplified by Purkinje cells in the cerebellar
cortex, this group of GABA neurons expressed predominantly
GABABR1b mRNA variant. IV: GABA neurons in the dorsal lateral
geniculate nucleus lacked significant expression of both GABABR1a
and GABABR1b mRNA variants. GABABR(1a⫹2), GABAB receptor
heterodimer formed by GABABR1a and GABABR2 subunits.
GABABR(1b⫹2), GABAB receptor heterodimer formed by GABABR1b
and GABABR2 subunits.
may be restricted to certain regions of the CNS and at a
level much lower than in neurons because a previous study
did not find significant GABABR1 gene expression in glial
cells of hippocampal CA3 region (Kaupmann et al., 1997).
The present study, as well, did not detect significant levels
of GABABR1a, 1b, or 1p hybridization signal in glia cells in
a majority of rat CNS structures other than the spinal
white matter.
It is unlikely that the widespread distribution of
GABABR1a and 1p was an artifact caused by nonspecific
hybridization or cross-hybridization of the probes with
other types of mRNA. In a series of pilot experiments,
probe concentrations ranging from 0.07 to 0.7 µg/ml for
GABABR1a and GABABR1p were tested. Although probe
concentrations above 0.4 µg/ml gave rise to certain background staining for GABABR1p and strong Purkinje cell
positivity for GABABR1a hybridization, probe concentrations ranging from 0.07–0.3 µg/ml produced consistent
hybridization patterns for different brain structures and
cell types. In addition, hybridization with sense control
probes gave no positive signal except for the very weak
hybridization for GABABR1b sense probe in the hippocampus. Finally, the present results on the distribution of the
two mRNA variants in the cerebellar cortex agree with
recently published data in this structure (Kaupmann et
al., 1998b).
septum expressed both GABABR1a and GABABR1b variants. This finding is in strong contrast to GABA neurons in
most subcortical structures. GABA neurons in thalamic
reticular nucleus, ventral lateral geniculate nucleus, caudate putamen, and GABAergic stellate/basket cells in the
cerebellar cortex hybridized mainly for GABABR1a antisense probe without detectable GABABR1b mRNA signal.
Cerebellar Purkinje cells showed mainly GABABR1b mRNA
expression. Finally, GABA neurons in the dorsal lateral
geniculate nucleus showed little expression of GABABR1
mRNA variants.
Presynaptic GABABR-mediated autoinhibition of GABA
release has been extensively studied and many important
neurophysiological and pathologic processes such as nociception, use-dependent depression of inhibition, LTP, and
epileptogenesis have been associated with this selfcontrolling mechanism (Floran et al., 1988; Giralt et al.,
1990; Seabrook et al., 1991; Morishita and Sastry, 1995;
Bonanno et al., 1997; for reviews, see Bowery, 1997; Deisz,
1997; Bettler et al., 1998). GABABR-mediated postsynaptic responses in GABA neurons have been less well characterized. Nevertheless, this disinhibitory mechanism by
other GABA neurons or by recurrent collaterals of the
GABA neurons themselves would also reduce GABAergic
inhibitory output. Thus, activation of GABABR-mediated
events in GABA neurons, either pre- or postsynaptic,
would cause increased excitation of the integrated network, contrary to the effects of GABABR activation in
excitatory neurons.
The present findings suggest that the GABABR-mediated late IPSP and presynaptic autoinhibition of GABA
release are differentially disposable to different GABA
neurons. Those GABA neurons in the dorsal lateral genicu-
Differential expression of GABABR1 mRNA
variants in GABA neurons
As illustrated schematically in Figure 14, four expression profiles of GABABR1a and/or GABABR1b mRNA
variants were distinguished in different GABA neurons.
Many GABA neurons in the neocortex, hippocampus and
EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS
late nucleus seemed lacking both mechanisms, in sharp
contrast to most GABA neurons in the neocortex and
hippocampus. In the context of these results, it is interesting to note that little GABABR-mediated responses have
been detected in the intrinsic GABAergic interneurons of
dorsal lateral geniculate nucleus of both cats and rats
(Pape and McCormick, 1995; Williams et al., 1996). These
interneurons, capable of firing trains of action potentials
at a frequency exceeding 500 Hz in the cat, seemingly use
alternative mechanisms, such as muscarinic acetylcholine
receptors, for modulating their capability to gate the
transmission of visual information from retina to visual
cortex (McCormick and Pape, 1988; Pape and McCormick,
1995).
The GABABR1a-positive GABA neurons such as those in
the striatum, thalamic reticular nucleus, and cerebellar
stellate/basket cells may also have physiological and pharmacologic properties very different from the GABA neurons in the cerebral cortex that expressed both GABABR1a
and GABABR1b, or from cerebellar Purkinje cells that
mainly expressed GABABR1b. It has been shown previously that GABAB autoreceptors in the cerebral cortex and
spinal cord represent pharmacologically distinct receptor
subtypes (Bonanno and Raiteri, 1993a). In view of the
proposition that the two variants might be differentially
located to axon terminals and somatodendritic domains of
neurons (see below), it remains to be determined how these
findings relate to the functions of individual GABA neuron
cell types and to their positioning in the integrated neural
network.
Margeta-Mitrovic and coworkers (1999) have found very
few neurons that coexpressed GABABR1 immunoreactivity and glutamic acid decarboxylase-67, in contrast to the
present results showing expression of either or both of the
GABABR1a and 1b mRNA variants in GABA neurons of
most CNS structures. It remains unknown whether this
discrepancy with the present results is caused by extremely low level of GABABR1 mRNA translation, limited
sensitivity of the antibody, or unrecognized GABABR variants/subunits in GABA neurons. Of particular relevance to
the last point, for example, the antibody used by MargetaMitrovic et al. (1999) would not be able to detect the
recently cloned GABABR1d (Isomoto et al., 1998). The
present GABABR1b antisense probe, in contrast, would
also hybridize with GABABR1c and 1d mRNA variants as
the two share the same 58 end with GABABR1b (Isomoto et
al., 1998).
mRNA variants for receptor subtypes?
The differential but partly overlapping expression of the
two mRNA variants in various neuronal cell types and
brain structures as shown in the present study, may
contribute to the production of diverse subtypes of GABAB
receptors. In line with this molecular diversity, variable
physiological and pharmacologic profiles of GABABR agonist and antagonist actions have been demonstrated previously in different CNS structures and cell types (for
reviews see, Bowery, 1997; Deisz, 1997; Bettler et al.,
1998). However, the differential and variable expression
patterns of GABABR1a and GABABR1b mRNA variants in
both GABA and non-GABA neurons deny the possibility of
separation of each variant into either GABA or non-GABA
category. Differences in intracellular signal transduction
cascades and effector systems may greatly enhance the
functional heterogeneity and specificity derived from varia-
493
tions in GABABR variant expression in different neuronal
cell types.
It has been recently proposed for the cerebellar cortex
and retina that GABABR1a and GABABR1b variants might
target pre- and postsynaptic GABABR subtypes, respectively (Bettler et al., 1998; Kaupmann et al., 1998b; Zhang
et al., 1998). Although the present study showed diverse
patterns of expression of the two variants in the central
nervous system, a detailed correlation of the present data
with previous electrophysiological and pharmacologic studies seems to extend the postulation to several other brain
regions. The present study demonstrated, for example, the
predominant GABABR1a expression in the striatum, ventral lateral geniculate nucleus, and the thalamic reticular
nucleus. In the same brain regions, electrophysiological
studies have failed to detect significant late IPSPs mediated by postsynaptic GABABRs (Soltesz et al., 1989b;
Calabresi et al., 1991; Crunelli and Leresche, 1991; Nisenbaum et al., 1993; Ulrich and Huguenard, 1996; SanchezVives and McCormick, 1997). Thus, it seemed conceivable
that the mature translation product of GABABR1a mRNA
in neurons of these structures was mainly for locations at
presynaptic axon terminals. Both pre- and postsynaptic
GABABR-mediated events have been demonstrated for the
neocortex, hippocampus, and most dorsal thalamic nuclei
(Bowery, 1997; Deisz, 1997; Bettler et al., 1998). Again,
this agrees with the present findings of coexpression of the
two variants by these structures. In accordance with the
present results showing abundant expression of GABABR1b
and very weak expression of GABABR1a mRNA by cerebellar Purkinje cells, previous studies have reported high
levels of postsynaptic GABABRs on Purkinje cell dendrites
in molecular layer of the cerebellar cortex and low levels of
presynaptic GABABRs on Purkinje axon terminals in deep
cerebellar nuclei (Bowery et al., 1987; Chu et al., 1990;
Turgeon and Albin, 1993; Morishita and Sastry, 1995;
Mouginot et al., 1998). Likewise, it may be deduced that
most brain stem and spinal neuronal cell types, which
mainly expressed the GABABR1a variant as shown in the
present study, would manifest primarily a presynaptic
GABABR-mediated effect.
Except in the retina, no definite evidence has been
presented for presynaptic GABABR1 immunoreactivity,
either on GABA or non-GABAergic axon terminals, despite
the vast number of electrophysiological reports on the
matter. A direct immunohistochemical demonstration of
GABABR1a peptide distribution in the CNS, for example,
has not been possible because of the lack of suitable
specific antibodies (Koulen et al., 1998; Margeta-Mitrovic
et al., 1999; Fritschy et al., 1999). In view of the recent
cloning of GABABR1c/1d and the possibility of other unidentified GABABR1 variants, the subtraction strategy
(GABABR1p ⫺ GABABR1b ⫽ GABABR1a distribution) as
used by Fritschy and coworkers (1999) is no longer tenable. Because peptides are mostly synthesized in the cell
bodies and subsequently transported to their final destinations, immunoreactivity detected in the cell bodies and
proximal dendrites might represent immature, preassembling forms of the receptor subunit and, therefore,
could not rule out that the functioning form is located at
axon terminals. It awaits further studies, especially those
at electron microscopic level by using variant-specific
antibodies, to resolve the veracity of the postulation.
494
F. LIANG ET AL.
ACKNOWLEDGMENTS
We thank Dr. Katsuyoshi Ishii for many helpful discussions and Kiseko Shionoya, Takumi Agaki, Yasuhiro
Yamazaki, Chishiro Wakabayashi, and Yuki Hasegawa for
technical assistance.
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