Volume 16, Number 11,
Issue of June 1, 1996
pp. 3571-3589
Copyright ©1996 Society for Neuroscience
Nucleus-Specific Expression of GABAA Receptor Subunit
mRNAs in Monkey Thalamus
M. M. Huntsman,
M. G. Leggio, and
E. G. Jones
Department of Anatomy and Neurobiology, University of California,
Irvine, California 92717
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Expression of 10 GABAA receptor subunit genes
was examined in monkey thalamus by in situ hybridization
using cRNA probes specific for 
1,
2, 3,
4, 5,
1, 2,
3, 
1, and
2 subunit mRNAs. These displayed unique
hybridization patterns with significant differences from rodents.
1, 2, and
2 transcripts were expressed at high levels in
all dorsal thalamic nuclei, but expression was significantly higher in
sensory relay nuclei
especially the dorsal lateral geniculate nucleus.
Other transcripts showed nucleus-specific differences in levels of
expression and in the range expressed. 5 and
4 subunit transcripts were expressed in all
nuclei except the intralaminar nuclei. Levels of
2, 3,
1, 3, and
1 expression were very low, except in
intralaminar nuclei. In the reticular nucleus, most subunit transcripts
were not expressed, and only 2 transcripts
were consistently detected at modest levels. Thalamic
GABAA receptors may be assembled from
nucleus-specific groupings of subunit polypeptides.
Key words:
in situ hybridization;
, , and
subunits;
inhibition;
chromosomes;
sensory nuclei;
intralaminar
nuclei
INTRODUCTION
GABAergic inhibition in the mammalian thalamus
affects rates and temporal patterns of discharge of relay neurons
(Curtis et al., 1972
; Duggan and McLennan, 1971; Curtis and
Têbêcis, 1972; Hicks et al., 1986), receptive field
properties (Sillito and Kemp, 1983; Pape and Eysel, 1986; Eysel et al.,
1987; Sillito, 1992), and state-dependent rhythmic oscillations of the
thalamic network (Steriade et al., 1991; Warren et al., 1994; Bal et
al., 1995).
Thalamic inhibition is mediated by GABAergic neurons located in the
reticular nucleus and by GABAergic intrinsic interneurons (Houser et
al., 1980; Montero and Scott, 1981; Oertel et al., 1983; Spreafico et
al., 1983; Montero and Singer, 1985; Yen et al., 1985; DeBiasi et al.,
1986; Ohara et al., 1989; Cucchiaro et al., 1991; Ralston, 1991; Ohara
and Lieberman, 1993; Liu et al., 1995). In cats and monkeys,
interneurons constitute ~25% of dorsal thalamic neurons (LeVay and
Ferster, 1979; Weber and Kalil, 1983; Fitzpatrick et al., 1984; Montero
and Zempel, 1985, 1986; Rinvik et al., 1987; Benson et al., 1991b;
Hendry, 1991; Hunt et al., 1991).
The GABAA receptor, a fast-acting,
bicuculline-sensitive chloride channel is a heteropentameric complex
composed of combinations of five or fewer polypeptide subunits
(Macdonald and Olsen, 1994). The receptor has binding sites for
benzodiazepines, barbiturates, and neurosteroids, all of which modify
GABA-mediated chloride currents (DeLorey and Olsen, 1992). At least 15 genes encode GABAA receptor subunits that share a
high degree of sequence identity, classified as , , , 
, and
classes with subclass variants (1-6;
1-3, 1-3, ,
1,2). 1,
2, and 2 subunits are
expressed at high levels in most brain regions (Benke et al., 1991;
Fritschy et al., 1992; Benke et al., 1994; Fritschy and Möhler,
1995) of rodents (Gambarana et al., 1991; Persohn et al., 1992; Wisden
et al., 1992) and monkeys (Huntsman et al., 1994, 1995a,b).
GABAA receptors may be assembled from specific
combinations of individual subunits (Lüddens and Wisden, 1991;
Angelotti et al., 1993; Angelotti and Macdonald, 1993; Backus et al.,
1993; Perez-Velazquez and Angelides, 1993), but combinations of ,
, and subunits are necessary to produce fully functional
GABAA receptors (Pritchett et al., 1989a; Siegel
et al., 1990; Verdoorn et al., 1990; Angelotti and Macdonald,
1993).
Previous radioligand binding studies of GABAA
receptor localization in the thalamus were confined to rats (Palacios
et al., 1981; Unnerstall et al., 1981; Bowery et al., 1987; Olsen et
al., 1990; Bureau and Olsen, 1993) in which intrinsic interneurons are
largely absent (for review, see Benson et al., 1992), and to isolated
nuclei in other species (Shaw and Cynader, 1986; Kultas-Ilinsky et al.,
1988). Subunit-specific protein and mRNA expression has been briefly
described in rat thalamus (Wisden et al., 1992; Fritschy and
Möhler, 1995) but only in the dorsal lateral geniculate nucleus
of monkeys (Huntsman et al., 1995a).
Preliminary reports of the present study have appeared elsewhere
(Huntsman and Jones, 1993, 1995).
Abbreviations
| AM |
anteromedial nucleus of the thalamus
|
| AV |
anteroventral nucleus of the thalamus
|
| CeM |
central medial nucleus of the thalamus
|
| CL |
central lateral nucleus of the thalamus
|
| CM |
centre médian nucleus of the thalamus
|
| CN |
caudate nucleus
|
| Gpi |
globus pallidus, internal segment
|
| H |
habenular nuclei
|
| Hbm |
medial habenular nucleus
|
| L |
limitans nucleus of the thalamus
|
| LD |
lateral dorsal nucleus of the thalamus
|
| LGd |
dorsal lateral geniculate nucleus of the thalamus
|
| LGmc |
dorsal lateral geniculate nucleus of the thalamus, magnocellular
layers
|
| LGpc |
dorsal lateral geniculate nucleus of the thalamus, parvocellular
layers
|
| LP |
lateral posterior nucleus of the thalamus
|
| MD |
mediodorsal nucleus of the thalamus
|
| MG |
medial geniculate nucleus of the thalamus
|
| MGd |
medial geniculate nucleus, dorsal
|
| MGv |
medial geniculate nucleus, ventral
|
| Pc |
paracentral nucleus
|
| Pf |
parafascicular nucleus of the thalamus
|
| Pla |
anterior pulvinar nucleus of the thalamus
|
| Pli |
inferior pulvinar nucleus of the thalamus
|
| Pll |
lateral pulvinar nucleus of the thalamus
|
| Plm |
medial pulvinar nucleus of the thalamus
|
| Po |
posterior nucleus of the thalamus
|
| Prg |
pregeniculate nucleus
|
| PT |
pretectum
|
| Pv |
paraventricular nucleus of the thalamus
|
| R |
reticular nucleus of the thalamus
|
| Rh |
rhomboid nucleus of the thalamus
|
| Sb |
subthalamic nucleus
|
| SG |
suprageniculate nucleus of the thalamus
|
| SNr |
substantia nigra, pars reticulata
|
| VA |
ventral anterior nucleus of the thalamus
|
| VL |
ventral lateral nucleus
|
| VLa |
anterior ventral lateral nucleus of the thalamus
|
| VLp |
posterior ventral lateral nucleus of the thalamus
|
| VMb |
basal ventral medial nucleus of the thalamus
|
| VPI |
ventral posterior inferior nucleus of the thalamus
|
| VPL |
ventral posterior lateral nucleus of the thalamus
|
| VPM |
ventral posterior medial nucleus of the thalamus
|
| ZI |
zona incerta |
MATERIALS AND METHODS
Generation of cRNA probes. Complementary RNA
(cRNA) probes were prepared from monkey-specific cDNAs for localization
of the 1, 2,
3, 4,
5, 1,
2, 3,
1, and 2
GABAA receptor subunit mRNAs and for 67 kDa
glutamic acid decarboxylase (GAD) mRNA. These include the majority of
the GABAA receptor subunits that are expressed in
the CNS. The methods used to subclone, purify, and characterize the
cDNAs, including Northern blot analysis, were identical to those used
in a previous study (Huntsman et al., 1994). Synthetic oligonucleotide
primers were designed to flank a heterogeneous domain of each chosen
GABAA receptor subunit DNA that was then
amplified against a monkey cDNA template using the PCR. The cDNA was
sequenced and inserted into the pBS II cloning vector (Stratagene, La
Jolla, CA). The monkey-specific cDNA for GAD encodes the 67 kDa protein
product (GAD67) and was generated and fully
characterized in a previous study (Benson et al., 1991a).
Three additional monkey-specific cDNA subclones with sequences
corresponding to the intracellular cytoplasmic loops of
3, 3, and
1 GABAA receptor subunit
polypeptides were isolated and characterized using the same methods
used to isolate 1, 2,
4, 5,
1, 2, and
2 cDNAs in the previous study (Huntsman et
al., 1994). The cDNAs were then used as templates for the generation of
riboprobes for in situ hybridization histochemistry as
described previously (Benson et al., 1994; Huntsman et al., 1994,
1995a,b).
All the monkey-specific subclones used were similar in length (341-497
bp) and in G/C content and shared a high degree of sequence identity
with previously cloned cDNAs of human (where available) and rat
sequences, as determined by Genbank searches. Radiolabeled RNA probes
for in situ hybridization histochemistry were generated by
in vitro transcription of the linearized, subcloned cDNAs in
the presence of [-33P]UTP, using either T3
or T7 RNA polymerase for the production of antisense and sense
strands.
The riboprobes had similar specific activities. Northern blot analysis
(Huntsman et al., 1994) using the same cDNA templates identified
transcripts with molecular weights that were the same as those reported
in other studies (Garrett et al., 1988; Lolait et al., 1989; Ymer et
al., 1989a,b; Khrestchatisky et al., 1989, 1991; Malherbe et al.,
1990). Further verification of probe specificity is seen in the
distinct patterns of expression of the subunit variants. The
riboprobes that identified the 1,
2, 3,
4, and 5 subclass
variants gave very distinctive and sometimes nonoverlapping patterns of
labeling in the dorsal thalamus (e.g., 2 and
4) even though they share >70% sequence
identity in pairwise residue comparisons (Seeburg et al., 1990).
In situ hybridization histochemistry. The brains of
four monkeys were used: one Macaca fuscata, one Macaca
fascicularis, and two Macaca mulatta. All animals were
given an overdose of Nembutal and perfused through the ascending aorta
with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were blocked
and post-fixed overnight in 4% paraformaldehyde and then infiltrated
with 30% sucrose in 4% paraformaldehyde. The blocks were frozen in
dry ice and those containing the diencephalon were cut at 25 µm on a
sliding microtome. Sets of eleven sections were collected in cold,
sterile 4% paraformaldehyde for in situ hybridization
histochemistry; the two succeeding sections were collected in cold
phosphate buffer for Nissl and cytochrome oxidase (CO) (Wong-Riley,
1979) staining. Ten of each set of 11 sections collected in
paraformaldehyde were hybridized with radiolabeled antisense cRNA
probes for the GABAA receptor subunit mRNAs. The
remaining section of each set was used for repeat runs, if necessary,
for hybridization with the GAD67 riboprobe, or
for hybridization with sense control riboprobes.
Free-floating sections were prepared for in situ
hybridization by washing in 0.1 M glycine in 0.1 M phosphate buffer, pH 7.4, followed by
incubation in 1 mg/ml of proteinase K in 0.1 M
Tris-HCl buffer, pH 8.0, containing 0.05 M EDTA
for 30 min at 37°C; they were then washed in 0.25% acetic anhydride
in 0.1 M triethanolamine, pH 8.0, and in 2× SSC.
Sections were next incubated in the hybridization solution containing
50% formamide, 10% dextran sulfate, 0.7% Ficoll, 0.7% polyvinyl
pyrolidone, 0.5 mg/ml yeast tRNA, 0.33 mg/ml denatured herring sperm
DNA, and 20 mM dithiothreitol (DTT) for 1 hr at
60°C, then transferred to fresh hybridization solution containing an
additional 20 mM DTT and 1 × 106 cpm/1 ml of the
33P-radiolabeled sense or antisense
riboprobe.
After at least 20 hr of hybridization at 60°C, sections were washed
in 4× SSC at 60°C, digested with 20 mg/ml ribonuclease A, pH 8.0, for 30 min at 45°C, and washed through descending concentrations of
SSC with 5 mM DTT to a final stringency of 0.1×
SSC at 60°C for 1 hr. Sections were mounted on gelatin-coated slides,
dried, and exposed to Amersham -max film for 8 d. After development
of the film, the sections were lipid-extracted in chloroform, dipped in
Kodak NTB2 emulsion (diluted 1:1), exposed for 8 d at 4°C, developed
in Kodak D-19, fixed, and counterstained with cresyl violet.
Quantitative analysis. Film autoradiograms of
hybridized sections were quantified by taking optical density readings
using a microcomputer imaging device (MCID/M4; Imaging Research, St.
Catherine's, Ontario, Canada). Quantitative sampling was obtained by
calibrating the optical system to a set of radioactive standards before
reading density values. The imaging system functions by sampling mean
gray-level values of all the pixels within a defined sampling area and
converts them to values of concentration termed integrated optical
density (IOD) values. The IOD value is converted to units of
radioactivity by taking readings from 14C
reference standards (Amersham) exposed on the same sheet of film as the
sampled sections and converting the values from these standards to
units of radioactivity (nCi/gm) within the sampled area. Multiple
samples were taken from individual thalamic nuclei using a sampling
tool adjusted to fit within the borders of the nucleus, as confirmed
from the adjacent Nissl- and CO-stained sections (Fig.
1A). Each unit sampling was tracked and
recorded on the digitized image to ensure that the samples taken within
a single thalamic nucleus did not overlap. The threshold level for
reporting the presence of hybridization signal was set at 50 nCi/gm,
because at this level nuclear borders could not be discerned. Sections
hybridized with sense strand subunit riboprobes showed no labeling
above background levels (Fig. 1B). Nuclear delineations are
those of Jones (1985). The nuclear distribution patterns were compared
with the patterns of expression of GAD67, the
details of which were described by Benson et al. (1991b).
Fig. 1.
A, Digital scan of a section hybridized
with a cRNA probe specific for 1 subunit mRNA.
Each sampled area is represented by a box within the borders
of the relevant nucleus, which were confirmed from an adjacent
Nissl-stained section. B, Section hybridized with
sense-strand subunit riboprobe for 1 subunit
transcripts showed no labeling above background levels. Scale bar, 2 mm. C, Dark-field photomicrograph from an emulsion-dipped
autoradiograph showing 2 probe hybridization
in the reticular nucleus (R) and lateral posterior nucleus
(LP). Scale bar, 100 µm. D, Bright-field
photomicrograph showing labeling of large and small cells in the
mediodorsal nucleus with the 2 riboprobe;
nuclei of neuroglial cells are not labeled. Scale bar, 10 µm.
[View Larger Version of this Image (85K GIF file)]
RESULTS
Distribution of subunit mRNAs
The dorsal thalamus
The 10 GABAA receptor subunit mRNAs examined
in this study (1, 2,
3, 4,
5, 1,
2, 3,
1, and 2) had a wide
range of expression patterns in terms of both density and distribution
throughout the dorsal thalamus. The distribution of most of the subunit
mRNAs was overlapping. However, when examined in detail, each
transcript displayed a characteristic pattern that differed from that
of the other subunit mRNAs. The intensity of hybridization, reflecting
different levels of expression, specifically varied for each subunit
mRNA. In most dorsal thalamic nuclei, the 1,
2, and 2 subunit
transcripts were expressed at higher levels than the
5 transcript, which was in turn expressed at
higher levels than the 2,
3, 4,
1, 3, and
1 transcripts. Among each of the nuclei there
were individual differences, as described in detail below. Some nuclei
expressed a wide variety of subunit transcripts, whereas other nuclei
expressed a more limited range. In certain groups of nuclei, there was
a complementarity of expression in the sense that expression of one set
of transcripts was associated with nonexpression of another set. These
differences indicate nucleus-specific patterns of gene expression for
each of the subunits. The distribution and density of labeling for
individual transcripts is summarized in Table 1. The
following section deals with some of the unique features of the
localization patterns.
Cellular localization of mRNAs
Cell labeling with radioactive riboprobes consisted of
clusters of silver grains overlying the somata of individual cells.
Specific labeling consisted of compact accumulations of grain clusters
over cell somata, yielding a clear definition of individually labeled
cells, contrasting with the light scattering of silver grains
associated with background labeling (Fig. 1C,D). The sizes
of the grain clusters could therefore be used as a guide to the
relative sizes of the cell bodies, based on known sizes of the major
population of cells within particular thalamic nuclei (Benson et al.,
1991b). An example of hybridization of GABAA
receptor riboprobes specific for the 2 subunit
in the reticular nucleus and neighboring lateral posterior nucleus
(Fig. 1C) shows grain accumulations over Nissl-stained cell
bodies in which the grain clusters in the lateral posterior nucleus
range in size from small (10-15 µm) to large (20-25 µm). This was
typical of all dorsal thalamic nuclei (Fig. 1D). It is
therefore assumed that both relay cells and interneurons, which are
consistently smaller, were labeled with the subunit riboprobes. Those
in the reticular nucleus were all large, reflecting the unimodal size
range of cells in this nucleus. Qualitatively, the number of labeled
neurons detectable with the different riboprobes varied. In general, in
nuclei showing a high density of labeling for a particular subunit
mRNA, most of the neurons were labeled. In those showing weak labeling
for a particular subunit mRNA, few individually labeled cells could be
detected. All riboprobes labeled only neurons, except for the
4, 1, and
1 riboprobes in which labeling of significant
numbers of neuroglial cells could also be detected, leading to
darkening of the internal capsule in some of the autoradiograms (data
not shown).
Nuclear localization of mRNAs
In what follows, a nucleus is said to be labeled for subunit
transcripts if integrated optical density measurements translated to
more than the 50 nCi/gm threshold level.
The anterior nuclear complex
The AM, AV, and AD nuclei expressed all the subunit mRNAs with
varying degrees of intensity (Figs. 2, 3, 4, 5). The most
abundant mRNAs in order of intensity were 2,
5, 1,
3, and 2 (Figs.
2E,A,C,H, 3B). In these
nuclei, 5 transcripts reached higher levels of
hybridization than in any other nuclear group in the dorsal thalamus.
Modest levels were observed for the 3 and
1 mRNAs (Fig. 3C,A) with still
lower levels observed for 4,
2, and 1 mRNAs (Fig.
2D,B,G).
Fig. 2.
Photomicrographs from film autoradiograms of
adjacent sections through anterior levels of the thalamus hybridized
with the 1, 2,
3, 4,
5, 1, and
2 GABAA receptor
subunit-specific cRNA probes (A-E, G, H), a
GAD67 cRNA probe (F), and a
photomicrograph of an adjacent Nissl-stained section (I).
These show the patterns of distribution of 7 of the 10 subunit-specific
mRNAs in relation to each nucleus. All autoradiograms were exposed for
the same time. Scale bar, 2 mm.
[View Larger Version of this Image (132K GIF file)]
Fig. 3.
Photomicrographs from film autoradiograms of
sections adjacent to those in Figure 2 through anterior levels of the
thalamus, hybridized with 1,
2, and 3
GABAA receptor subunit-specific cRNA probes
(A-C). Scale bar, 2 mm.
[View Larger Version of this Image (66K GIF file)]
Fig. 4.
Photomicrographs from film autoradiograms of
adjacent sections through levels of the thalamus posterior to those
represented in Figures 2 and 3. Sections were hybridized with the
1, 2,
3, 4,
5, 1, and
2 GABAA receptor
subunit-specific cRNA probes (A-E, G, H) or with a
GAD67 riboprobe (F). All
subunit mRNA distribution patterns can be compared with an adjacent
Nissl-stained section (I). Note increased levels of
2 transcripts in VLa (H).
Scale bar, 2 mm.
[View Larger Version of this Image (141K GIF file)]
Fig. 5.
Photomicrographs from film autoradiograms of
sections adjacent to those shown in Figure 4. Adjacent sections were
hybridized with 1, 2,
and 3 (A-C) subunit-specific cRNA
probes. Scale bar, 2 mm.
[View Larger Version of this Image (66K GIF file)]
The ventral group of nuclei
Moderate levels of expression were evident for the
1, 2, and
2 mRNAs in the VA nucleus (Figs.
2A,H, 3B, 4A,H, 5B), with
slightly lower levels for 5 mRNAs (Figs.
2F, 3F) and reduced levels for the other subunit
mRNAs. The VL nucleus had a similar distribution pattern with
noticeably increased levels of 2 subunit
transcripts in the VLa (see Fig. 5H). In the ventral
posterior nucleus, the 1,
2, 2, and
5 subunit transcripts were the most prominent
subunit mRNAs expressed (see Figs. 6A,E,H, 7B,
8A,E,H, 9B). 3 and
4 transcripts were also labeled but with lower
intensity (see Figs. 6C,D, 8C,D), and even lower
levels of hybridization were observed for 2,
1, 3, and
1 mRNAs (see Figs. 6B,G,
7A,C, 8B,G, 9A,C). In the VPL and VPM
nuclei, 8 of the 10 subunit mRNAs showed increased levels in VPM
compared with VPL, but 4 and
2 levels did not (see Figs. 6D,H).
The VPI nucleus typically had moderate levels of hybridization with
noticeable decreases for 1 and
2 in relation to neighboring VPL and VPM (see
Figs. 6A, 7B). The VMb nucleus had the highest
expression levels of the ventral group for almost all of the subunits
examined, exceeding those for both VPM and VPL, with the exception of
the 1 subunit transcripts, the levels of which
decreased (see Fig. 6A).
Fig. 6.
Photomicrographs from film autoradiograms of
adjacent sections through middle levels of the thalamus. Each section
was hybridized with the 1,
2, 3,
4, 5,
1, and 2
GABAA receptor subunit-specific cRNA probes
(A-E, G, H) or with a GAD67 riboprobe
(F). mRNA distribution patterns can be compared with an
adjacent Nissl-stained section (I). Note intensity of
1 transcript levels in LGd and in the ventral
nuclear group (A) in addition to the restricted
hybridization of 3 transcripts in the
magnocellular layers of the LGd, indicated by an arrow
(C). Scale bar, 2 mm.
[View Larger Version of this Image (139K GIF file)]
Fig. 7.
Photomicrographs from film autoradiograms of
sections adjacent to those in Figure 6, hybridized with
1, 2, and
3 (A-C)
GABAA receptor subunit-specific cRNA probes.
Scale bar, 2 mm.
[View Larger Version of this Image (71K GIF file)]
Fig. 8.
Photomicrographs from film autoradiograms of
adjacent sections through posterior levels of the thalamus. Each
section was hybridized with the 1,
2, 3,
4, 5,
1, and 2
GABAA receptor subunit-specific cRNA riboprobes
(A-E, G, H) or with a GAD67 riboprobe
(F). Patterns can be compared with an adjacent Nissl-stained
section (I). Scale bar, 2 mm.
[View Larger Version of this Image (139K GIF file)]
Fig. 9.
Photomicrographs from film autoradiograms of
sections adjacent to those in Figure 8 hybridized with
1, 2, and
3 (A-C)
GABAA receptor subunit-specific cRNA probes.
Scale bar, 2 mm.
[View Larger Version of this Image (72K GIF file)]
The MD nucleus
The 2, 2,
5, 3, and
1 mRNAs were present in the mediodorsal
nucleus at moderate levels, with slightly lower levels observed for
1, 4,
3, 1, and
2 subunit mRNAs (Figs.
4, 5, 6, 7, 8, 9).
No obvious subnuclear differences could be detected.
The lateral group of nuclei
The LD nucleus expressed high levels of most subunit transcripts,
especially 2, 1,
5, and 2 (see Figs.
6A,E,H, 7B, 8A,E,H, 9B),
along with moderately high levels of 3,
1, and 1 (see Figs.
6C,G, 7A, 8C,G, 9A) but
weaker levels of 2,
4, and 3 mRNAs (see
Figs. 6B,D, 7C, 8B,D, 9C).
The LP nucleus similarly displayed high hybridization levels of
2, 1,
5, and 2 mRNAs (see
Figs. 8A,E,H, 9B, 10A,E,H,
11B) that approached those observed in the LGd nucleus.
Overall, LP maintained high levels of the remaining subunit
transcripts, except for 2,
3, 1, and
3 mRNAs (see Figs. 8B,C,
9A,C, 10B,C, 11A,C). In the nuclei of
the pulvinar, the Pla, Pli, Plm, and Pll nuclei showed a heterogeneous
distribution of receptor subunit transcripts in which
1 clearly dominated, reaching high levels in
Pli, Pll, and Plm (see Fig. 10A), although
levels were still lower than in the adjacent lateral geniculate and
lateral posterior nuclei. High levels of 5,
2, and 2 transcripts
were also observed in Pli and Plm (see Figs. 10E,H,
11A), along with moderate levels of
2, 3,
4, 1,
3, and 1 mRNAs,
primarily in Pli and Plm, in which levels never dipped below the weakly
positive range (see Figs. 10B,C,D,G, 11A,C).
Overall, Pla had the lowest transcript levels for all subunits examined
in the pulvinar, except for 3 and
1, whose levels in Pll were slightly less (see
Figs. 9C,G, 10C,G).
Fig. 10.
Photomicrographs from film autoradiograms of
adjacent sections through the posterior pole of the thalamus, including
all subdivisions of the pulvinar except Pla. Sections were hybridized
with the 1, 2,
3, 4,
5, 1, and
2 GABAA receptor
subunit-specific cRNA riboprobes (A-E, G, H) or with a
GAD67 riboprobe (F). Note increased
levels in LP for the 1 (A) and 2
(H) transcripts. Patterns can be compared with an adjacent
Nissl-stained section (I). Scale bar, 2 mm.
[View Larger Version of this Image (115K GIF file)]
Fig. 11.
Photomicrographs from film autoradiograms of
sections adjacent to those shown in Figure 10 and hybridized with
1, 2, and
3 (A-C)
GABAA receptor subunit-specific riboprobes. Scale
bar, 2 mm.
[View Larger Version of this Image (63K GIF file)]
The intralaminar nuclei
The intralaminar nuclei showed moderate-to-high levels of almost
all of the receptor subunit transcripts examined. The moderate levels
of hybridization observed for the 2,
3, 1,
3, and 1 mRNAs were
unusual because these transcripts were only detected at very low levels
in the majority of thalamic nuclei. The rostral group of intralaminar
nuclei in particular displayed moderately high levels of the
1, 2,
3, 5,
1, 2,
3, 1, and
2 transcripts, especially in Rh and CeM nuclei
(Figs. 2, 3, 4, 5, 6, 7). There were also very high levels in the Pf nucleus of the
caudal group of intralaminar nuclei (see Figs. 8, 9). Labeling of
4 transcripts was noticeably absent from every
nucleus in the intralaminar group with the Rh and CeM nuclei being
minor exceptions, showing just weakly positive levels (Fig.
4D). 5 transcripts were also
decreased in the caudal intralaminar group from the usual moderately
high levels of expression seen in other dorsal thalamic nuclei; there
was a dramatic drop in hybridization levels for
5 transcripts in the CM and CL nuclei (see
Figs. 6E, 8E). Another unusual feature of the
intralaminar nuclei compared with other nuclei was the complementary
nature of expression of 4 and
5 mRNAs and of 2,
3, 1, and
1 mRNAs in the CM and CL nuclei. The
4 and 5 subunit
transcripts were present in many of the dorsal thalamic nuclei, with
the exception of the intralaminar nuclei in which there was a dramatic
drop in hybridization levels for them (Figs. 4D,E,
6D,E, 8D,E). This was in sharp contrast to the
expression of 2, 3,
1, and 1 subunit
mRNAs, which was virtually absent in most other nuclei but present at
moderate-to-high levels in the CeM, CM, Pc, Pf, and CL nuclei of the
intralaminar group (Figs. 4B,C,G, 5A,
6B,C,G, 7A).
The medial geniculate complex
All of the subunit transcripts were present at moderate
levels in the medial geniculate complex. High levels of hybridization
were observed for the 1,
5, 2, and
2 subunit transcripts in both the MGv and MGd
nuclear divisions, with a slight increase in signal in MGv (see Figs.
8A,E,H, 9B). A distinctive increase in
hybridization levels was also observed for 1
subunit transcripts in the magnocellular nucleus (see Fig.
8A). Moderate levels were detected for
1 and 1 subunit
transcripts (see Figs. 8G, 9A) with slightly
lower levels for 3,
3, 4, and
2 transcripts (see Figs. 8B,C,D,
9C). The dorsal nucleus of the complex showed increased
levels over the ventral nucleus for 3,
4, and 5 subunit
transcripts.
The LGd nucleus
Of all nuclei in the thalamus, the LGd nucleus had the highest
levels of hybridization for every subunit transcript. The expression
patterns in this nucleus have been described in detail elsewhere
(Huntsman et al., 1995a). Expression of the 1,
2, and 2 subunit
mRNAs was particularly high. Moreover, the LGd nucleus also expressed
all other transcripts at moderate-to-high levels, including transcripts
that were usually expressed at low levels in other nucleia similar
pattern observed for the other sensory relay nuclei. Expression of
1, 5,
2, and 2 mRNA was
slightly higher in the magnocellular layers (Figs.
6A,H, 7B), whereas that of the
2, 4,
1, 3, and
1 mRNAs was equally high in all six layers
(Figs. 6B,D,G, 7A,C). The distribution of
3 mRNAs was the most unique because
hybridization for this subunit mRNA was primarily located in the
magnocellular layers (Fig. 6C).
The posterior group of nuclei
The posterior group had overall low levels of expression of all
receptor subunit transcripts, even those normally expressed at high
levels, such as 1, 5,
2, and 2 mRNAs. The
SG-L nuclei expressed a wide variety of receptor subunit transcripts,
including 1, 2,
3, 1,
2, 1, and
2 (see Figs. 10A,B,C,G,H,
11A,B). The distribution pattern and variety of transcripts
expressed in SGL was similar to that observed in the intralaminar
nuclei, with a comparable increase in hybridization levels for the
otherwise rare 2, 3,
1, and 1 subunit
mRNAs and a reduction to negligible levels of
4 subunit mRNAs.
The epithalamus
In the habenular nuclei, the most prominently expressed subunit
mRNAs were the two representatives of the class,
1 and 2 (Figs.
8G,H), and 3 (Fig. 8C).
1, 2,
1, and 2 subunit
mRNAs were present, but at much lower levels (Figs. 8A,B,
9A,B), and 4,
5, and 3 mRNAs
hybridized just above background (Figs. 8D,E,
9C). The highest transcript levels detected in the
paraventricular nuclei were 2,
1, 1,
3, 5, and
1 and, to a lesser degree,
2, 2,
3, and 4 (Figs. 2,
3). All of the subunit mRNAs examined were present at relatively high
levels, similar to those observed in the intralaminar nuclei. The
paraventricular nuclei also had the highest expression levels of
1 transcripts: almost double that observed in
any other nucleus of the thalamus (Fig. 2G).
The ventral thalamus
The reticular nucleus was unique. Of all the transcripts studied,
only the 2 subunit mRNA was expressed in
significant amounts. 2,
3, 5,
1, 3, and
1 subunit expression could sometimes be
identified at extremely low levels, but this was not consistent and
always bordered on undetectability. 3 was
detected more often than the others. Labeling for the
1, 4, and
2 transcripts was never detected in the
reticular nucleus. In the fields of Forel and the ZI, mRNAs for
2, 1,
2, and 3 subunits
were present at relatively low levels, and other transcripts were
absent (Figs. 4A,C,H, 5B). The small
dorsal extension of the ZI into the posteroventral part of the
reticular nucleus showed expression typical of the rest of the ZI
(Figs. 4, 5). The pregeniculate nucleus expressed most of the subunit
transcripts at moderate levels, especially 2
and 1 (Figs. 6A,H).
Quantitative observations
Relative levels of mRNA expression were determined by measuring
the density of hybridization from film autoradiograms and converting
them to units of radioactivity (nCi/gm). Density readings for each cRNA
probe were collected and compared across thalamic nuclei to determine
which groups of subunit mRNAs were expressed in particular nuclei and
which nuclei maintained the highest levels of each subunit mRNA (Table
2). Optical density readings for all
GABAA receptor transcripts verified the
observations shown in Figures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and were the source of numerical
data used to construct Figures 12, 13, 14 and Tables 1 and 2. The
1, 2, and
2 subunit transcripts were present at
extremely high levels in the sensory relay nuclei (Fig.
12) and exhibited high levels in most of the other
nuclei (Fig. 13). The other transcripts showed varying
patterns of which the greatest variations were found in the
intralaminar nuclei (Fig. 13). In the reticular nucleus, levels of most
subunit mRNAs did not reach threshold, except
2 and occasionally 3
subunit transcripts (Fig. 14).
Fig. 12.
Histogram of mean levels of receptor subunit mRNA
distribution for all the subunit mRNAs examined in selected nuclei of
the dorsal thalamus, including the sensory relay nuclei. There is a
noticeable increase in subunit mRNA levels within sensory-related
nuclei, especially for the 1,
2, and 2 subunit
mRNAs. Optical density readings converted to measures of radioactivity
(nCi/gm) by reference to 14C standards exposed on
the same sheet of film.
[View Larger Version of this Image (39K GIF file)]
Fig. 13.
Histogram of mean density of hybridization taken
from intralaminar nuclei for all subunit transcripts examined. Note the
decreased levels of 4 and
5 subunit mRNAs when compared with neighboring
nuclei of the ventral group. Density readings were converted to units
of radioactivity (nCi/gm) by reference to 14C
standards exposed on the same sheet of film.
[View Larger Version of this Image (42K GIF file)]
Fig. 14.
Histogram of mean density of hybridization taken
from the reticular nucleus at all levels of the thalamus for all
subunit transcripts examined. Note the absence of
1 and 2 subunit mRNAs
in this nucleus and the background levels of other subunit mRNAs,
except for 2 and 3.
Density readings were converted to units of radioactivity (nCi/gm) by
reference to 14C standards exposed on the same
sheet of film.
[View Larger Version of this Image (15K GIF file)]
DISCUSSION
Ten GABAA receptor subunit mRNAs were
expressed throughout the thalamus in overlapping yet distinct
nucleus-specific patterns. Overall, 1,
2, and 2 subunit
mRNAs were expressed at much higher levels than
2-5,
1, 3, and
1. Sensory relay nuclei (LGd, VP, MG) and the
pulvinar showed increased levels compared with other nuclei and
expressed a greater variety of transcripts. In intralaminar nuclei,
transcripts such as 2,
3, 3, and
1, with low levels of expression in most other
nuclei, were expressed at moderate-to-high levels, whereas others, such
as
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