H . Meyer et al. Brain Research 880 2000 147 –158
149
2.4. In situ hybridization labeled neuron was subtracted from specific labeling.
Sections of a control and a stressed animal mounted on the To identify the neurons expressing a
-adrenoceptor same slide were analyzed pair wise for details see Section
2A
mRNA, antibody-stained paraffin sections were pretreated 3. For statistical evaluation, data were subjected to
before the in situ hybridization. Briefly, slides were ANOVA and Newman–Keuls post hoc test.
hydrated in graded alcohols, rinsed in 0.9 NaCl and phosphate-buffered saline PBS for 5 min each and post-
fixed in 4 PFA 10 min. They were then treated with
3. Results
proteinase K 20 mg ml; Boehringer, Mannheim, Ger- many at 378C, 60 min, washed in PBS, acetylated in 0.1
3.1. a -cDNA and cRNA probe
2 A
M triethanolamine, and processed for in situ hybridization see below.
Three clones of the tree shrew a -adrenoceptor gene
2A
For quantification of receptor expression in stressed were obtained which yielded identical DNA-sequences
animals and controls, frozen brains were cut on a cryostat. representing 89 of the expected receptor coding sequence
Sections 10 mm were thaw-mounted on gelatin-coated Fig. 1. Alignment of the tree shrew cDNA sequence with
slides. Sections from one subordinate and one control those from the human clone C10 and the rat gene clone
animal were mounted pair wise on the same slide. The RG20 showed 92 DNA sequence identity with the
cryostat sections were then dried at room temperature for human and 88 identity with the rat sequence. Antisense
20 min, fixed in 4 PFA, rinsed in PBS, dehydrated run-off transcription of the linearized cDNA-clone in the
35
through graded alcohols, air-dried and stored at 2808C. presence of [ S]UTP generated a 1057-nucleotide cRNA
Before the in situ hybridization, cryostat sections were probe complementary to nucleotides 238–1219 of the
re-hydrated, post-fixed a second time with 4 PFA, and cloned cDNA fragment and 75 nucleotides of the vector
then acetylated. For in situ hybridization, all sections were sequence Fig. 1. In situ hybridization of brain sections
processed as described with two modifications: 1 the with this probe resulted in a specific hybridization pattern
hybridization was carried out at 608C for 18 h, and 2 visualized by film autoradiography Fig. 2. At the level of
when washing the sections, the RNase treatment was the LC, the most pronounced hybridization was detected in
followed by a high stringency wash 0.23 SSC, 60 min, the LC itself and scattered labeling was observed in the
658C [21]. area of the noradrenergic cell group A5. Moderate labeling
To obtain an overview of the hybridization pattern, was observed in the solitary tract nucleus. The lateral
labeled sections were exposed on BioMax film Kodak, reticular nucleus was strongly labeled. Hybridization with
Rochester, NY, USA for 7 days at 48C. To visualize the sense probe yielded only a faint and diffuse au-
hybridization on the cellular level, slides were coated with toradiographic image with no distinct labeling of brain
NTB 2 nuclear emulsion Kodak, Rochester, NY, USA stem structures Fig. 2.
and exposed for 25 days at 48C. Silver grains were developed in D 19 Kodak, slides were rinsed in water
3.2. a -Adrenoceptor RNA expressing neurons
2 A
and fixed with Unifix Kodak. Sections were faintly counter stained with 0.05 toluidine blue in 0.1 di-
Neurons expressing a -adrenoceptor mRNA were first
2A
sodium tetraborate. For quantification of silver grains over visualized on cryostat sections that had been hybridized
35
single neurons in cryostat sections from stressed tree with the
S-labeled cRNA probe and lightly counter shrews and controls, emulsion-coated sections were in-
stained with toluidine blue Fig. 3. Cells were classified as spected under a microscope with a CCD camera connected
being specifically labeled when the number of silver grains to an image analysis system using the grain counting
over the cell was more than 5 times the number of grains
35
program MCID M1 4.2 Imaging Research, Ontario, in the surrounding area. Hybridization with the
S-labeled Canada. For this purpose, slides had been labeled with a
sense probe yielded no cells in the respective brain areas code unknown to the investigator. The specificity of the
that fulfilled this criterion. antisense hybridization signal was checked by comparing it
The largest number of silver grains was detected over to sections hybridized with the sense probe. Quantification
LC neurons, which could be easily identified because of was performed within an observation area of 140390 mm
their characteristic elongated shape. Nuclei of these neu- magnification of objects on the screen, 315 000. Within
rons displayed 4665 silver grains mean6S.E.M.; n550, this area, labeled cells were defined as those displaying at
and the cytoplasm 10668 grains background was sub- least 5 times more silver grains sample area compared to
tracted; see Section 2. In the solitary tract nucleus, only the surrounding area. Labeled neurons were sampled by
nuclei of cells were visible because of the faintness of the manual outlining, and the relative number of silver grains
counter stain that was necessary to yield a high contrast over the cells was determined as number of pixels mean
between the background and silver grains. Only few cells size of a silver grain, 14.3 pixels. Background labeling
in the solitary tract nucleus showed accumulations of silver mean number of silver grains in the surrounding of the
grains 4062 grains per cell; Fig. 3. These cells were
150 H
Fig. 1. Nucleic acid sequence of the tree shrew a -adrenoceptor cDNA representing 89 of the expected receptor coding sequence, and alignments with
2A
the partial a -adrenoceptor cDNAs of human clone C10; nucleotides 88–1306 [15] and rat clone RG20; 88–1306 [16]. Dashes represent identical
2A
nucleotides, asterisks mark missing nucleotides. Underlined letters denote the forward and reverse oligonucleotide primers used for PCR-amplification of the tree shrew receptor cDNA.
primarily located in the medial subdivision of the solitary noradrenergic cell group A1 and the adrenergic cell group
tract nucleus, and around the subnucleus gelatinosus which C1, there were only very few cells displaying low numbers
is a prominent structure in the tree shrew solitary tract of silver grains over their nuclei 1465; Fig. 3. Silver
nucleus. In the ventrolateral medulla, in the areas of the grains over C1 cells were diffusely distributed and not as
H . Meyer et al. Brain Research 880 2000 147 –158
151
sibly glial cells sometimes showed slight accumulations of silver grains.
3.3. Immunocytochemcal identification of antisense labeled cells
To identify the neurons that expressed mRNA for the a
-adrenoceptor, paraffin
sections were
immuno-
2A
cytochemically stained with antibodies, either against tyrosine hydroxylase TH, the rate-limiting enzyme of the
noradrenaline biosynthesis
pathway, against
phenyl- ethanolamine-N-methyltransferase PNMT, the enzyme
generally used to visualize adrenergic neurons, or against glutamate or GABA. After the immunocytochemical pro-
cedure, sections were processed for in situ hybridization.
The noradrenergic neurons in the LC were strongly stained by the TH antibody and showed pronounced
accumulations of silver grains corresponding to a -ad-
2A
renoceptor RNA in cell nuclei and the cytoplasm Fig. 4. In the solitary tract nucleus, only few TH-labeled cells
showed minor accumulations of silver grains not shown. PNMT immunopositive neurons in the solitary tract nu-
cleus were not labeled by the a
-adrenoceptor probe Fig.
2A
4. Instead, the hybridization signal was seen over toluidine blue stained nuclei of cells that were not labeled
by the PNMT antibody. In the area of the adrenergic cell group C1, PNMT-immunopositive neurons showed only
slight accumulations of silver grains Fig. 4. In the vicinity of PNMT-positive neurons, immunonegative struc-
tures displayed high numbers of silver grains. On sections stained with the antibody against glutamate, these struc-
tures were identified as large immunopositive neurons that showed pronounced accumulations of silver grains with
numbers of grains varying from cell to cell Fig. 4. Cells stained with the GABA antibody were never labeled by the
receptor antisense probe, neither in the LC, nor in solitary tract nucleus and lateral reticular nucleus. No specific
labeling of antibody-stained cells was detected with the
35
S-labeled sense probe in the analyzed areas. 3.4. Quantification of a -adrenoceptor expression
2 A
a -Adrenoceptor expression in brain stem nuclei of
2A
chronically stressed tree shrews and controls was quan-
35
Fig. 2. Autoradiograms showing hybridization with the S-labeled
tified by determining the numbers of silver grains over
antisense riboprobe generated from the a -adrenoceptor cDNA A,B,
2A 35
single neurons on cryostat sections coated with photo-
and with the S-labeled sense probe C. The neuroanatomical levels of
the sections are P 2.0 A,C and P 7.5 B according to the tree shrew
graphic emulsion. For quantification, the microscopic
atlas [36]. Abbreviations: A5, noradrenergic cell group A5. LC, locus
picture was projected onto a computer screen, labeled cells
coeruleus; LRt, lateral reticular nucleus; Sol, solitary tract nucleus. Bar
in the observation area were manually delineated, and
represents 4 mm.
silver grains were counted by computer-based image analysis yielding pixels representing relative numbers of
concentrated over the cell nuclei as was the case in the LC silver grains per cell. Only those cells were analyzed that
and the solitary tract nucleus. In contrast, in the lateral showed at least 5 times more silver grains than surround-
reticular nucleus, large neurons with high numbers of ing areas.
silver grains over the cytoplasm and nucleus were detected For the LC, only sections from the anatomical level P
9166 grains cell. In all areas investigated, small pos- 2.0 were inspected that corresponds to the center of the
152 H
Fig. 3. Expression of a -adrenoceptor RNA in neurons of the locus coeruleus LC, the solitary tract nucleus Sol, the area of the noradrenergic cell
2A
group A1, and of the adrenergic cell group C1. Sections were lightly counterstained with toluidine blue to visualize cell nuclei. Filled arrows indicate labeled cells, open arrows denote unlabeled cells. Note that in the LC, many silver grains are located over neurons, whereas in the solitary tract nucleus,
only a few neurons are labeled with low numbers of silver grains. In the areas of the noradrenergic cell groups A1 and the adrenergic cell group C1, there are only minor accumulations of silver grains over cell nuclei. Bar represents 30 mm.
nucleus where noradrenergic cells are loosely clustered animal no. 2 versus 3, P,0.05, but overall, this was not
Fig. 4. At this level, seven to 10 labeled cells per significant one-way ANOVA, P50.066. Also two-way
hemisphere were analyzed 14–20 cells per section. There ANOVA showed a reliable effect of the treatment F5
were no reliable differences in numbers of labeled neurons 138.2, P50.0013, and a difference between individuals
between the hemispheres. Since stress had the same effect F517.6, P50.045. Therefore, in the LC of subordinates,
on silver grain numbers in the nuclei as in the cytoplasm of the number of silver grains was significantly lower than in
neurons, silver grains over these cellular compartments controls 224.0; Fig. 5.
were analyzed together. As demonstrated in Fig. 3, back- In the medial subdivision of the solitary tract nucleus,
ground labeling in the surrounding of the labeled cells was only silver grains over cell nuclei were counted because
very low. the cytoplasm could not be delineated Fig. 3. Also in this
Evaluation of labeled neurons in the LC of psycho- area, subordinates showed reduced numbers of silver
socially stressed and control animals demonstrated a stress- grains 231; Fig. 5. Repeated measures ANOVA yield-
dependent reduction in silver grain numbers Table 1. ed a reliable effect of the treatment P,0.0001 with the
One-way ANOVA for repeated measures yielded a signifi- post hoc test showing differences between each pair of
cant effect P,0.0001 with the post hoc test showing animals Table 1. Individual differences were also de-
reliable differences between each pair of animals each pair tected within the control group P50.0001; animal no. 1
representing one control and one subordinate; Table 1. versus all three other animals, P,0.01 in each case.
Differences were also detected between individual animals Furthermore, the post hoc test detected differences within
within the control group P50.014; differences between the stress group animal no. 4 versus the three others;
animal no. 2 and all three other animals, P,0.05 in each P,0.001 in each case. Two-way ANOVA yielded a
case. Furthermore, the post hoc test detected a slight reliable effect of the treatment F512.1, P50.04, but no
difference between individual animals of the stress group difference between individuals F51.4.
H . Meyer et al. Brain Research 880 2000 147 –158
153
Fig. 4. TH-immunopositive neurons in the locus coeruleus LC, PNMT-immunopositive neurons in the solitary tract nucleus Sol, PNMT-immuno- positive neurons in area C1, and glutamate Glu immunopositive neurons in the lateral reticular nucleus LRt. Paraffin sections were first stained with the
respective antibody and then processed for in situ-hybridization to visualize a -adrenoceptor expression silver grains. LC: note that the TH-
2A
immunopositive neurons show high numbers of silver grains representing RNA encoding the receptor filled arrow. Sol: section was counterstained with toluidine blue to visualize cell nuclei. Note that PNMT-immunopositive cells show only background levels of silver grains open arrow, whereas toluidine
35
blue stained nuclei of immunonegative cells are labeled by the S-labeled cRNA probe arrow head. C1: note that PNMT-immunopositive cells show only
slight accumulations of silver grains open arrow. Instead, the surrounding of the PNMT cell reveals patches of silver grains arrow head. LRt: note that the large glutamate-immunopositive neurons in the lateral reticular nucleus display high numbers of silver grains filled arrow. Bar represents 30 mm.
In the lateral reticular nucleus, only large, presumptive effect of the treatment F522.2, P50.018, but no signifi-
glutamatergic neurons were analyzed. In situ hybridization cant difference between individuals F56.8, P50.075.
showed reduced numbers of silver grains over these In the dorsal motor nucleus of vagus, no significant
neurons in
stressed animals
compared to
controls differences between stress animals and controls were
218.8, Fig. 5. Repeated measures ANOVA yielded a detected. Repeated measures ANOVA yielded P,0.0001,
reliable effect of the treatment P,0.0001 with the post but this was due to differences within groups, whereas
hoc test revealing differences between each pair of animals differences between the pairs of animals were not reliable
Table 1. Differences were also detected within the Table 1. Individuals within the control group showed
control group P50.0003; animal no. 1 versus 2, P,0.01; reliable differences animal no. 4 versus 1, P,0.05; no. 4
no. 2 versus 4, P,0.001. Furthermore, within the stress versus 2, P,0.001; no. 4 versus 3, P,0.01, and also
group, there were significant differences between indi- within the stress group, individual differences were de-
viduals P50.012; animal no. 4 versus all three others, tected animal no. 4 versus 2 and 3, respectively; no. 1
P,0.05 in each case. Two-way ANOVA yielded a reliable versus 2 and 3, P,0.05 in all cases. Two-way ANOVA
154 H
Table 1
3.5. Peripheral reactions during chronic subordination
Effect of chronic stress on numbers of silver grains expressed as
stress
a
pixels cell in brain stem nuclei n5number of cells Mean
n S.D.
S.E.M. P
In accordance with our previous reports, the chronic
Locus coeruleus
psychosocial stress exposure significantly reduced body
Control 1 2383
43 730
111
weight of subordinate males to 91.763.9 of the initial
Control 2 2808
43 783
119
value P,0.05. Body weight of control animals remained
Control 3 2352
43 644
98
constant during the whole experimental period. Cortisol in
Control 4 2450
43 822
125
urine of subordinates was significantly increased mean of
All controls 2504
4 203
101
all days during the stress period: 0.2060.01 ng mmol
Stress 1 1907
43 492
75 ,0.01
creatinine versus 0.0860.01 ng mmol creatinine during the
Stress 2 2059
43 580
88 ,0.001
no-stress period; P,0.01, whereas urinary cortisol in
Stress 3 1765
43 465
71 ,0.001
Stress 4 1920
43 542
83 ,0.001
control animals remained low throughout the experimental
All stressed 1903
4 128
64
period 0.0960.03 ng mmol creatinine.
Solitary tract n .
Control 1 742
55 227
31
4. Discussion
