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
Control 2 681
60 192
25 Control 3
597 50
188 27
Control 4 669
50 202
29
The present study shows that chronic subordination
All controls 672
4 59
30
stress reduces expression of the a -adrenoceptor mRNA
2A
Stress 1 559
55 225
30 ,0.001
in noradrenergic neurons of the locus coeruleus, in solitary
Stress 2 503
60 155
20 ,0.001
tract neurons that might be noradrenergic and in the
Stress 3 502
50 179
25 ,0.05
glutamatergic neurons of the lateral reticular nucleus.
Stress 4 292
50 93
13 ,0.001
All stressed 464
4 118
59
4.1. Sequence of the tree shrew a -adrenoceptor cDNA
2 A
Dorsal motor n .
Control 1 538
50 205
29
The cloned 1.22-kb a -adrenoceptor cDNA-fragment
2A
Control 2 616
50 192
27
of the tree shrew contained 89 of the expected receptor
Control 3 571
50 177
25
coding sequence. Sequence alignment of this fragment
Control 4 446
50 163
23 All controls
543 4
72 36
with clones from human and rat showed a higher extent of identity with the human receptor cDNA 92 than with
Stress 1 506
50 190
27 n.s.
the rat 88 [15,16]. At position 515, the tree shrew
Stress 2 608
50 172
24 n.s.
Stress 3 586
50 162
23 n.s.
a -adrenoceptor sequence displays a guanine nucleotide
2A
Stress 4 498
50 158
22 n.s.
which corresponds to the same nucleotide at position 602
All stressed 550
4 56
28
in the human receptor gene, whereas the rat cDNA contains a cytosine at the corresponding site. In the human
Lateral reticular n .
and tree shrew a -adrenoceptor proteins this encodes a
Control 1 1514
50 509
72
2A
Control 2 1180
50 465
66
cysteine at amino acid position 201; the corresponding
Control 3 1391
50 453
64
amino acid in the rat receptor referred to as the a -
2D
Control 4 1644
50 690
98
adrenoceptor subtype is serine. The pharmacological
All controls 1432
4 197
99
consequences of this amino acid substitution have been
Stress 1 1146
50 360
51 ,0.01
discussed elsewhere [20].
Stress 2 1073
50 366
52 n.s.
Stress 3 1112
50 326
46 ,0.05
4.2. Combination of immunocytochemistry and in situ
Stress 4 1320
50 506
72 ,0.01
All stressed 1163
4 109
55
hybridization
a
Sections of stressed animals and controls had been mounted on the same
The combination of immunocytochemistry and in situ
slide and were analyzed pair wise. Significant differences P between the groups were calculated by one-way ANOVA followed by Newman Keuls
hybridization allows detection of receptor mRNA expres-
post hoc test n.s., not significant.
sion in cytochemically identified neurons. However, using this technique control experiments have to be performed to
find out whether antibody-stained cells are specifically labeled by the cRNA probe because it has been reported
showed no reliable effect of the treatment P50.73, F5 that radio-labeled nucleotide probes may non-specifically
0.14, but differences between individuals P50.036, F5 stick to the peroxidase–anti-peroxidase complex [29]. In
11.9. the present experiments, the in situ hybridization con-
H . Meyer et al. Brain Research 880 2000 147 –158
155
Fig. 5. Silver grains in single neurons of chronically stressed male tree shrews expressed as percentages of controls mean6S.E.M.. Differences between groups are significant see Table 1.
ditions had to be modified to suppress non-specific label- was not detectable in PNMT-containing cells of the Sol.
ing. This was achieved by treating the antibody-stained There might be two explanations for this: i the expres-
paraffin sections with proteinase K prior to the hybridiza- sion level of the a
-adrenoceptor gene in these cells is
2A
tion see Section 2. As a control experiment, we also low, or ii the turnover of the receptor-specific mRNA is
35
hybridized antibody-stained sections with the S-labeled
higher than in the LC neurons. Since in the other adren- sense probe. In these sections, no cells in the LC, the
ergic cell group, C1 in the ventrolateral medulla, PNMT- solitary tract nucleus, and the lateral reticular nucleus were
immunopositive neurons were also only lightly labeled by radio-labeled. However, throughout the rostral medulla
the antisense probe, one has to conclude that adrenergic oblongata, there were some very large neurons that showed
neurons in the Sol either contain no a -adrenoceptor gene
2A
accumulation of silver grains presumably motor neurons; transcript, or only at a very low level. Similar results were
diameter of cell body approximately 35 mm. obtained for the TH-immunopositive neurons in Sol and
the A1 cell group, respectively. In the Sol, TH-containing 4.3. a -Adrenoceptor expression in nor adrenergic and
neurons displayed scant but specific antisense hybridiza-
2 A
glutamatergic neurons tion, but A1 neurons displayed no specific antisense
labeling. Therefore, the expression level of a -adreno-
2A
A specific in situ hybridization pattern was obtained ceptors in medullary noradrenergic neurons at the level of
with the antisense riboprobe generated from the tree shrew the obex might by lower or the RNA turnover might be
a -adrenoceptor cDNA. Like in other species, this re-
faster than in the LC.
2A
ceptor subtype is strongly expressed in the main norad- A pronounced antisense hybridization was detected in
renergic cell group in the brain, the LC [25,33,39]. Within the large neurons of the lateral reticular nucleus LRt that
LC neurons, silver grains were found over nucleus and are known to be glutamatergic [14,24]. These findings
cytoplasm. The nuclear labeling probably represents hy- show that in the brain stem, the a
-adrenoceptor is not
2A
bridization with the primary gene transcript heteronuclear only expressed in noradrenergic neurons, which agrees
RNA because the coding region of the a -adrenoceptor
with previous electron microscopic studies on the ventrola-
2A
gene is not interrupted by introns [15]. Cytoplasmic teral medulla where a
-adrenoceptor immunoreactivity
2A
labeling presumably represents hybridization with mRNA. was not limited to catecholaminergic neurons [23]. Hybrid-
In accordance with previous reports indicating that the ization of the receptor-specific probe was never observed
a -adrenoceptor is expressed in noradrenergic neurons,
in cells that were stained by the GABA antibody in LC,
2A
the antisense-labeled cells in the LC of the tree shrew were Sol and LRt, indicating that at least in these nuclei,
immunoreactive for tyrosine hydroxylase [2,17,32]. a
-adrenoceptor expressing cells are not GABAergic.
2A
In contrast to the LC, the solitary tract nucleus Sol showed only few cells that were moderately labeled by the
4.4. Quantification of a -adrenoceptor RNA in stressed
2 A
receptor probe. These labeled cells were loosely arranged animals
in the medial part of the nucleus and around the subnu- cleus gelatinosus which is a pronounced structure in the
Male tree shrews have formerly been shown to be a tree shrew [22]. Since PNMT-immunopositive neurons in
suitable model to investigate the consequences of chronic the
tree shrew
Sol also
surround the
subnucleus subordination stress [9]. The decrease in body weight and
gelatinosus, it could be assumed that the a -adrenoceptor
the increase in urinary cortisol of subordinates reflect the
2A
expressing cells might be adrenergic. However, in the persistent activation of the sympathetic nervous system and
double-labeling experiments the receptor gene transcript of the HPA-axis.
156 H
Our earlier observations indicated that a -adrenergic elucidated. Besides the regulation of gene transcription,
2
radioligand binding is reduced in the brain stem of tree altered degradation of receptor mRNA might also be
shrews subjected to subordination stress [6]. In the present involved in stress-mediated down-regulation of a
-ad-
2A
study, quantification of hybridization signals over neurons renoceptor expression.
in the LC and in the solitary tract nucleus showed a significant stress-dependent reduction of a
-adrenoceptor 4.6. Implications of stress-reduced a -adrenoceptor
2A 2 A
RNA expression. It therefore appears that the reduced expression
3
[ H]RX821002 binding observed earlier in brain stem nuclei of subordinate tree shrews is due to reduced
Although the present results show that that not only receptor gene expression [6]. In this former study, the
noradrenergic but also glutamatergic neurons in the brain extent of down-regulation of a -adrenergic binding sites
stem express the a -adrenoceptor, they support the view
2 2A
was only 10 in the LC, whereas in the present analysis, that this receptor subtype is an autoreceptor in noradrener-
the receptor RNA levels were reduced by 24 in the gic neurons, at least in the LC [2,17,32]. The stress-
stressed animals. One possible reason for this discrepancy induced down-regulation of autoreceptor expression proba-
is that the a -adrenoceptor radioligand labeled all three bly contributes to an imbalance in the noradrenergic
2
a -adrenoceptor subtypes A, B, C leading to an undere- system which, during periods of chronic stress, is persis-
2
stimation of the stress effect on a -adrenoceptor expres-
tently or repetitively activated [1]. This chronic activation
2A
sion. may be due to the low autoreceptor expression that results
In the Sol, a -adrenoreceptor expression was quan-
in reduced capacity for negative feed-back and increased
2A
tified in neurons located in the medial part of the nucleus firing rates of noradrenergic LC neurons [1], thus enhanc-
and around the subnucleus gelatinosus. The distribution ing release of noradrenaline at projection sites of the LC
pattern of these cells indicated that they might represent [26]. Since the LC is an important integrator of the stress
noradrenergic and or adrenergic neurons. However, since response that participates in the regulation of sleep,
the double-labeling experiments showed co-expression arousal, learning and memory, and controls endocrine and
with TH in only very few cells, and not at all with PNMT, autonomic nervous functions through a widespread system
no definitive conclusion may be made concerning the of efferent fibers, the autoreceptor down-regulation has
identity of the a -adrenoceptor expressing cells in the Sol
consequences for many brain functions [37]. In the
2A
where receptor expression is reduced during stress. In areas subordinate male tree shrews, the resulting noradrenergic
A1 and C1, the number of antisense-labeled cells was too hyperactivity may contribute to the affective state of
low for a statistically relevant quantification of silver subordination stress resulting in behavioral changes and in
grains. In addition, the hybridization signal of the strongly an impairment of cognitive performance [9,27].
antisense-labeled LRt neurons tended to mask the hybridi- The co-expression of a
-adrenoceptors and glutamate
2A
zation signals from the A1 and C1 cells. in LRt neurons agrees with pharmacological results in-
dicating that a -adrenoceptor agonists such as clonidine
2
4.5. Mechanisms of a -adrenoceptor down-regulation may at least in part exert their hypotensive effects via LRt
2 A
neurons [10]. Iontophoretically applied noradrenaline and Previous investigations on a -adrenoceptor regulation
systemically injected clonidine depressed LRt neuronal
2
have focused on mechanisms of rapid minutes and long- firing [3]. Also, a -adrenoceptors in the LRt modulate
2
term hours agonist-induced receptor desensitization in spinal antinociception in rats [13,19]. The stress-induced
cell culture [5]. Little is known about the effects of chronic changes in a
-adrenoceptor expression in the LRt might
2A
stress and the accompanying persistent or repetitive agonist therefore affect cardiovascular as well as nociceptive
exposure on these receptors in brains of mammals. Several functions in tree shrews, although the physiological conse-
lines of evidence emphasize the importance of agonists in quences of the receptor changes have not yet been ana-
transcriptional and post-transcriptional control, but hetero- lyzed in detail. In addition, since the LRt projects to the
logous regulation could also explain the present findings cerebellar cortex, an impairment of cerebellar functions
[12]. Recently it was shown that a -adrenoceptor RNA
should also be considered [38,41].
2A
transcription in rat astroglial cultures is down-regulated by increased levels of intracellular cyclic AMP and by protein
kinase C PKC activation [31]. The promoter region of
5. Conclusions
