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Neurophysiology, Basic and Clinical NeuroReport
0959-4965 # Lippincott Williams & Wilkins
5-HT
1A
autoreceptor
desensitization by chronic
ultramild stress in mice
Laurence Lanfumey,
CA
Marie-Christine Pardon,
1
Nora Laaris,
Chantal Joubert, Naima Hanoun,
Michel Hamon and Charles Cohen-Salmon
1
INSERM U 288 and
1
CNRS 7593, IFR des
Neurosciences, CHU Pitie
Â
-Salpe
Ã
trie
Á
re, 91 Bd de
l'Ho
Ã
pital, 75634 Paris cedex 13, France
CA
Corresponding Author
ELECTROPHYSIOLOGICAL and biochemical approaches
were used to assess possible changes in central 5-HT
neurotransmission in mice that had been subjected to
chronic ultramild stress for 8 weeks. This treatment
produced a signi®cant decrease in the potency of the 5-
HT
1A
agonist ipsapirone to inhibit the electrical activity
of serotoninergic neurons in the dorsal raphe nucleus,
without modifying 5-HT
1A
receptor binding in various
brain areas. These data demonstrate that chronic ultra-
mild stress triggers a long term and durable functional
desensitization of somatodendritic 5-HT
1A
autorecep-
tors in mice. NeuroReport 10:3369±3374 # 1999 Lippin-
cott Williams & Wilkins.
Key words: Chronic ultramild stress; Electrophysiology;
5-HT
1A
autoreceptors; Serotonin; Slice
Introduction
It is well documented that stress induces alterations
in brain monoaminergic systems, and there is now a
large body of evidence supporting the idea that
serotoninergic neurons are especially affected by
stress and related psychopathological disorders [1].
Indeed, marked changes in brain serotonin (5-HT)
turnover have been shown to occur in association
with the activation of the hypothalamo-pituitary-
adrenal (HPA) axis under various stressful condi-
tions [1,2]. However, the effects of stress on 5-HT
neurotransmission appear to depend on its time-
related characteristics. Whereas acute stress enhances
5-HT system activity [1], chronic stress seems to
reduce it [3±5]. Among key proteins for 5-HT
neurotransmission, 5-HT receptors have been re-
ported to exhibit adaptive changes in response to
stress [1]. In particular, immobilization stress pro-
duces a decrease in hippocampal 5-HT
1A
receptor
density [6], and stress due to exposure to novel
environment triggers functional desensitization of 5-
HT
1A
autoreceptors in the dorsal raphe nucleus
(DRN) [7,8]. These ®ndings are of particular interest
because (i) clear-cut links exist between brain 5-HT
neurotransmission, especially 5-HT
1A
receptors, and
the pathogenesis of depression [9] and (ii) stress has
long been regarded as a major factor in the develop-
ment of a variety of psychopathologies, including
depression and post-traumatic stress disorders [10].
Further assessment of the latter hypothesis could
notably be made in a relevant animal model where
rats subjected to chronic mild stress exhibit beha-
vioral alterations that can be reversed by antidepres-
sant drugs [11,12]. Adaptation of this model to mice
allowed us to investigate whether exposure to
chronic ultramild stress (CUMS) [13] actually pro-
duces alterations in central 5-HT neurotransmission.
Both electrophysiological and biochemical ap-
proaches were used for this purpose.
Materials and Methods
Procedures involving animals and their care were
conducted in conformity with the institutional
guidelines that are in compliance with national and
international laws and policies (Council directive
87-848, October 19, 1987, MinisteÁre de l'Agriculture
et de la ForeÃt, Service VeÂteÂrinaire de la SanteÂetdela
Protection Animale, permissions 0299 to M.H. and
6269 to L.L.).
Because genetic factors are thought to play a role
in the inter-individual variability of stress responses
[14], experiments were performed on B6D2F1 mice,
corresponding to the ®rst generation issued from
crossing C57BL6J (B6) with DBA/2 (D2) animals.
Indeed, all B6D2F1 subjects are genetically identical,
and therefore differences observed between stressed
and paired control animals can only be attributed to
the stress effect.
Experiments on the effects of stress in rodents
generally used males as subjects. However, it has
been shown that female rats are more vulnerable to
stressors than males [15] and depression, possibly
NeuroReport 10, 3369±3374 (1999)
Vol 10 No 16 8 November 1999
3369
triggered by repeated stress, is more frequent in
women. These observations led us to use female
mice in our studies.
Female B6D2F1 mice (Iffa-Credo, Lyon, France),
3 months of age, were brought into the laboratory 1
month before the start of the experiment. On arrival,
the animals were housed in groups of ®ve per cage
(20 3 32 3 12 cm) in the animal research facility and
maintained under standard laboratory conditions:
12:12 hr light:dark cycle (with lights on at 07.30 h),
22 28C, 60% relative humidity, ad lib access to
food and water. At the start of the experiment, the
stressed animals were housed singly (except where
indicated) in a separate room and had no contact
with the paired control animals, which were main-
tained under the same standard conditions as for the
preceding month.
Stress procedure: The stress regimen used for the
study is a modi®cation of the chronic mild stress
procedures described by Willner et al. [11] and
Moreau et al. [12] for the rat. In contrast to these
procedures, nociceptive stressors and food and water
deprivations were excluded and only environmental
and social disturbances were applied. The stress
procedure consisted of various mild stressors such as
repeated periods of 308 cage-tilt, con®nement to
small cages (11 3 8 3 8 cm), two 2 h periods of
paired housing, one overnight period of dif®cult
access to food (without any reduction in the actual
food ration), one period of continuous overnight
illumination, and one overnight period in a soiled
cage (50 ml of water/l of sawdust bedding). Animals
were also placed on a reversed light/dark cycle from
Friday evening to Monday morning. These stressors
were scheduled over a 1 week period and repeated
throughout the 8 week experiment (see [13] for
details).
Biochemical measurements: For the corticosterone
assay, mice were killed by decapitation at 09:00 h
and blood from trunk vessels was collected in chilled
tubes for the measurement of serum corticosterone
levels. Corticosterone was quanti®ed by radio-
immunoassay after extraction in ethanol [16]. Anti-
corticosterone antiserum was generously given by F.
HeÂry (INSERM U 297, Marseille, France). Corti-
costerone (Sigma, St Quentin Fallavier, France) was
used as standard and [
3
H]corticosterone (87 Ci/
mmol, Amersham, Les Ulis, France) as radiotracer
[16].
Immediately after death, the brain was removed
and the hippocampus, brain stem and cerebral cortex
were dissected on ice. Tissues were homogenized in
40 vol. (v/w) ice-cold 50 mM Tris±HCl, pH 7.4,
with a Polytron (type PT10 OD) tissue disrupter.
The resulting homogenates were centrifuged at
40 000 3 g for 20 min at 48C, and the pellets were
washed twice by resuspension in 100 vol. ice-cold
buffer, followed by centrifugation. The sedimented
material was then resuspended in 40 vol. Tris±HCl
buffer and incubated at 378C for 10 min to allow the
dissociation of endogenous 5-HT. Membranes were
centrifuged and washed three more times as above,
and the ®nal pellet was resuspended in 10 vol. Tris±
HCl buffer. Aliquots (50 ìl, corresponding to
0.25 mg protein) of membrane suspensions were
incubated at 258C for 60 min in 0.5 ml (®nal vol.)
50 mM Tris±HCl, pH 7.4, containing either 0.67 nM
[
3
H]alnespirone [17] (92 Ci/mmol, Amersham) with
0.05% bovine serum albumin, or 0.20 nM [
3
H]WAY
100635 [18] (85 Ci/mmol, Wyeth-Ayerst). Non-
speci®c binding was determined in the presence of
10 ìM 5-HT. Assays were stopped by rapid ®ltra-
tion through GF/B ®lters, and entrapped radio-
activity was quanti®ed by scintillation counting (see
[17,18] for details).
Tissue levels of 5-HT and 5-HIAA were meas-
ured in animals which had been decapitated and
their brains immediately dissected at 08C. Brain
structures (brain stem, hippocampus, striatum and
cerebral cortex) were homogenized in 10 vol. (v/w)
0.1 N HCIO
4
containing 0.5% Na
2
S
2
O
5
and 0.5%
disodium EDTA. Homogenates were centrifuged at
30 000 3 g for 15 min at 48C and the supernatants
were neutralized with 2 M KH
2
PO
4
/K
2
HPO
4
,pH
7.0. After a second centrifugation as above, the clear
supernatants were saved and aliquots (10±20 ìl)
were injected directly into a high-performance liquid
chromatography column (Ultrasphere IP, 25 cm,
0.46 cm o.d. 5 ìm) protected with a Brownlee pre-
column (3 cm, 5 ìm). The mobile phase consisted of
70 mM KH
2
PO
4
, 2 mM triethylamine, 0.1 mM dis-
odium EDTA, 1.25 mM octane sulfonic acid and
15% methanol, adjusted to pH 2.78 with solid citric
acid. The elution rate was set at 1 ml/min and
electrochemical detection of 5-HT and 5-hydroxyin-
doleacetic acid (5-HIAA) was performed at a poten-
tial of 0.65 V. Quantitative determinations were
made with a CR3A Shimadzu integrator using
appropriate standards [19].
Electrophysiology: Mice were killed by decapita-
tion and the brain was rapidly removed and placed
in ice-cold oxygenated (95% O
2
/5% CO
2
) arti®cial
cerebrospinal ¯uid (aCSF). A block of tissue con-
taining the DRN was cut into 400 ìm sections using
a vibratome, while immersed in ice-cold ACSF.
After sectioning, slices were kept in oxygenated
ACSF for at least 1 h at room temperature (20±
238C). A single slice was then placed on a nylon
mesh, completely submerged in a small chamber and
3370
Vol 10 No 16 8 November 1999
NeuroReport L. Lanfumey et al.
superfused with oxygenated aCSF (348C) at a con-
stant ¯ow rate of 2 ml/min. Drugs were admin-
istered through a three-way tap system, and
complete exchange of ¯uid in the chamber occurred
within 2 min [20]. Extracellular recordings of the
®ring of DRN neurons were made using glass
microelectrodes ®lled with 2 M NaCl (12±15 MÙ).
Cells were identi®ed as 5-HT neurons according to
described criteria [20]. Electrical signals were fed
into a high-input impedance ampli®er (VF 180,
Biologic, France), an oscilloscope and an electronic
ratemeter triggered by individual action potentials
connected to an A/D converter and a personal
computer. Using a dedicated software, the integrated
®ring rate was recorded, computed, and displayed
on a chart recorder as consecutive 10 s samples.
Baseline activity was recorded for at least 10 min
before application of various concentrations of the
5-HT
1A
agonist ipsapirone [9] and the 5-HT
1A
antagonist WAY 100635 [18]. The effects of ipsapir-
one were evaluated by comparing the mean dis-
charge frequency recorded during the 2 min that
preceded its application with that recorded at the
peak action of the drug. Data are expressed as
percentages of the baseline ®ring rate s.e.m. Statis-
tical analyses were made using one-way ANOVA
and, in case of signi®cance ( p , 0.05), the F-test for
signi®cant treatment effects was followed by the
two-tailed Student's t-test to compare the experi-
mental groups with their controls.
Results
Biochemical measurements: Morning levels of cor-
ticosterone in control mice (6.98 1.91 ìg/100 ml
serum, n 12) were slightly higher than those
usually found in normal rodents for this period of
the day and this was attributed to the transfer of
animals from the housing room to the experimental
room. Indeed, corticosterone levels found here are
comparable to those reported in the relevant litera-
ture dealing with similar protocols [24] and can be
considered more as a HPA response to the mice
transfer than as really basal levels. However, under
these conditions, a marked and signi®cant increase
(350%) in serum corticosterone levels was ob-
served in mice that had been subjected to the CUMS
protocol (24.06 3.56 ìg/100 ml serum, n 12, p ,
0.01).
Exposure to CUMS for 8 weeks changed neither
the speci®c binding of [
3
H]alnespirone to 5-HT
1A
receptors in hippocampal, cortical and brain stem
membranes nor that of [
3
H]WAY 100635 in cortical
membranes (Table 1).
Data in Table 2 show that both 5-HT and 5-
HIAA levels were generally decreased (up to
ÿ33%) in brain areas from mice subjected to CUMS
compared to paired controls. A signi®cant reduction
(18%) in the 5-HIAA/5-HT ratio was also noted
in the brain stem, hippocampus and striatum, but
not in the cerebral cortex in stressed mice (Table 2).
Electrophysiology: DRN 5-HT neurons recorded
in mouse brain stem slices displayed the character-
istic slow (1.69 0.18 spikes/s, n 12) and regular
(Fig. 1) discharge pattern previously described for
rats under similar conditions [8,20]. CUMS affected
neither the frequency (1.58 0.21 spikes/s, n 16)
nor the pattern (Fig. 1) of the discharge of DRN 5-
HT neurons.
Addition of the 5-HT
1A
receptor agonist, ipsapir-
one (10 nM±1 ìM), to the ACSF superfusing brain
Table 1. Effect of chronic ultramild stress on 5-HT
1A
receptor
binding in various brain areas
5-HT
1A
receptor binding (fmol/mg protein)
Control mice Stressed mice
[
3
H]alnespirone
Hippocampus 72.7 6.9 68.5 3.6
Cerebral cortex 45.9 3.0 43.1 1.8
Brain stem 29.5 1.7 28.9 2.4
[
3
H]WAY00635
Cerebral cortex 65.6 1.5 62.8 2.4
Chronic ultramild stress sessions were applied for 8 weeks.
Binding assays were carried out with 0.67 nM [
3
H]alnespirone or
0.20 nM [
3
H]WAY 100635. Each value is the mean s.e.m. of
6±8 independent determinations (one determination per
mouse). No signi®cant differences were noted between control
and stressed mice.
Table 2. Effects of chronic ultramild stress on 5-HT and 5-
HIAA levels in various brain areas
Control mice Stressed mice
Brain stem
5-HT (ìg/g) 0.584 0.036 0.463 0.015
5-HIAA (ìg/g) 0.437 0.012 0.292 0.009
5-HIAA/5-HT 0.75 0.04 0.63 0.03
Hippocampus
5-HT (ìg/g) 0.545 0.026 0.481 0.024
5-HIAA (ìg/g) 0.444 0.018 0.329 0.011
5-HIAA/5-HT 0.82 0.05 0.68 0.04
Striatum
5-HT (ìg/g) 0.433 0.014 0.407 0.017
5-HIAA (ìg/g) 0.308 0.015 0.229 0.006
5-HIAA/5-HT 0.71 0.04 0.56 0.03
Cerebral cortex
5-HT (ìg/g) 0.442 0.025 0.363 0.011
5-HIAA (ìg/g) 0.170 0.011 0.124 0.004
5-HIAA/5-HT 0.38 0.02 0.34 0.02
Chronic ultramild stress sessions were applied for 8 weeks.
Each value is the mean s.e.m. of at least six independent
determinations.
p , 0.05 compared with respective control values.
Vol 10 No 16 8 November 1999
3371
Chronic ultramild stress and 5-HT
1A
autoreceptor NeuroReport
stem slices from control mice resulted in a concen-
tration-dependent inhibition of the ®ring of DRN
5-HT neurons, with complete blockade at 300 nM of
the ligand (Fig. 2). The concentration-response curve
of ipsapirone-induced inhibition in the control
group indicated an EC
50
value of 38.2 1.6 nM
(n 10), similar to that previously found in the rat
under similar in vitro conditions [8,20] (Fig. 2). As
shown in Fig. 2, ipsapirone was signi®cantly less
potent in inhibiting the ®ring of DRN 5-HT cells in
stressed animals. In particular, only 30% inhibition
was noted in the latter group with 100 nM ipsapir-
one, whereas complete blockade of the discharge
was achieved at this concentration in the control
group (Fig. 2). Thus, CUMS produced a shift to the
right of the ipsapirone concentration±response
curve, with an EC
50
value of 99.5 7.6 nM, 2.5 times
higher than that determined for paired-control ani-
mals ( p , 0.001, two-tailed t-test, t 4.7, df 22).
In both control and stressed mice, the inhibitory
effect of ipsapirone (100 nM) could be prevented by
the selective 5-HT
1A
antagonist, WAY 100635
(3 nM; not shown), as expected from its mediation
through 5-HT
1A
autoreceptor stimulation.
Discussion
The present study showed that, in mice, a 8-week
exposure to a chronic ultramild stress procedure
reduced the potency of an agonist, ipsapirone, to
inhibit the discharge of DRN 5-HT cells via the
stimulation of somatodendritic 5-HT
1A
autorecep-
tors. However, 5-HT
1A
autoreceptor desensitization
was apparently not associated with any change in 5-
HT
1A
receptor binding in the brain stem (as well as
the cerebral cortex and the hippocampus) after this
long lasting stress. These data indicated that the 5-
HT system adapts to chronic mild stress, in contrast
80
60
50
40
30
20
10
0
70
0 100 200 300 400 500 600 700 800 900 1000
control mouse
A
60
40
30
20
10
0
50
0 100 200 300 400 500 600 700 800 900 1000
stressed mouse
B
Number of events
interspike interval (ms)
FIG. 1. Effect of chronic ultramild stress on the discharge character-
istics of DRN 5-HT cells in mice. Interspike interval histogram (n 31
intervals, 400 bins, bin width 0.025 ms) illustrating the similarity in the
discharge characteristics of DRN 5-HT cells in a control mouse (A) and
in a mouse subjected to an 8-week CUMS session (B).
FIG. 2. Effect of chronic ultramild stress on the inhibitory effect of ipsapirone on the ®ring of DRN 5-HT neurons. (A) Integrated ®ring rate histograms
(in spikes/10 s) showing the effect of 300 nM ipsapirone on the electrical activity of a DRN 5-HT neuron in a control mouse and a mouse that had been
exposed to CUMS for 8 weeks. (B) Concentration-dependent inhibition by ipsapirone of the ®ring of DRN 5-HT neurons in brain stem slices from control
or stressed mice. Stress consisted of CUMS for 8 weeks. Ipsapirone-induced inhibition is expressed as percentage of the baseline ®ring rate. Each point
is the mean s.e.m. of data obtained from 8±16 individual cells. The dotted lines illustrate the increased EC
50
value of ipsapirone (abscissa) in CUMS-
exposed mice compared with controls.
P , 0.01 compared with the respective inhibition in control mice.
20
0
20
0
10 30 60 100 300
Ipsapirone (nM)
10 30 60 100 300
3 min
A
firing rate (spikes / 10 s)
control mouse
Ipsapirone (nM)
stressed mouse
100
75
50
25
0
29 28 27 26 25
log [ipsapirone]
M
stressed mice
control mice
*
*
firing inhibition (%)
B
3372 Vol 10 No 16 8 November 1999
NeuroReport L. Lanfumey et al.
to that observed after an intense acute stress such as
immobilization, where no modi®cations of DRN 5-
HT
1A
autoreceptor regulation could be found [8].
Such a functional desensitization of DRN 5-HT
1A
autoreceptors with no modi®cation of their density
has already been found in rats subjected to various
types of chronic stress [8,22]. However, we showed
here for the ®rst time that 5-HT
1A
autoreceptor
desensitization is a long lasting adaptive phenomen-
on, with no escape even after 8 weeks of stress in
mice. Interestingly, such a chronic ultramild stress
alters decision-making behaviors [13], and whether
adaptive desensitization of DRN 5-HT
1A
autorecep-
tors underlies these behavioral changes has to be
addressed in future investigations.
We also saw a marked increase (350%, p , 0.01)
in serum corticosterone levels in mice subjected to
CUMS. If the circulating levels of corticosterone
actually re¯ected activation of the HPA axis, it can
be inferred that chronic exposure to different stres-
sors such as those used in the present CUMS
procedure probably renders the HPA axis super-
sensitive to each consecutive unpredictable stress
session. Indeed, repeated exposure to the same stress
such as immobilization does not increase basal
corticosterone levels [23], but enhances the cortico-
sterone response to another stress [24].
Previous studies in rats have shown that elevated
corticosterone levels probably play a major role in
stress-induced DRN 5-HT
1A
autoreceptor desensiti-
zation, because this phenomenon can be prevented
by adrenalectomy and is mimicked by the direct
application of the hormone onto brain stem slices
[7,8]. It can therefore be surmised that CUMS-
induced desensitization of DRN 5-HT
1A
autorecep-
tors in mice also results from a direct action of
corticosterone at glucocorticoid receptors in seroto-
ninergic neurons [25]. The lack of changes in 5-
HT
1A
receptor binding in mice subjected to CUMS
suggests that glucocorticoid receptor activation does
not affect 5-HT
1A
autoreceptors per se but alters
their transduction mechanisms. Similar conclusions
were drawn in a model of psychosocial stress, where
a small transient decrease in the af®nity of 5-HT
1A
autoreceptors for [
3
H]8-OH-DPAT with no change
in their density were observed in the DRN of
stressed male tree shrews [22].
Since activation of DRN 5-HT
1A
autoreceptors
triggers a negative feed-back control of 5-HT turn-
over, the inhibitory in¯uence of corticosterone upon
5-HT
1A
autoreceptor functioning might contribute
to the well established enhancement of 5-HT synth-
esis and release under acute stressful conditions [1].
However, under CUMS conditions, a decrease in 5-
HT and 5-HIAA levels and the 5-HIAA/5-HT ratio
was generally observed in the four brain areas
examined, possibly re¯ecting a decreased 5-HT
neurotransmission in stressed mice. Indeed, previous
studies in rats [3,5] and monkeys [4] showed that
chronic exposure to stressful conditions actually
reduced the activity of central 5-HT systems.
Although direct determination of 5-HT turnover
has to be made in mice subjected to CUMS for 8
weeks, it can already be emphasized that 5-HT
1A
autoreceptor desensitization was not associated with
increased 5-HT synthesis and utilization under such
chronic stress conditions. Other adaptive changes,
possibly involving auto- (via 5-HT
1B
autoreceptors)
and/or hetero- (via afferent pathways controlling
DRN 5-HT neurons) regulatory mechanisms might
compensate for the expected increase in 5-HT
neurotransmission due to the 5-HT
1A
autoreceptor
desensitization in CUMS-exposed mice.
Conclusion
Corticosteroids have been shown to modify the
responsiveness of hippocampal neurons to 5-HT
1A
receptor stimulation and to attenuate 5-HT auto-
inhibition in the DRN [8,21], leading to an increase
5-HT tone under non pathological conditions. In
contrast, hypercorticism combined with apparent
hypoactivity of 5-HT systems have been reported in
depressed subjects. CUMS might be a relevant
model of depression in mice since it produces both a
hyperactive HPA axis and an apparent decrease in
5-HT neurotransmission. Whether 5-HT
1A
autore-
ceptor desensitization observed in this model re¯ects
a similar change in human depression is an interest-
ing question to be addressed in future investigations.
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ACKNOWLEDGEMENTS: This research was supported by grants from INSERM, IFR
des Neurosciences, European Community (Biotech B104 CT960752), CNRS, Bayer
Pharma and IPSEN Foundation. The generous gifts of drugs (ipsapirone, WAY
100635) and radioligands ([
3
H]alnespirone, [
3
H]WAY 100635) by pharmaceutical
companies (Bayer-Pharma, Servier, Wyeth-Ayerst) are gratefully acknowledged.
Received 5 August 1999;
accepted 20 August 1999
3374
Vol 10 No 16 8 November 1999
NeuroReport L. Lanfumey et al.