It is well known that the pituitary
adrenocortical axis is activated in response to
stressful stimuli in animals and man. In
addition, it is well recognized that the limbic
system is involved in both neuroendocrine
regulation and emotional responsivity to stress.
Accordingly, a
limbic-hypothalamic-pituitary-adrenocortical
system bas been conceptualized as an integrating
unit which controls the physiological and
behavioral response to stress. Little is known
about the neuroanatomical basis for this
response in the forebrain. However, evidence
from psychopharmacological and lesion studies
have suggested that the septo-hippocampal
cholinergic system is an important part of a
neuronal network in the brain which controls the
response to stress, a response which is adaptive
in nature. Indeed, we have recently demonstrated
that in rats the septo-hippocampal cholinergic
system is actively involved in the response to
stress. This is expressed by a reduction in high
affinity choline uptake and increased muscarinic
binding capacity in the hippocampus. These
changes are more pronounced and occur carlier in
rat strains which are more reactive to stress.
Furthermore, it has been demonstrated that
the hippocampus is a main target region for
corticosterone and that the electrical activity
of pyramidal neuror in the hippocampus is
affected by corticosterone. These data indicate
that the hippocampus can integrate both
endocrine and neural signals of stress, possibly
through participation of the cholinergic system.
In the present study therefore we have examined
the effects of stress, ACTH and corticosterone
treatments on the dynamics of hippocampal
cholinergic synaptic mechanisms.
Sprague-Dawley female rats, 10-12 weeks old,
were housed 5 to a cage, supplied freely with
food and water and maintained at 24°C with
a 12 h light-dark cycle. Stress consisted of
immobilization in a prone position for 10 min,
as reported before. Rats left undisturbed in
their home cage served as controls.
For adrenalectomy, rats were anesthetized
with halothane and the two adrenal glands were
removed through small incisions made on both
sides of the back. Operated rats were maintained
on 0.9% NaCl (saline) drinking water. Sham
operated rats served as controls.
Drugs were injected intraperitoneally (i.p.)
in 200 µl of solvent (vehicle):
corticosterone (35 mg/kg) in peanut oil and ACTH
(1.5 u/kg) in saline. Vehicle injected rats
served as controls.
After 10 min of stress, or 10 min after drug
treatments rats were decapitated, their brains
rapidly excised and the hippocampus dissected.
The dissected tissues were immediately
homogenized in 10 vols. (w/v) of ice-cold 0.32 M
sucrose in a glass homogenizer fitted with a
Teflon pestle. Homogenates were centrifuged at
1000 g for 10 min and the resulting supernatant
centrifuged at 17,000 g for 20 min. The rude
synaptosomal pellet (P2 fraction) was
resuspended in the original volume of 0.32 M
sucrose and used for biochemical assays.
High affinity sodium-dependent uptake of
[3H]choline (0.4 µCi, spec. act. 80
Ci/mmol), measured for 4 min, was assayed
according to the method of Atweh et al.
Spontaneous release of newly synthesized
[3H]aceiylcholine (ACh) was measured in
synaptosomal fractions following incubation for
4 min in the presence of [3H]choline
(0.4,uCi, spec. act. 80 Ci/mmol). Immediately
after the incubation 3 ml of ice-cold
Krebs-Ringer phosphate buffer, pH 7.4, were
added and synaptosomal fractions were centriuged
at 20,000 g for 15 min. The pellets were washed
,wice with 1 ml and then resuspended in 1 ml of
the same buffer containing 5µm eserine
sulfate. Release of [3H]ACh was measured
at 37 'C for up to 8 min. The tubes were then
rapidly centrifuged at 20,000 g for 15 min at 4
'C and the supernatant removed for
[3H]ACh measurements according to the
method of McCaman and Stetzler.
To assess muscarinic cholinergic binding, the
antagonist [3H]quinuclidinyl benzilate
(ONB) (spec. act. 33 Ci/mmol) served as a
ligand. Binding was assayed according to
Yamamura and Snyder in P2 fractions, using 10
µM atropine sulfate to determine
non-specific binding. The values of QNB binding
measurements were plotted according to
Scatchard.
Protein content was measured by the method of
Lowry et al. Effects of stress, ACTH or
corticosterone treatments. Injections of high
doses of ACTH (1.5 u/kg) or corticosterone (35
mg/kg) led 10 min later to increases in both
[3H]choline uptake and in newly
synthesized [3H]ACh release, as did a 10
min session of immobilization stress. Analysis
of the variance indicated that there was no
difference between treatments with regard to
[3H]choline uptake elevations (FO.05
(3,28)=2.4).
Effects of adrenalectomy. The effects of
adrenalectomy were examined 2 days after the
operation. Both [3H]choline uptake and
[3H]ACh release were increased as
compared to unhandled or sham operated controls.
Adrenalectomy led to the largest observed
increase in 13HIACh release. Interestingly,
[-3H]ACh release but not
[-3H]choline uptake, was increased in
sham operated animals (2 days postoperative) as
compared to unhandled controls. This could have
been the result of the long term stress caused
by the operation.
Effects of stress, ACTH or corticosterone
treatments on adrenalectomized rats. The effects
of stress, or single injections of ACTH or
corticosterone on [3H]choline uptake and
[3H] ACh release were examined after 10
min in animals which were adrenalectomized 2
days earlier.
Uptake of [3H] choline was elevated
after all treatments to the same extent (F0, 05
(6,40) = 2. 1). The increases in [3H]ACh
release observed after adrenalectomy alone were
not altered by acute stress or ACTH treatments.
However, corticosterone treatment of
adrenalectomized rats did result 10 min later in
a significant reduction; yet these reduced
levels were still significantly higher than
unhandled controls. In contrast, corticosterone
did not alter choline uptake after
adrenalectomy.
The present data demonstrate that the
hippocampal cholinergic synaptic mechanisms are
activated within minutes after stress and that
similar changes can be produced shortly after
treatments with high doses of ACTH or
corticosterone. An increase in [3H]
choline uptake was observed before in
hippocampal synaptosomes 3 h after a single
intravenous injection of a high dose of
corticosteroids. These findings indicate that
the septo-hippocampal cholinergic system can
be activated secondary to an activation of the
pituitary-adrenocortical axis, an activation
which occurs rapidly when stressful conditions
are encountered.
In contrast, ACTH, which can produce similar
effects as does corticosterone in intact
animals, has no effect on the cholinergic
induced changes after adrenalectomy. Comparable
sensitivity to alterations in the
pituitary-adrenocortical hormonal status was
demonstrated by Azmitia et al. for the septal
driving of hippocampal thetarhythm. These
authors have attributed the change. to be the
result of interaction between the serotonine
input to the hippocampus and hippocampal neurons
which are able to concentrate
corticosterone8,21. Our findings demonstrate
that stress-induced activation of the
pituitary-adrenocortical axis, which results in
enhanced circulating corticosterone or treatment
with high doses of corticosterone, can in turn
activate the presynaptic cholinergie terminals
in the hippocampus. This indicates that
septo-hippocampal cholinergic neurons themselves
are sensitive to major changes in circulating
levels of corticosterone. Overproduction of
corticosterone can occur after stress or in
disease (e.g. Cushing's syndrom) while
deficiency may occur in adrenocortical
insufficiency (e.g. Addison's syndrom).
The finding that corticosterone treatment
reduces the adrenalectomy-induced increase in
ACh release, with no significant effect on the
enhanced choline uptake levels, indicates that
these two mechanisms which control presynaptic
cholinergic activity are differentially
sensitive to corticosterone. So far
corticosterone effects in the brain were
correlated mostly with intracellular binding
within neurons. However, the rapid effects
demonstrated in the present study indicate that
corticosterone may act directly on these
presynaptic cholinergic mechanisms in the
hippocampus. This possibility is currently being
examined by measuring the effects of
corticosterone on synaptosomal preparations in
vitro.
Preliminary data indicate that values of QNB
binding to hippocampal synaptosomal preparations
were not altered after acute stress. ACTH, or
corticosterone treatments and remained at levels
of unhandled control values which were: Bmax =
2.35 ± 0.12 (pmol/mg protein); Ad = 0.61
± 0.03 (nM). At 2 days after adrenalectomy,
however, Kd values were increased to 124.6% of
control, with no change in Bmax Mis change
together with the previous demonstration that
chronic intermittent stress leads to increased
Bmax values indicate that muscarinic receptors
can be modified when circulating corticosteroid
levels are altered over prolonged periods. In
support of this view is the recent report on
corticosteroid iodulation of muscarinic
receptors in rat myocardial membranes.
In summary, the present study demonstrates
that after short term stress the
septo-hippocampal cholinergic system is
activated secondary to activation of the
pituitary-adrenocortical axis. The demonstration
that major changes in circulating levels of
corticosteroids can modulate the activity of the
hippocampal cholinergic synapse implies that the
septo-hippocampal system may be a common output
for various neuronal and endocrine systems which
convey signals of stress.
