Heterogeneous neurochemical responses to different
stressors: a test of Selye’s doctrine of nonspecificity
KAREL PACAK,1,2 MIKLOS PALKOVITS,3 GAL YADID,4
RICHARD KVETNANSKY,5 IRWIN J. KOPIN,1 AND DAVID S. GOLDSTEIN1
1Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke and
3Laboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892; 2Department of Medicine, Washington Hospital Center,
Washington, District of Columbia 20010; 4Department of Life Sciences, Bar-Ilan University,
Ramat-Gan, Israel 52100; and 5Institute of Experimental Endocrinology, Slovak Academy
of Sciences, Bratislava, Slovak Republic 81000
ACTH; norepinephrine; epinephrine
HANS SELYE in 1936 (31) reported that exposure to any of
several noxious agents produced the same syndrome:
adrenal enlargement, gastrointestinal ulceration, and
thymicolymphatic involution. From this pathological
triad, he developed a theory of stress that attained wide
popularity and aroused intense research interest but
also incited controversy that persists to this day (32).
He defined stress as the nonspecific response of the
body to any demand (33), emphasizing that the same
pathological triad would result from exposure to any
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
stressor. In this report, we call this the doctrine of
nonspecificity.
Selye focused on the hypothalamic-pituitary-adrenocortical (HPA) system as a key effector of the stress
response. The HPA activation attending stress was
thought to be part of a constellation of stereotypical
neural and endocrine responses. Administration of
ACTH can elicit all three components of the pathological triad, and some have even defined stress empirically as that which increases plasma ACTH concentrations (8). Selye did not claim that ACTH, or high
circulating levels of adrenocortical steroids, can elicit
the pathological triad.
Numerous studies, however, have demonstrated differential neuroendocrine responses during exposure to
different stressors. For instance, glucopenia evokes
selective adrenomedullary and pituitary-adrenocortical activation (10, 22, 23); orthostasis, hyperthermia,
and cold exposure evoke selective sympathoneural activation (16, 27, 37); water deprivation evokes selective
vasopressinergic activation (29); and manipulations of
dietary salt intake produce selective effects on reninangiotensin-aldosterone system activity (17). Moreover, during exposure to different stressors, sympathetic responses exhibit pronounced heterogeneity among
different organs (6, 36); response patterns also depend on
the duration and intensity of the stressor (24, 30) and
vary with the experience of the individual (13).
This patterning does not of itself refute the doctrine
of nonspecificity, because the different patterns could
result from superimposition of different stressorspecific homeostatic responses on the same nonspecific
stress response. According to Selye’s theory, stress
refers only to the nonspecific component revealed after
subtraction of the specific components from the total
response. As demonstrated mathematically in the DISCUSSION, it is not possible to test Selye’s theory by
comparing effector responses to single intensities of
different stressors.
Acceptance of certain assumptions, however, renders
the doctrine of nonspecificity amenable to experimental
testing. This testing was the main purpose of the
present experiments. We assessed plasma ACTH and
catecholamine responses to different intensities of various stressors, all of which are known to evoke increases
in circulating ACTH levels. By comparing ratios of
differences in responses to low- and high-intensity
R1247
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
Pacak, Karel, Miklos Palkovits, Gal Yadid, Richard
Kvetnansky, Irwin J. Kopin, and David S. Goldstein.
Heterogeneous neurochemical responses to different stressors: a test of Selye’s doctrine of nonspecificity. Am. J. Physiol.
275 (Regulatory Integrative Comp. Physiol. 44): R1247–
R1255, 1998.—Selye defined stress as the nonspecific response of the body to any demand. Stressors elicit both
pituitary-adrenocortical and sympathoadrenomedullary responses. One can test Selye’s concept by comparing magnitudes of responses at different stress intensities and assuming that the magnitudes vary with stress intensity, with the
prediction that, at different stress intensities, ratios of increments neuroendocrine responses should be the same. We
measured arterial plasma ACTH, norepinephrine, and epinephrine in conscious rats after hemorrhage, intravenous
insulin, subctaneous formaldehyde solution, cold, or immobilization. Relative to ACTH increments, cold evoked large
norepinephrine responses, insulin large epinephrine responses, and hemorrhage small norepinephrine and epinephrine responses, whereas immobilization elicited large increases in levels of all three compounds. The ACTH response
to 25% hemorrhage exceeded five times that to 10%, and the
epinephrine response to 25% hemorrhage was two times that
to 10%. The ACTH response to 4% formaldehyde solution was
two times that to 1%, and the epinephrine response to 4%
formaldehyde solution exceeded four times that to 1%. These
results are inconsistent with Selye’s doctrine of nonspecificity and
the existence of a unitary ‘‘stress syndrome,’’ and they are more
consistent with the concept that each stressor has its own central
neurochemical and peripheral neuroendocrine ‘‘signature.’’
R1248
TESTING OF STRESS THEORY
stressors above a threshold level of stressor intensity,
we examined the hypothesis that all stressors evoke
the same nonspecific response pattern, a hypothesis
that has survived more than half a century without
undergoing a direct test.
METHODS
RESULTS
At their highest intensities, all the stressors increased levels of ACTH, NE, and Epi significantly
compared with levels after intravenous Sal (Figs. 1–5).
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
All experiments described in the present study were approved by the National Institute of Neurological Disorders
and Stroke Animal Care and Use Committee.
Male Sprague-Dawley rats (Taconic Farms, Germantown,
NY), weighing 280–370 g, were maintained in an animal
housing room at 22 6 2°C and 40% humidity, with lights on
from 0600 to 1800. The rats were fed a normal laboratory diet.
Tap water was provided ad libitum. At least 4 days for
acclimatization in the housing room elapsed before the start
of the experiment.
Conscious animals with an indwelling arterial catheter
were exposed to one of the following stressors: insulin (Ins);
hemorrhage (Hem); immobilization (Immo); formaldehyde
solution (Form) injected subcutaneously to produce pain and
tissue damage; cold exposure; physiological saline (Sal) injected
subcutaneously after handling in preparation for the injection;
and painless intravenous Sal (without handling) as a control.
Preparation of animals. Twenty-four hours before the acute
experiments, each rat was anesthetized with pentobarbital
sodium (40 mg/kg ip). Polyethylene catheters (PE-50) filled
with heparinized 0.9% Sal (50 IU/ml) were inserted into a
femoral artery and, in some experiments, into a femoral vein.
The catheters were sutured in place and tunneled subcutaneously to the nape, where they were attached to a metal spring.
The cannulas were flushed with heparinized 0.9% Sal (100
IU/ml, 0.2 ml) at the end of light cycles (1700–1800).
Acute experiments. All acute experiments began between
0800 and 0900 on the day after operation. Each rat was
assigned randomly to one of the experimental treatments
described below.
Blood samples (0.4 ml each) were collected into tubes
containing heparin (5 ml, 1,000 IU/ml) for catecholamine
determinations or tubes containing EDTA for ACTH determinations. Sal was added to the blood cells, and the same
volume as had been drawn was injected into the animal
immediately after each blood sample was collected. Blood
samples for catecholamine determinations were centrifuged
immediately at 13,000 rpm for 90 s to separate the plasma.
Blood samples for ACTH determinations were centrifuged at
2,900 rpm for 20 min at 4°C to separate the plasma. Plasma
samples were stored at 270°C and analyzed within 3 wk.
Intravenous Sal. The neurochemical results for the various
stressors were compared with those after intravenous injection of Sal as a control, as described below.
Handling and subcutaneous Sal. Even brief, gentle handling of conscious rats produces marked increases in plasma
levels of catecholamines (18). Subcutaneous injection would
also be expected to evoke a catecholaminergic response. Some
animals underwent subcutaneous injection of physiological
Sal, after handling to position the animal for the injection, as
described below.
Insulin-induced hypoglycemia. After the baseline collection, the animals received 0.3 (n 5 7; 313 6 9.4 g final body
wt), 1.0 (n 5 5), or 3.0 (n 5 7) IU/kg of insulin (Eli Lilly,
Indianapolis, IN) or physiological Sal intravenously (0.1 ml
solution/100 g body wt). Arterial blood samples were obtained
at 15, 45, 75, and 105 min after insulin administration.
Formaldehyde solution-induced pain and tissue damage.
After baseline samples were collected as described above, the
animals received 1% formaldehyde solution (Baker, Phillips-
burg, NJ; n 5 6), 4% formaldehyde solution (n 5 7; 344 6
11.3 g final body wt), or Sal (n 5 5) subcutaneously into the
right leg (0.1 ml/100 g body wt). Arterial blood samples were
obtained at 15, 45, 75, 105, and 135 min after formaldehyde
solution administration.
Nonhypotensive and hypotensive hemorrhage. The experiment began with collection of a baseline arterial blood
sample. The animals were bled to either 10 (n 5 6) or 25%
(n 5 7) of estimated blood volume (EBV), with EBV calculated
from the equation, EBV 5 0.06 3 body weight (g) 6 0.77 (21).
Arterial blood samples were obtained 15, 45, 105, and 135
min after hemorrhage.
Cold exposure. The experiment began with collection of a
baseline arterial blood sample at room temperature (24 6
2°C) outside the cold chamber. The unshaved animal was
then carefully transferred (,30 s) from its home cage (placed
next to cold chamber) into the cold chamber [chamber temperature 4 (n 5 9) or 23°C (n 5 7)] and kept in the chamber for 3 h.
Arterial blood samples were collected during exposure to cold
at 15, 45, 105, and 165 min. After 3 h, the cooling system in
the cold chamber was switched off, the doors of the cold
chamber were opened, and room temperature was attained
inside the chamber after #10 min. Arterial blood samples
were obtained 15, 45, and 105 min after cold exposure. To
assess effects of the transfer itself, three rats in a control
group were exposed to room temperature for 1 h in the
chamber.
Immobilization. The experiment began with collection of a
baseline arterial blood sample. Each animal then underwent
2 h of Immo, which consisted of taping the rat’s limbs to a
metal frame with hypoallergic tape (18, 19). Arterial blood
samples samples were obtained at 15, 30, 45, 75, 105, and 120
min during Immo.
Assays. Plasma concentrations of norepinephrine (NE) and
epinephrine (Epi) were assayed using reverse-phase liquid
chromatography with electrochemical detection after partial
purification by adsorption on alumina (5, 12, 14). Catechol
concentrations in each sample were corrected for recovery of the
internal standard, dihydroxybenzylamine. The limits of detection
for NE and Epi were 2–4 pg (30 fmol) per volume injected.
Plasma levels of ACTH-(1—39) were measured in duplicate
without prior extraction, using a commercially available RIA
kit (ICN Biomedicals) (26). The intra-assay coefficient of
variation was ,5%.
Data analyses. Results are presented as means 6 SE. P ,
0.05 defined statistical significance.
Plasma levels of ACTH, NE, and Epi were assayed before,
during, and after exposure to the stressor. Changes in levels
of ACTH, Epi, and NE were analyzed either by one- or
two-way ANOVA for repeated measures. An area under the
curve (AUC) was calculated based on concentration 3 time as
a measure of the magnitude of the response. Thus each
animal provided one data point, the AUC, for each dependent
variable. Total AUCs for plasma catecholamines and plasma
ACTH were calculated by rectangular integration. Net AUCs
from the baseline were calculated as the difference between
total AUCs and basal AUCs. For statistical purposes, the
length of testing for all stressors was limited to the duration
of Immo (2 h). Differences among stressors or neuroendocrine
measures were also assessed by ANOVA as appropriate, as
were responses to different stressor intensities. Independentmeans t-tests were used for examining differences in responses to stressors at two intensities.
TESTING OF STRESS THEORY
R1249
tively were similar to those previously reported in
conscious, unrestrained rats (18).
The 4% Form concentration elicited about a twofold
larger plasma ACTH response and about a fourfold
larger plasma Epi response than did the 1% concentration, the differences for both variables being statistically significant (P , 0.05 for ACTH, P , 0.01 for Epi).
Hem evoked small NE and Epi responses relative to
ACTH responses. The 25% Hem elicited about a fivefold
larger ACTH response than did the 10% Hem (P ,
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
Fig. 1. Effects of subcutaneous administration of formaldehyde
solution (Form; 1 or 4%) or physiological saline (Sal) on plasma levels
of ACTH, norepinephrine (NE), and epinephrine (Epi) in conscious
rats. Results are expressed as means 6 SE.
Although Immo evoked large increases in plasma levels
of ACTH, NE, and Epi, other stressors induced disproportionately large NE or Epi responses compared with
ACTH responses. Thus cold evoked large NE responses, whereas low-dose Ins evoked large Epi responses. There was no effect of the transfer of rats into
the cold chamber on plasma NE, Epi, or ACTH (data
not shown). The plasma catecholamine and ACTH
results in the rats receiving intravenous Sal postopera-
Fig. 2. Effects of intravenous administration of insulin (Ins; 0.1, 1.0,
or 3.0 IU/kg) or Sal on plasma levels of ACTH, NE, and Epi in
conscious rats. Results are expressed as means 6 SE.
R1250
TESTING OF STRESS THEORY
dent measures, demonstrate the marked heterogeneity
of neuroendocrine responses. Ins, regardless of dose,
markedly stimulated adrenomedullary secretion, as
reflected by plasma Epi levels, with much less intense
sympathoneural activation, as reflected by plasma NE
levels; cold exposure produced much larger proportionate increments in plasma NE levels than in plasma Epi
or ACTH levels; Hem produced large ACTH responses
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
Fig. 3. Effects of immobilization (Immo; 2 h) on plasma levels of
ACTH, NE, and Epi in conscious rats. Results are expressed as
means 6 SE.
0.01). There were no differences in plasma NE or Epi
responses to 10 and 25% Hem.
DISCUSSION
The present results, based on several experiments
involving various intensities of different stressors and
multiple peripheral and central neurochemical-depen-
Fig. 4. Effects of cold stress (4 or 23°C) on plasma levels of ACTH,
NE, and Epi in conscious rats. Results are expressed as means 6 SE.
R1251
TESTING OF STRESS THEORY
but relatively small NE and Epi responses; and Immo
increased plasma levels of all three compounds. In
addition, although ACTH and Epi responses to Ins were
highly correlated, ACTH and Epi responses to other
stressors were more weakly correlated or were not
related at all, despite significant increases in ACTH
levels and therefore HPA activation in response to all
the stressors.
A x 5 x · a n 1 x · a x;
Bx 5 x · bn 1 x · bx
A y 5 y · a n 1 y · a y;
By 5 y · bn 1 y · by
(1)
The doctrine of nonspecificity predicts that the ratio
an/bn for each stressor is the same, i.e., that
anx /bnx 5 any /bny 5 an /bn
(2)
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
Fig. 5. Effects of hemorrhage (Hem; 10 or 25% of estimated blood
volume) on plasma levels of ACTH, NE, and Epi in conscious rats.
Results are expressed as means 6 SE.
Heterogeneity does not disprove the doctrine of nonspecificity. As noted above, this heterogeneity does not
of itself support or refute Selye’s stress theory. The
stress response was thought to occur in three stages,
the ‘‘general adaptation syndrome’’ (GAS), consisting of
‘‘alarm,’’ ‘‘resistance,’’ and ‘‘exhaustion.’’ Selye (33) wrote
that the GAS denoted the whole response, of which the
alarm, resistance, and exhaustion stages were merely
successive phases. In other words, although during the
different stages of the GAS the intensity of the response
might vary, the neural and endocrine pattern would be
the same, regardless of the stage. The stressors used by
Selye and his students (33) included acute manipulations, several of which are used in the present study,
hemorrhage, cold, insulin, and immobilization.
To explain the heterogeneity of neuroendocrine responses to stressors, Selye hypothesized the existence
of specific reactions. Only after subtraction of these
from consideration could one identify the core, nonspecific, shared element, the stress, and its stereotypical
consequence, the stress response.
The doctrine of nonspecificity cannot be disproved. No
one appears to have considered formally the possibility
or impossibility of disproving the doctrine of nonspecificity.
In a well-known review, Munck et al. (25) argued
persuasively against Selye’s concept of ‘‘diseases of
adaptation,’’ pointing out correctly that glucocorticoids
produce potent anti-inflammatory effects, in contrast
with Selye’s view that collagen diseases, allergy, and
rheumatic diseases result from an abnormal or excessive GAS. Munck also correctly noted that, after the
1960s, the concept of diseases of adaptation was largely
ignored. The review did not consider the validity of
Selye’s doctrine of nonspecificity.
It can be shown mathematically that, without simplifying assumptions, the doctrine of nonspecificity cannot
be disproved.
Consider responses of effector systems, A and B, e.g.,
ACTH and Epi, to the different stressors, x and y. The
overall magnitude of the response of effector system A
to stress x (Ax ) is the sum of the nonspecific component
(x · an ) and the specific component (x · ax ), where x is a
particular intensity of the stressor and an and ax are
constants relating the intensity of the stress to the
nonspecific and specific components of the response.
(Most stimulus-response curves approximate a linear
relationship between the magnitude of the response
and the logarithm of the stimulus intensity.) The
nonspecific and specific components of the response of
effector system B can be represented similarly. Thus,
for stressors x and y and responses A and B
R1252
TESTING OF STRESS THEORY
A X 5 X · an 1 X · ax
(3)
and
Ax 5 x · a n 1 x · a x
For the other stressor, Y, the constant for the specific
response is ay. If there were a threshold stressor
intensity, t, for eliciting the stress response (nonspecific
component), and if X . x . t, then for effector systems A
and B
AX 5 (X 2 t) · an 1 (X 2 t) · ax
Ax 5 (x 2 t) · an 1 (x 2 t) · ax
(4)
Assumptions rendering the doctrine of nonspecificity
testable. Inspection of Eq. 4 indicates that if the constants ax, bx, ay, and by were all equal to zero (or were
very small with respect to an and bn ), then one could
test the doctrine of nonspecificity. Assuming the constants for the specific components (ax, bx, ay, and by )
were small relative to the constants for the nonspecific
component (an and bn ) would be tantamount to assuming that above the threshold intensity, t, the specific
components would contribute negligibly to the overall
responses of the effector systems. In this situation,
regardless of the stressor, the ratio of the intensityrelated increment in the value for effector system A to
the intensity-related increment in the value for effector
system B would be a constant, namely, an /bn.
Another assumption would also enable testing of the
doctrine of nonspecificity. This assumption is that the
magnitudes of both the specific and nonspecific components vary directly with the intensity of the stressor
over the whole range of stressor intensities, i.e., that
there is no ceiling for the specific component and no
threshold for the nonspecific component. The reasoning
is as follows. If one considers ratios of values for the
dependent variables at different stressor intensities
AX/Ax 5 [(X 2 t) · an 1 X · ax]/[(x 2 t) · an 1 x · ax]
Equation 6 cannot be solved; however, if t 5 0, then Eq.
6 is reduced to
AX /Ax 5 (X · an 1 X · ax)/(x · an 1 x · ax)
5 X (an 1 ax)/x(an 1 ax) 5 X/x
BX 5 (X 2 t) · bn 1 (X 2 t) · bx
Similarly, for stressor Y
Bx 5 (x 2 t) · bn 1 (x 2 t) · bx
If one assumes that the terms X · ax, x · ax, X · bx, and
x · bx, corresponding to the specific components, either
reach a maximal value or are negligible at stressor
intensities eliciting the stress syndrome, then X · ax 5
x · ax.
From these equations, it can be shown that
(AX 2 Ax )/(BX 2 Bx) 5 an/bn
(6)
(5)
and analogously for stressor y
(AY 2 Ay )/(BY 2 By) 5 an/bn
The doctrine of nonspecificity predicts that the neuroendocrine pattern corresponding to the nonspecific
component is the same for all stressors and is therefore
the same for stressors x and y, i.e., for all stressors
k 5 an /bn
Inspection of the four parts of Eq. 4, however, demonstrates that if one does not accept either of the above
assumptions, then even measuring responses of different effector systems to different intensities of different
stressors could not solve the equations and therefore
could not test the prediction of the doctrine of nonspecificity.
AY /Ay 5 Y/y
Analogously, for dependent measure B
BX /Bx 5 X/x;
BY /By 5 Y /y
leading to the testable predictions, BX /Bx 5 AX /Ax and
BY /By 5 AY /Ay.
A test of the doctrine of nonspecificity. Even with
simplifying assumptions, appropriate testing of the
doctrine of nonspecificity would require a very complex
study, involving multiple stressors at different intensities and multiple simultaneously assessed dependent
measures. Although an enormous literature describes
effects of graded intensities of stressors on neuroendocrine-dependent measures, none would apply to the
issue of the validity of the doctrine of nonspecificity.
As demonstrated mathematically above, the doctrine
of nonspecificity could be tested, given one of two
assumptions about the existence of a threshold stressor
intensity for the nonspecific response or the magnitude
of the specific response above the threshold stressor
intensity. In the present study, for some stressors, the
data obtained were inconsistent with both assumptions
and therefore irrelevant as tests of the doctrine of
nonspecificity. For instance, there appeared to be a
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
Note that the four parts of Eq. 1 contain eight
variables. From a comparison of the responses to a
single intensity of two (or any number) of different
stressors, it is theoretically impossible to test the
prediction in Eq. 2.
Concepts about the neuroendocrinology of stress
have continued Selye’s line of reasoning, proposing that
above a threshold stressor intensity, a ‘‘stress syndrome’’ occurs (3, 15). If the magnitude of the specific
component were directly related to the intensity of the
stressor and if the nonspecific component were expressed after a threshold of stressor intensity, this
would not render the above equations solvable, as
follows.
One can obtain data for different intensities of the
same stressor. If x is the low-intensity value of stressor
x and X the high-intensity value, then applying Eq. 1
TESTING OF STRESS THEORY
Fig. 6. Tests of doctrine of nonspecificity. A: ratios (greater stress/less
stress) of responses of plasma levels of NE, Epi, and ACTH for Hem
(H) and Form (F). B: increments in area under curve (DAUC) for
plasma NE, Epi, and ACTH for Form and Hem. Doctrine of nonspecificity would predict that arrows should be parallel to each other and
same length.
summary data in forms related to the theoretical
predictions. As shown in Fig. 6A), for plasma Epi, the
ratio of the response for the more severe Hem to the less
severe Hem was smaller than the ratio of the response
for the more severe Form to the less severe Form. The
doctrine of nonspecificity would predict that the difference between Hem and Form would also obtain for
plasma ACTH; in fact, however, for plasma ACTH, the
ratio of the response for the more severe Hem to the less
severe Hem was much larger than the ratio for the
more severe Form to the less severe Form. As shown in
Fig. 6B), the increment in plasma Epi levels between
the two intensities of Form was larger than the increment in plasma ACTH levels; yet the increment in
plasma Epi levels between the two intensities of Hem
was smaller than the increment in plasma ACTH
levels. Thus, by both tests, the doctrine of nonspecificity
failed to predict the experimental results for Hem and
Form.
With regard to the doctrine of nonspecificity, we
therefore reach the following conclusions. Without simplifying assumptions, which may or may not be acceptable for particular stressors, the doctrine of nonspecificity is impossible to disprove and is therefore of little
scientific value. Yet with acceptable simplifying assumptions, the doctrine of nonspecificity fails to predict the
obtained experimental results. Given the simplifying
assumptions, then, the data are inconsistent with
Selye’s stress theory and refute the existence of a
unitary stress syndrome.
An alternative proposal: primitive specificity of stress
responses. The present results are more consistent with
an alternative proposal: that each stressor has a neurochemical ‘‘signature,’’ with quantitatively if not qualitatively distinct central and peripheral mechanisms.
These neurochemical changes would occur not in isolation but in concert with physiological, behavioral, and
even experiential changes. In evolutionary terms, natural selection would have favored this patterning of
stress responses by enhancing the protection and propagation of genes (4). We call this ‘‘primitive specificity.’’
In line with the notion of stressor-specific alterations
in central neurotransmission, Lachuer et al. (20) reported differential early time course activation of brain
stem catecholaminergic groups in response to various
stressors. Ceccatelli and Orazzo (2) reported increased
expression of enkephalin mRNA and neurotensin mRNA
in the paraventricular nucleus after ether exposure
and after immobilization but not after swimming or
exposure to cold; none of the stressors increased corticotropin-releasing hormone (CRH) mRNA expression.
Romero et al. (28) reported stressor-specific responses
of oxytocin, CRH, and vasopressin accumulation after
colchicine blockade of axonal transport. Gaillet et al. (7)
noted that the involvement of noradrenergic ascending
pathways in stress-induced HPA activation depends on
the type of stressor.
Possible limitations of present experiments. Assessing the extent of experienced ‘‘distress’’ in laboratory
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
threshold for enhanced ACTH responses to Ins; yet Epi
responses continued to increase substantially above
the threshold. This would be reasonable, since one
might expect an intensity-related increase in the need
for a specific, hormonal adrenergic response as a glucose counterregulatory mechanism; however, the Ins
results could not be used to test the doctrine of nonspecificity, because the obtained data did not fit either
assumption.
The results about Immo also could not be applied,
because the intensity of the stressor was not varied
quantitatively. The results about cold could not be
applied, because the specific noradrenergic component
appeared to predominate regardless of stressor intensity. This would be consistent with a specific, noradrenergic neuronal response to conserve heat and expend
energy; however, the responses of ACTH levels were so
small, the nonspecific component could have contributed only negligibly to the overall response.
The data about ACTH and Epi responses to Hem and
Form did seem to fit the assumptions required to test
the doctrine of nonspecificity. Figure 6 depicts the
R1253
R1254
TESTING OF STRESS THEORY
Perspectives
The present findings emphasize the stressor specificity of neuroendocrine response patterns. Although Selye
defined stress as the nonspecific response of the body to
any demand, the present analysis has demonstrated
the untestability of this idea, unless one accepts certain
assumptions; yet given these assumptions, the doctrine
of nonspecificity did not predict the present empirical
results. According to an alternative ‘‘homeostatic’’ theory
of stress (9), survival advantages afforded by multiple,
shared effectors have led to the evolution of primitively
specific generators for patterned neuroendocrine responses to different stressors. Studies relating neurochemical alterations in specific brain pathways with
simultaneously monitored dependent neuroendocrine,
physiological, and behavioral variables should enable
identification of the components and elucidate the
regulation of these patterns. Moreover, studies of appropriate animal models and human pedigrees should
elucidate the genetic bases of these patterns and provide insights about the mechanisms underlying the
predisposition to develop clinical stress-related disorders.
Address for reprint requests: D. S. Goldstein, National Institute of
Neurological Disorders and Stroke, National Institutes of Health,
9000 Rockville Pike, Bldg. 10, Rm. 6N252, Bethesda, MD 20892–
1424.
Received 30 March 1998; accepted in final form 2 June 1998.
REFERENCES
1. Benveniste, H., and P. C. Huttemeier. Microdialysis—theory
and application. Prog. Neurobiol. 35: 195–215, 1990.
2. Ceccatelli, S., and C. Orazzo. Effect of different types of
stressors on peptide messenger ribonucleic acids in the hypothalamic paraventricular nucleus. Acta Endocrinol. 128: 485–492,
1993.
3. Chrousos, G. P., and P. W. Gold. The concepts of stress and
stress system disorders. J. Am. Med. Assoc. 267: 1244–1252,
1992.
4. Dawkins, R. The Selfish Gene. New York: Oxford Univ. Press,
1989.
5. Eisenhofer, G., D. S. Goldstein, R. Stull, H. R. Keiser, T.
Sunderland, D. L. Murphy, and I. J. Kopin. Simultaneous
liquid chromatographic determination of 3,4-dihydroxyphenylglycol, catecholamines, and 3,4-dihydroxyphenylalanine in plasma
and their responses to inhibition of monoamine oxidase. Clin.
Chem. 32: 2030–2033, 1986.
6. Fagius, J., and C. Berne. Changes of sympathetic nerve
activity induced by 2-deoxy-D-glucose infusion in humans. Am. J.
Physiol. 256 (Endocrinol. Metab. 19): E714–E720, 1989.
7. Gaillet, S., J. Lachuer, F. Malaval, I. Assenmacher, and A.
Szafarczyk. The involvement of noradrenergic ascending pathways in the stress-induced activation of ACTH and corticosterone
secretions is dependent on the nature of stressors. Exp. Brain
Res. 87: 173–180, 1991.
8. Ganong, W. F. Review of Medical Physiology. Norwalk, CT:
Appleton & Lange, 1995, p. 327–351.
9. Goldstein, D. S. Stress, Catecholamines, and Cardiovascular
Disease. New York: Oxford Univ. Press, 1995.
10. Goldstein, D. S., A. Breier, O. M. Wolkowitz, D. Pickar, and
J. W. M. Lenders. Plasma levels of catechols and ACTH during
acute glucopenia in humans. Clin. Auton. Res. 2: 359–366, 1992.
11. Goldstein, D. S., M. Garty, G. Bagdy, K. Szemeredi, E. M.
Sternberg, S. Listwak, K. Pacak, A. Deka-Starosta, A.
Hoffman, P. C. Chang, R. Stull, P. W. Gold, and I. J. Kopin.
Role of CRH in glucopenia-induced adrenomedullary activation
in rats. J. Neuroendocrinol. 5: 475–3486, 1993.
12. Goldstein, D. S., R. Stull, R. Zimlichman, P. D. Levinson, H.
Smith, and H. R. Keiser. Simultaneous measurement of DOPA,
DOPAC, and catecholamines in plasma by liquid chromatography with electrochemical detection. Clin. Chem. 30: 815–816,
1984.
13. Graessler, J., R. Kvetnansky, D. Jezova, M. Dobrakovova,
and G. R. Van Loon. Prior immobilization stress alters adrenal
hormone responses to hemorrhage in rats. Am. J. Physiol. 257
(Regulatory Integrative Comp. Physiol. 26): R661–R667, 1989.
14. Holmes, C., G. Eisenhofer, and D. S. Goldstein. Improved
HPLC method for plasma dihydroxyphenylacetic acid and other
catechols. J. Chromatogr. B 653: 131–138, 1994.
15. Johnson, E. O., T. C. Kamilaris, G. P. Chrousos, and P. W.
Gold. Mechanisms of stress: a dynamic overview of hormonal
and behavioral homeostasis. Neurosci. Biobehav. Rev. 16: 115–
130, 1992.
16. Kim, Y. D., C. R. Lake, D. E. Lees, W. H. Schuette, J. M. Bull,
V. Weise, and I. J. Kopin. Hemodynamic and plasma catecholamine responses to hyperthermic cancer therapy in humans.
Am. J. Physiol. 237 (Heart Circ. Physiol. 6): H570–H574, 1979.
17. Krieger, J. E., J. F. Liard, and A. W. Cowley, Jr. Hemodynamics, fluid volume, and hormonal responses to chronic high-salt
intake in dogs. Am. J. Physiol. 259 (Heart Circ. Physiol. 27):
H1629–H1636, 1990.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
animals is at best difficult. If one accepts that emotional
distress increases plasma levels of Epi and ACTH, then
prior experience with the preparation has indicated
little if any distress in conscious, unrestrained animals
1 day after insertion of an indwelling arterial cannula.
Studies over the past 5 years have involved preparations for stress testing and blood and microdialysate
sampling beginning 24 h after surgical placement of
catheters and a microdialysis probe (1). After this
period of recovery, food and water intake and plasma
levels of catecholamines, corticosterone, and ACTH
averaged about the same to those in animals studied 48
h after surgery (34). In humans, arterial plasma concentrations of Epi and ACTH return postoperatively to
preoperative values within 24 h of surgery, without
further changes during the subsequent 2 days (35).
Hypothermia induced by pentobarbital sodium anesthesia and heat loss during catheter implantation
probably did not affect responses of levels of NE, Epi, or
ACTH during cold exposure beginning 22–24 h after
the surgery. Body temperature was maintained by
using a heating lamp throughout the surgery, and there
was no evidence that by the time of the acute study the
animals were hypothermic (36.8 6 0.2°C). Moreover,
arterial plasma levels of ACTH and catecholamines at 1
day after catheter implantation under halothane anesthesia (11) averaged about the same as at 1 day after
catheter implantation under pentobarbital sodium anesthesia in the present study (60.8 6 7.6 vs. 49.5.8 6
5.9 pg/ml).
Future studies in this area should focus on further
examination of the notion of stressor-specific patterns
of central neurotransmitter release and regional neuronal activation and on altered responsiveness of organisms with specific genetic changes. Meanwhile, we hope
that the present findings spur attempts to elaborate
concepts that incorporate the specific and nonspecific
elements of stress responses and that yield testable
hypotheses.
TESTING OF STRESS THEORY
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
of stimuli that release neuronal and adrenomedullary catecholamines in man. Circulation 59: 637–643, 1979.
Romero, L. M., P. M. Plotsky, and R. M. Sapolsky. Patterns of
adrenocorticotropin secretagog release with hypoglycemia, novelty, and restraint after colchicine blockade of axonal transport.
Endocrinology 132: 199–204, 1993.
Ryan, K. L., R. M. Thornton, and D. W. Proppe. Vasopressin
contributes to maintenance of arterial blood pressure in dehydrated baboons. Am. J. Physiol. 256 (Heart Circ. Physiol. 25):
H486–H492, 1989.
Schadt, J. C., and J. Ludbrook. Hemodynamic and neurohormonal responses to acute hypovolemia in conscious mammals.
Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H305–H318, 1991.
Selye, H. A syndrome produced by diverse nocuous agents.
Nature 138: 32, 1936.
Selye, H. The Stress of Life. New York: McGraw-Hill, 1956.
Selye, H. Stress. Montreal, Canada: Acta, 1950.
Szemeredi, K., G. Bagdy, R. Stull, A. E. Calogero, I. J.
Kopin, and D. S. Goldstein. Sympathoadrenomedullary inhibition by chronic glucocorticoid treatment in conscious rats. Endocrinology 123: 2585–2590, 1988.
Udelsman, R., J. A. Norton, S. E. Jelenich, D. S. Goldstein,
W. M. Linehan, D. L. Loriaux, and G. P. Chrousos. Responses
of the hypothalamic-pituitary-adrenal, and renin-angiotensin
axes and the sympathetic system during controlled surgical and
anesthetic stress. J. Clin. Endocrinol. Metab. 64: 986–994, 1987.
Victor, R. G., P. Thoren, D. A. Morgan, and A. L. Mark.
Differential control of adrenal and renal sympathetic nerve
activity during hemorrhagic hypotension in rats. Circ. Res. 64:
686–694, 1989.
Wittert, G. A., H. K. Or, J. H. Livesey, A. M. Richards, R. A.
Donald, and E. A. Espiner. Vasopressin, corticotrophinreleasing factor, and pituitary adrenal responses to acute cold
stress in normal humans. J. Clin. Endocrinol. Metab. 75: 750–
755, 1992.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.5 on June 14, 2017
18. Kvetnansky, R., D. S. Goldstein, V. K. Weise, C. Holmes, K.
Szemeredi, G. Bagdy, and I. J. Kopin. Effects of handling or
immobilization on plasma levels of dopa, catecholamines, and
metabolites in rats. J. Neurochem. 58: 2296–2302, 1992.
19. Kvetnansky, R., and L. Mikulaj. Adrenal and urinary catecholamines in rats during adaptation to repeated immobilization
stress. Endocrinology 87: 738–743, 1970.
20. Lachuer, J., S. Gaillet, B. Barbagli, M. Buda, and M.
Tappaz. Differential early time course activation of the brainstem catecholaminergic groups in response to various stresses.
Neuroendocrinology 53: 589–596, 1991.
21. Lee, H. B., and M. D. Blaufox. Blood volume in the rat. J. Nucl.
Med. 26: 72–76, 1985.
22. Mitrakou, A., M. Mokan, G. Boli, T. Veneman, T. Jenssen, P.
Cryer, and J. Gerich. Evidence against the hypothesis that
hyperinsulinemia increases sympathetic nervous system activity
in man. Metabolism 41: 198–200, 1992.
23. Mitrakou, A., C. Ryan, T. Veneman, T. Mokan, P. Jenssen, I.
Kiss, J. Durrant, P. Cryer, and J. Gerich. Hierarchy of
glycemic thresholds for counterregulatory hormone secretion,
symptoms, and cerebral dysfunction. Am. J. Physiol. 260 (Endocrinol. Metab. 29): E67–E74, 1991.
24. Morita, H., and S. F. Vatner. Effects of volume expansion on
renal nerve activity, renal blood flow and sodium and water
excretion in conscious dogs. Circ. Res. 57: 788–793, 1985.
25. Munck, A., P. M. Guyre, and N. J. Holbrook. Physiological
functions of glucocorticoids in stress and their relation to pharmacological actions. Endocrin. Rev. 5: 25–44, 1984.
26. Pacak, K., R. Kvetnansky, M. Palkovits, K. Fukuhara, G.
Yadid, I. J. Kopin, and D. S. Goldstein. Adrenalectomy
augment in vivo release of norepinephrine in the paraventricular
nucleus during immobilization stress. Endocrinology 133: 1404–
1410, 1993.
27. Robertson, D. A., G. A. Johnson, R. M. Robertson, A. S.
Nies, D. G. Shand, and J. A. Oates. Comparative assessment
R1255