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Gender differences in sympathoadrenal activity in rats at rest and in response to footshock stress
\
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 05\ No[ 2:3\ pp[ 178Ð184\ 0887 Þ 0887 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:87 ,08[99¦9[99
PII ] S9625Ð4637"87#99910Ð4
GENDER DIFFERENCES IN SYMPATHOADRENAL ACTIVITY IN RATS AT REST AND IN RESPONSE TO FOOTSHOCK STRESS MARTA WEINSTOCK\ $ MICHAL RAZIN\$ DONNA SCHORER!APELBAUM\$ DISHENG MEN% and RICHARD McCARTY% $ Department of Pharmacology\ Hebrew University Hadassah Medical Centre\ Ein Kerem\ Jerusalem\ 80019\ Israel ^ % Department of Psychology\ Gilmer Hall\ University of Virginia\ Charlottesville\ Virginia 11892\ U[S[A[ "Received 14 September 0886 ^ revised 07 November 0886 ^ accepted 08 November 0886# Abstract*A comparison was made of the dynamics of sympathoadrenal activity in 00 age!matched male and female rats\ under basal conditions and after exposure to footshock[ Rats were prepared with indwelling catheters in the tail artery 13 h before the experiment[ Measurements were made of plasma corticosterone "COR#\ norepinephrine "NE#\ epinephrine "EPI#\ dihydroxyphenylalanine "DOPA#\ dihydroxyphenylglycol "DHPG# and dihydroxyphenylacetic acid "DOPAC# under resting conditions\ after transfer to the shock box "novelty# and at various times after footshock[ Under basal conditions\ males have signi_cantly higher blood pressure and plasma DHPG:NE ratios but lower plasma levels of COR\ NE and DOPAC than females[ Three min after exposure to the shock chamber "novelty stress# there were signi_cant increases in COR\ EPI\ NE and DHPG in both sexes\ while DOPA increased only in females and DOPAC remained unchanged in both sexes[ Footshock produced a further increase in EPI\ NE and DOPAC within 1 min\ which lasted about 04 min[ There were signi_cant sex di}erences in the extent and duration of the response of COR\ EPI and DHPG[ The data show that the female sympathoadrenal system is more reactive than that of the male to the stresses of a novel environment and footshock[ The smaller DHPG:NE ratios in females at rest and after stress suggest that neuronal uptake of NE is lower in females than in males[ The _nding that stress produces larger increments of plasma DOPA and DOPAC in female rats indicates that tyrosine hydroxylase in the sympathetic nerve terminals and adrenal medulla may also be higher than in males[ Þ 0887 ISDN[ Published by Elsevier Science Ltd Key words] plasma corticosterone\ norepinephrine\ epinephrine\ novelty stress\ footshock\ males\ females[
Activation of the hypothalamic!pituitary adrenal "HPA# and sympathetic nervous systems occurs in response to emotional and intimidating stimuli[ The rate and intensity of activation of these systems can be followed by measuring corticosterone "COR# and catecholamines in the peripheral circulation[ The levels of norepinephrine "NE# in plasma are the net e}ect of transmitter release and its removal by neuronal uptake and clearance[1 An assessment of the in~uence of neuronal uptake can be made by measurement of the change in plasma levels of dihydroxyphenylglycol "DHPG#\ which is derived from capture of released NE into the neuron and from some leakage from storage vesicles[1 The degree of activation of tyrosine hydroxylase in nerve terminals and the adrenal medulla induced by the stressor may be re~ected by increases in plasma dihydroxyphenylalanine "DOPA#\ the precursor of dopamine\ NE and epinephrine "EPI#[3 Dihydroxyphenyl acetic acid "DOPAC# in plasma may depend on the synthesis of dopamine in nerve terminals and may also give some indirect indication of tyrosine hydroxylase activation[7 Although many studies have examined the response of rats to di}erent forms of stress\ such as exposure to a novel environment\ immobilization and footshock\ these have almost all been per! formed in males[ One study\ that compared changes in plasma COR and catecholamines in male and female SpragueÐDawley rats subjected to restraint\ found sex di}erences with marked individual variations in each group\ even though the animals were of the same weight and from the same supplier[8 The aim of the present study was to compare blood pressure\ plasma catechols and COR in male and female rats under basal conditions and in response to exposure to novelty and footshock[ The rats were of the same age and were housed and reared under identical conditions from weaning in an attempt to reduce potential sources of variation[
To whom all correspondence should be addressed[ Tel[ ] 99861 1 5647620 ^ Fax ] 99861 1 5320983[ 178
189
M[ Weinstock et al[
MATERIALS AND METHODS Animals Experiments were performed in 11 SpragueÐDawley rats\ aged 3[4Ð4 months\ of which 5 pairs of males and females were littermates\ while the remainder were born to di}erent mothers\ but were of the same age[ The rats were anesthetized with tribomoethanol "0 ml:kg# i[p4 and prepared with tail artery catheters as described by Chieuh and Kopin[0 Brie~y\ a small midline incision was made through the ventral tail sheath near the base of the tail[ The ventral tail artery was exposed and cannulated with PE 49 tubing which was led under the tail sheath and skin to exit in the midscapular region[ This was covered with a stainless steel extensions spring wire and secured to each rat with an adhesive tape collar[ The catheter was _lled with heparinized saline "299 I[U[:ml# and the end of the tubing attached to a 12 g needle and tuberculin syringe[ Rats were housed in individual cages\ the spring wire and catheter were fed out of the top of the cage at the center and secured to allow the rat complete freedom of movement[ Patency of the cannula was maintained by ~ushing with 9[4 ml of the heparinized saline early in the morning on the day of the experiment[ Experimental protocol A baseline blood sample "0[4 ml# was taken in each animal between 97]04 and 98]29 h\ with a minimum of disturbance[ The sample was centrifuged for 1 min at 01\999 rpm\ the plasma removed and stored at −69>C until assayed[ After this\ and each subsequent blood sample\ an equal volume of heparinized saline was added to the red cells which were slowly infused back into the rat to maintain blood volume[ The rats were transferred to the experimental room and allowed to rest for 04 min before their mean arterial blood pressure "MAP# and heart rates "HR# were measured[ They were then placed individually into the shock chamber and a further blood sample taken after 2 min[ Three footshocks 0[4 mA\ 3 s duration were delivered at 14 s intervals and another blood sample was taken immediately after the last shock[ Two further samples were collected 4 and 04 min later[ Each animal was then returned to its home cage and another blood sample collected 34 min after the footshock[ At 89 min after footshock a _nal blood sample of 9[4 ml was taken for measurement of COR only[ Vaginal smears were performed on all females at the end of the experiment to determine the stage of the estrous cycle[ Assays Catechols\ NE\ EPI\ DOPA\ DOPAC and DHPG were extracted from 9[1Ð9[4 ml plasma by batch alumina extraction and measured by high performance liquid chromatography with electrochemical detection[5 COR was measured by radio!immunoassay using a rabbit antiserum raised against corticosterone!10!thyroglobulin "Biomakor\ Kiryat Weizmann\ Rehovot\ Israel# and ð0\1\5\6Ł!2H! N!corticosterone\ "9[905 Ci:mmol# "New England Nuclear\ Boston\ MA\ USA#[ The sensitivity of the COR assay was 4 ng:ml[ Statistical analyses The results are expressed as means2sem[ Resting values of MAP\ HR\ and plasma levels of COR\ catecholamines and metabolites were analysed by ANOVA and Levine|s test for equality of variances[ Since it was found that the variances of the concentrations of COR and catecholamines were higher in the females than in the males\ gender di}erences between mean values were analysed by the non parametric Mann!Whitney test[ The response to transfer to the shock chamber and to footshock was analysed by ANOVA for repeated measures\ for TIME and GENDER[ The criterion of signi_cance was set at P ³ 9[94[ RESULTS Differences in baseline levels of catechols and corticosterone in males and females Although rats were the same age\ males were about 049 g heavier than females "Table 1#[ Seven of the females in the study were found to be in estrous and four in diestrous on the day of the study[
Stress\ gender and sympathoadrenal activity
180
Table 0[ Resting values for MAP\ HR and sympatho!adrenal indices in female rats at two di}erent stages of the estrous cycle Parameter
Estrous "6#
Diestrous "3#
MAP "mm Hg# HR "beats:min# Plasma COR "ng:ml# Plasma NE "pg:ml# Plasma EPI "pg:ml# DHPG "pg:ml# DOPA "pg:ml# DOPAC "pg:ml#
87[222[1 342202 150239 530252 149250 7622038 523274 509243
75[925[2 300216 035255 7612071 297282 8112113 5452010 570275
Table 1[ Resting values for MAP\ HR plasma catecholamines and corticosterone in male and female rats Parameter
Males "00#
Females "00#
Body weight "g# MAP "mm Hg# HR "beats:min# Plasma COR "ng:ml# Plasma NE "pg:ml# Plasma EPI "pg:ml# DHPG "pg:ml# DHPG:NE DOPA "pg:ml# NE:DOPA DOPAC "pg:ml#
39328 00123 308219 65[1202[4 221237 034225 627246 1[4229[17 412228 9[5129[00 470242
14122 8322 333201 085239 688279 168248 7582019 0[0629[10 534252 0[7229[15 798233
Signi_cantly di}erent from males\ P ³ 9[94[
No signi_cant di}erences were found in MAP\ HR or in the resting plasma levels of COR\ NE\ EPI\ DOPAC\ DOPA or DHPG in the females at the two stages of the estrous cycle "Table 0#[ The data for females were therefore pooled and compared with those of the males[ The resting values for all the above parameters in male and female rats are shown in Table 1[ MAP was signi_cantly higher in males than in females\ but HR did not di}er[ Plasma levels of COR\ NE and DOPAC were signi_cantly higher\ and the ratio of DHPG:NE lower in females than in males[ The scatter of the values of most of the substances except DOPAC was considerably greater in females than in males[ This may have been due to the four females that were in diestrous\ since the values for the rats in estrous were less variable and their standard deviations more closely resembled those in males[ The fact that it took several more minutes each time to collect su.cient blood for the assays in the females because of their smaller size may also have contributed to the higher variability[ Signi_cant correlations were only found between the resting plasma levels of COR and that of DHPG\ r 9[379\ P ³ 9[914 ^ and between plasma NE and EPI\ r 9[568\ P ³ 9[9994[ Sympathoadrenal activation in response to exposure to novelty Exposure of the rats to the novel chamber in which they were to received footshock resulted in signi_cant increases in NE\ EPI\ COR and DHPG in rats of both genders within 2 min[ DOPAC increased in females only and DOPA did not change signi_cantly in either group "Fig[ 2#[ The increments in the ratio of plasma DHPG:NE in response to the novel environment were 0[0129[98 in males and 9[4129[07 in females\ "P ³ 9[94#[ Sympathoadrenal activation in response to exposure to footshock Plasma concentrations of NE\ EPI and the catechols increased further within 1 min of the completion of footshock in both groups[ COR increased in some rats after 4 min and in others after 04 min[ The time course of the changes in plasma levels of all the substances are shown in Figs 0Ð
181
M[ Weinstock et al[
Fig[ 0[ Time course for plasma corticosterone in response to novelty and footshock stress in male and female rats[ Open circles\ males ^ closed circles\ females[ First arrow indicates time of transfer to shockbox ^ second arrow indicates application of footshock[ Repeated measures ANOVA\ TIME\ F5\05 20[3\ P ³ 9[990 ^ GENDER ( TIME\ F5\05 4[77\ P ³ 9[91[ Increment in COR in response to novelty stress is signi_cantly greater in females than in males\ P ³ 9[94[ No signi_cant di}erence in the increment of plasma COR at 4 min in response to footshock in the two sexes[
2[ Repeated measures ANOVA revealed a signi_cant e}ect of TIME for all substances measured and signi_cant TIME ( GENDER interactions for EPI\ DHPG\ and COR[ The areas under the curve of plasma COR over time were signi_cantly di}erent in males and females ^ 9[4829[97 and 0[5729[11 "mg:ml:89 min#\ respectively "P ³ 9[990#[ Those for NE were 0[8829[12 "ng:ml:34 min# for males and 1[8029[81 for females\ "NS# and for EPI\ 1[7229[17 for males and 3[5029[31 for females\ "P ³ 9[90#[ Exposure to the shock box\ resulted in a further signi_cant increment in plasma DHPG in males and of DOPA in females[ The increase in DOPAC was signi_cantly higher in females than in males "P ³ 9[94#[ There were signi_cant correlations between the increments in EPI and NE "r 9[649\ P ³ 9[9994#\ EPI and DHPG "r 9[363\ P ³ 9[94# and EPI and DOPAC "r 9[364\ P ³ 9[94# in response to footshock[ The resting levels of COR\ NE or EPI did not predict the magnitude of the response to shock or the area under the curve of plasma concentrations over time[ DISCUSSION The present study found that adult age!matched males had signi_cantly higher MAP than females at rest but lower concentrations of COR and NE[ This is at variance with the _nding of Livezey et al[8 in which there were no di}erences in basal COR and NE in males and females of a similar weight "but presumably of a di}erent age#[ However\ the resting plasma concentration of COR in their males was about three times higher than that in the present study and is in the range usually seen after stress[09\01 On the other hand\ their concentrations of NE and EPI were about half those in the present study[ This may have been due to the method of collection through the jugular vein\ since the values in our study are the same as those in a previous report in SpragueÐDawley rats in which the amines were measured in arterial blood[00 Like Livezey et al[\8 we found a signi_cantly greater variability in the concentrations of NE\ EPI and COR in females than in males[ This also applied to plasma levels of DHPG and DOPA\ which were measured in the present study as indices of NE reuptake and tyrosine hydroxylase activity\ respectively[7 This higher variability remained after excluding the four females that were in diestrous\ which showed higher variances than the majority of the rats which were in estrous[ Since DHPG levels did not di}er in the two sexes at rest\ while those of NE in males were more than double those in females\ it is possible that in the latter\
Stress\ gender and sympathoadrenal activity
182
Fig[ 1[ Time course for plasma NE and EPI in response to novelty and footshock stress in male and female rats[ Open circles\ males ^ closed circles\ females[ First arrow indicates time of transfer to shockbox ^ second arrow indicates application of footshock[ Repeated measures ANOVA\ NE ] TIME\ F4\05 10[8\ P ³ 9[990 ^ GENDER ( TIME\ F4\05 0[29\ NS[ EPI ] TIME\ F4\05 22[6\ P ³ 9[990 ^ GENDER ( TIME\ F4\05 1[77\ P ³ 9[94[ No signi_cant gender di}erence in plasma NE in response to novelty stress or to footshock[ No signi_cant gender di}erence in plasma EPI in response to novelty stress\ but the increment in EPI in response to footshock is insigni_cantly greater in females than in males\ P ³ 9[94[
leakage of NE from vesicles and:or its neuronal uptake may be lower[ This could be veri_ed by determining the e}ect of desipramine on plasma NE and DHPG levels in females[2 Handling of rats and transfer to a novel environment "the chamber in which they were to receive electric footshock# produced an increase of similar magnitude in NE and EPI within 2 min in rats of both sexes[ The _nding of a greater rise in COR in females than in males after exposure to novelty is in agreement with other reports[6\01 On the other hand\ the increment in DHPG was signi_cantly higher in males\ which suggests that a greater proportion of NE released in response to novelty was recaptured by nerve terminals than in females[ The plasma concentrations of NE and EPI were highly correlated after transfer to the shock box but bore no relationship to that of COR\ indicating a disparate activation of the sympathetic and HPA systems by novelty stress[ Plasma levels of the catechols and COR were still elevated following exposure to the shock box when electric footshock was applied to the rats[ This may be why we did not see clear increases in COR in response to the shock\ since its release is slower than that of the catecholamines and plasma
183
M[ Weinstock et al[
Fig[ 2[ Time course for plasma DOPA\ DOPAC and DHPG in response to novelty and footshock stress in male and female rats[ Open circles\ males ^ closed circles\ females[ First arrow indicates time of transfer to shockbox ^ second arrow indicates application of footshock[ Repeated measures ANOVA\ DHPG ] TIME\ F4\05 7[53\ P ³ 9[90 ^ GENDER ( TIME\ F4\05 1[57\ P 9[95[ DOPA ] TIME\ F4\05 6[93\ P ³ 9[94 ^ GENDER×TIME\ F4\05 9[55\ NS[ DOPAC ] TIME\ F4\05 03[36\[ P ³ 9[990 ^ GENDER ( TIME\ F4\05 0[76\ NS[ Signi_cantly greater increment in plasma DHPG in male than in females in response to novelty stress\ P ³ 9[94 ^ no di}erence in response to footshock[ Plasma DOPA levels only increased signi_cantly above after novelty stress and footshock in females[ Plasma DOPAC did not change signi_cantly in either sex in response to novelty stress but increased more in females than in males 4 min after footshock\ P ³ 9[94[
Stress\ gender and sympathoadrenal activity
184
levels were still increasing in response to the novel situation 4 min previously[ Thus further increases occurred in some of the rats 4 min\ and in others\ 04 min after shock\ and returned to baseline concentrations in most of them by 89 min[ By contrast\ footshock produced signi_cantly greater increments in EPI and DOPAC than after transfer\ in spite of the fact that their plasma concentrations were already elevated[ These increments in EPI and DOPAC were signi_cantly higher in females than in males[ Footshock resulted in further increments in plasma NE in both sexes that were similar to those seen after exposure to the novel situation[ This study demonstrates that female rats show greater individual variation than males in their concentrations of plasma catechols and COR\ and have higher basal levels of COR and NE[ On the other hand\ they have a lower DHPG:NE ratio\ which suggest that proportionally less NE is taken back into nerve terminals than in males[ In spite of this\ their blood pressures were lower[ The _nding of higher plasma concentrations of EPI and COR in females than in males indicated that the female adrenal gland is more reactive to the stress of a novel environment and footshock[ The higher increments of plasma DOPA and DOPAC produced by these stressors in female rats also supports the suggestion of increased activity of tyrosine hydroxylase in the sympathetic nerve terminals and adrenal medulla[ REFERENCES 0[ Chieuh\ C[ C[ and Kopin\ I[ J[\ Hyperresponsivity of spontaneously hypertensive rat to indirect measurement of blood pressure[ Am[ J[ Physiol[\ 0867\ 123\ H589ÐH584[ 1[ Eisenho}er\ G[\ Goldstein\ D[ S[ and Kopin\ I[ J[\ Plasma dihydroxyphenglycol for estimation of noradrenaline neuronal reuptake in the sympathetic nervous system in vivo[ Clin[ Sci[\ 0878\ 65\ 060Ð071[ 2[ Eisenho}er\ G[\ Pacak\ K[\ Goldstein\ D[ S[ and McCarty\ R[\ Neuronal uptake and sympathoadrenal release of catecholamines in aged rats[ In Stress ] Molecular Genetic and Neurobiolo`ical Advances\ ed R[ McCarty\ G[ Aguilera\ E[ Sabban and R[ Kvetnansky[ Gordon and Breach Science Publishers S[A[\ New York\ 0885\ pp[ 838Ð854[ 3[ Goldstein\ D[ S[\ Udelsman\ R[\ Eisenho}er\ G[\ Stull\ R[\ Keiser\ H[ R[ and Kopin\ I[ J[\ Neuronal sources of plasma dihydroxyphenylalanine[ J[ Clin[ Endocr[ Metab[\ 0876\ 53\ 745Ð750[ 4[ Herskowitz\ M[\ Eliash\ S[ and Cohen\ S[\ The muscarinic cholinergic receptors in the posterior hypothalamus of hypertensive and normotensive rats[ Eur[ J[ Pharmacol[\ 0871\ 75\ 118Ð125[ 5[ Holmes\ C[\ Eisenho}er\ G[ and Goldstein\ D[ S[\ Improved assay for plasma dihydroxphenylacetic acid and other catechols using high performance liquid chromatography with electrochemical detection[ J[ Liquid Chromato`r[\ 0883\ B542\ 020Ð027[ 6[ Kitay\ J[ I[\ Sex di}erences in adrenal cortical secretion in the rat[ Endocrinol[\ 0850\ 57\ 707Ð713[ 7[ Kvetnansky\ R[\ Armando\ I[\ Weise\ V[ K[\ Holmes\ C[\ Fukuhara\ K[\ Deka!Starosta\ A[\ Kopin\ I[ J[ and Goldstein\ D[ S[\ Plasma DOPA responses during stress ] dependence on sympathoneural activity and tyrosine hydroxylation[ J[ Pharmacol[ Exp[ Ther[\ 0881\ 150\ 788Ð898[ 8[ Livezey\ G[ T[\ Miller\ J[ M[ and Vogel\ W[ H[\ Plasma norepinephrine\ epinephrine and corticosterone stress responses to restraint in individual male and female rats\ and their correlations[ Neurosci[ Lett[\ 0874\ 51\ 40Ð45[ 09[ Maccari\ S[\ Piazza\ P[ V[\ Deminiere\ J[ M[\ Lemaire\ V[\ Mormede\ P[\ Simon\ H[\ Angelucci\ L[ and Le Moal\ M[\ Life events!induced decrease of corticosteroid type I receptors is associated with reduced corticosterone feedback and enhanced vulnerability to amphetamine self!administration[ Brain Res[\ 0880\ 436\ 6Ð01[ 00[ McCarty\ R[ and Kopin\ I[ J[\ Sympatho!adrenal medullary activity and behavior during exposure to footshock stress ] a comparison of seven rat strains[ Physiol[ Behav[\ 0867\ 10\ 456Ð461[ 01[ Weinstock\ M[\ Matlina\ E[\ Maor\ G[ I[\ Rosen\ H[ and McEwen\ B[ S[\ Prenatal stress selectively alters the reactivity of the hypothalamic!pituitary adrenal system in the female rat[ Brain res[\ 0881\ 484\ 084Ð199\
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 05\ No[ 2:3\ pp[ 178Ð184\ 0887 Þ 0887 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:87 ,08[99¦9[99
PII ] S9625Ð4637"87#99910Ð4
GENDER DIFFERENCES IN SYMPATHOADRENAL ACTIVITY IN RATS AT REST AND IN RESPONSE TO FOOTSHOCK STRESS MARTA WEINSTOCK\ $ MICHAL RAZIN\$ DONNA SCHORER!APELBAUM\$ DISHENG MEN% and RICHARD McCARTY% $ Department of Pharmacology\ Hebrew University Hadassah Medical Centre\ Ein Kerem\ Jerusalem\ 80019\ Israel ^ % Department of Psychology\ Gilmer Hall\ University of Virginia\ Charlottesville\ Virginia 11892\ U[S[A[ "Received 14 September 0886 ^ revised 07 November 0886 ^ accepted 08 November 0886# Abstract*A comparison was made of the dynamics of sympathoadrenal activity in 00 age!matched male and female rats\ under basal conditions and after exposure to footshock[ Rats were prepared with indwelling catheters in the tail artery 13 h before the experiment[ Measurements were made of plasma corticosterone "COR#\ norepinephrine "NE#\ epinephrine "EPI#\ dihydroxyphenylalanine "DOPA#\ dihydroxyphenylglycol "DHPG# and dihydroxyphenylacetic acid "DOPAC# under resting conditions\ after transfer to the shock box "novelty# and at various times after footshock[ Under basal conditions\ males have signi_cantly higher blood pressure and plasma DHPG:NE ratios but lower plasma levels of COR\ NE and DOPAC than females[ Three min after exposure to the shock chamber "novelty stress# there were signi_cant increases in COR\ EPI\ NE and DHPG in both sexes\ while DOPA increased only in females and DOPAC remained unchanged in both sexes[ Footshock produced a further increase in EPI\ NE and DOPAC within 1 min\ which lasted about 04 min[ There were signi_cant sex di}erences in the extent and duration of the response of COR\ EPI and DHPG[ The data show that the female sympathoadrenal system is more reactive than that of the male to the stresses of a novel environment and footshock[ The smaller DHPG:NE ratios in females at rest and after stress suggest that neuronal uptake of NE is lower in females than in males[ The _nding that stress produces larger increments of plasma DOPA and DOPAC in female rats indicates that tyrosine hydroxylase in the sympathetic nerve terminals and adrenal medulla may also be higher than in males[ Þ 0887 ISDN[ Published by Elsevier Science Ltd Key words] plasma corticosterone\ norepinephrine\ epinephrine\ novelty stress\ footshock\ males\ females[
Activation of the hypothalamic!pituitary adrenal "HPA# and sympathetic nervous systems occurs in response to emotional and intimidating stimuli[ The rate and intensity of activation of these systems can be followed by measuring corticosterone "COR# and catecholamines in the peripheral circulation[ The levels of norepinephrine "NE# in plasma are the net e}ect of transmitter release and its removal by neuronal uptake and clearance[1 An assessment of the in~uence of neuronal uptake can be made by measurement of the change in plasma levels of dihydroxyphenylglycol "DHPG#\ which is derived from capture of released NE into the neuron and from some leakage from storage vesicles[1 The degree of activation of tyrosine hydroxylase in nerve terminals and the adrenal medulla induced by the stressor may be re~ected by increases in plasma dihydroxyphenylalanine "DOPA#\ the precursor of dopamine\ NE and epinephrine "EPI#[3 Dihydroxyphenyl acetic acid "DOPAC# in plasma may depend on the synthesis of dopamine in nerve terminals and may also give some indirect indication of tyrosine hydroxylase activation[7 Although many studies have examined the response of rats to di}erent forms of stress\ such as exposure to a novel environment\ immobilization and footshock\ these have almost all been per! formed in males[ One study\ that compared changes in plasma COR and catecholamines in male and female SpragueÐDawley rats subjected to restraint\ found sex di}erences with marked individual variations in each group\ even though the animals were of the same weight and from the same supplier[8 The aim of the present study was to compare blood pressure\ plasma catechols and COR in male and female rats under basal conditions and in response to exposure to novelty and footshock[ The rats were of the same age and were housed and reared under identical conditions from weaning in an attempt to reduce potential sources of variation[
To whom all correspondence should be addressed[ Tel[ ] 99861 1 5647620 ^ Fax ] 99861 1 5320983[ 178
189
M[ Weinstock et al[
MATERIALS AND METHODS Animals Experiments were performed in 11 SpragueÐDawley rats\ aged 3[4Ð4 months\ of which 5 pairs of males and females were littermates\ while the remainder were born to di}erent mothers\ but were of the same age[ The rats were anesthetized with tribomoethanol "0 ml:kg# i[p4 and prepared with tail artery catheters as described by Chieuh and Kopin[0 Brie~y\ a small midline incision was made through the ventral tail sheath near the base of the tail[ The ventral tail artery was exposed and cannulated with PE 49 tubing which was led under the tail sheath and skin to exit in the midscapular region[ This was covered with a stainless steel extensions spring wire and secured to each rat with an adhesive tape collar[ The catheter was _lled with heparinized saline "299 I[U[:ml# and the end of the tubing attached to a 12 g needle and tuberculin syringe[ Rats were housed in individual cages\ the spring wire and catheter were fed out of the top of the cage at the center and secured to allow the rat complete freedom of movement[ Patency of the cannula was maintained by ~ushing with 9[4 ml of the heparinized saline early in the morning on the day of the experiment[ Experimental protocol A baseline blood sample "0[4 ml# was taken in each animal between 97]04 and 98]29 h\ with a minimum of disturbance[ The sample was centrifuged for 1 min at 01\999 rpm\ the plasma removed and stored at −69>C until assayed[ After this\ and each subsequent blood sample\ an equal volume of heparinized saline was added to the red cells which were slowly infused back into the rat to maintain blood volume[ The rats were transferred to the experimental room and allowed to rest for 04 min before their mean arterial blood pressure "MAP# and heart rates "HR# were measured[ They were then placed individually into the shock chamber and a further blood sample taken after 2 min[ Three footshocks 0[4 mA\ 3 s duration were delivered at 14 s intervals and another blood sample was taken immediately after the last shock[ Two further samples were collected 4 and 04 min later[ Each animal was then returned to its home cage and another blood sample collected 34 min after the footshock[ At 89 min after footshock a _nal blood sample of 9[4 ml was taken for measurement of COR only[ Vaginal smears were performed on all females at the end of the experiment to determine the stage of the estrous cycle[ Assays Catechols\ NE\ EPI\ DOPA\ DOPAC and DHPG were extracted from 9[1Ð9[4 ml plasma by batch alumina extraction and measured by high performance liquid chromatography with electrochemical detection[5 COR was measured by radio!immunoassay using a rabbit antiserum raised against corticosterone!10!thyroglobulin "Biomakor\ Kiryat Weizmann\ Rehovot\ Israel# and ð0\1\5\6Ł!2H! N!corticosterone\ "9[905 Ci:mmol# "New England Nuclear\ Boston\ MA\ USA#[ The sensitivity of the COR assay was 4 ng:ml[ Statistical analyses The results are expressed as means2sem[ Resting values of MAP\ HR\ and plasma levels of COR\ catecholamines and metabolites were analysed by ANOVA and Levine|s test for equality of variances[ Since it was found that the variances of the concentrations of COR and catecholamines were higher in the females than in the males\ gender di}erences between mean values were analysed by the non parametric Mann!Whitney test[ The response to transfer to the shock chamber and to footshock was analysed by ANOVA for repeated measures\ for TIME and GENDER[ The criterion of signi_cance was set at P ³ 9[94[ RESULTS Differences in baseline levels of catechols and corticosterone in males and females Although rats were the same age\ males were about 049 g heavier than females "Table 1#[ Seven of the females in the study were found to be in estrous and four in diestrous on the day of the study[
Stress\ gender and sympathoadrenal activity
180
Table 0[ Resting values for MAP\ HR and sympatho!adrenal indices in female rats at two di}erent stages of the estrous cycle Parameter
Estrous "6#
Diestrous "3#
MAP "mm Hg# HR "beats:min# Plasma COR "ng:ml# Plasma NE "pg:ml# Plasma EPI "pg:ml# DHPG "pg:ml# DOPA "pg:ml# DOPAC "pg:ml#
87[222[1 342202 150239 530252 149250 7622038 523274 509243
75[925[2 300216 035255 7612071 297282 8112113 5452010 570275
Table 1[ Resting values for MAP\ HR plasma catecholamines and corticosterone in male and female rats Parameter
Males "00#
Females "00#
Body weight "g# MAP "mm Hg# HR "beats:min# Plasma COR "ng:ml# Plasma NE "pg:ml# Plasma EPI "pg:ml# DHPG "pg:ml# DHPG:NE DOPA "pg:ml# NE:DOPA DOPAC "pg:ml#
39328 00123 308219 65[1202[4 221237 034225 627246 1[4229[17 412228 9[5129[00 470242
14122 8322 333201 085239 688279 168248 7582019 0[0629[10 534252 0[7229[15 798233
Signi_cantly di}erent from males\ P ³ 9[94[
No signi_cant di}erences were found in MAP\ HR or in the resting plasma levels of COR\ NE\ EPI\ DOPAC\ DOPA or DHPG in the females at the two stages of the estrous cycle "Table 0#[ The data for females were therefore pooled and compared with those of the males[ The resting values for all the above parameters in male and female rats are shown in Table 1[ MAP was signi_cantly higher in males than in females\ but HR did not di}er[ Plasma levels of COR\ NE and DOPAC were signi_cantly higher\ and the ratio of DHPG:NE lower in females than in males[ The scatter of the values of most of the substances except DOPAC was considerably greater in females than in males[ This may have been due to the four females that were in diestrous\ since the values for the rats in estrous were less variable and their standard deviations more closely resembled those in males[ The fact that it took several more minutes each time to collect su.cient blood for the assays in the females because of their smaller size may also have contributed to the higher variability[ Signi_cant correlations were only found between the resting plasma levels of COR and that of DHPG\ r 9[379\ P ³ 9[914 ^ and between plasma NE and EPI\ r 9[568\ P ³ 9[9994[ Sympathoadrenal activation in response to exposure to novelty Exposure of the rats to the novel chamber in which they were to received footshock resulted in signi_cant increases in NE\ EPI\ COR and DHPG in rats of both genders within 2 min[ DOPAC increased in females only and DOPA did not change signi_cantly in either group "Fig[ 2#[ The increments in the ratio of plasma DHPG:NE in response to the novel environment were 0[0129[98 in males and 9[4129[07 in females\ "P ³ 9[94#[ Sympathoadrenal activation in response to exposure to footshock Plasma concentrations of NE\ EPI and the catechols increased further within 1 min of the completion of footshock in both groups[ COR increased in some rats after 4 min and in others after 04 min[ The time course of the changes in plasma levels of all the substances are shown in Figs 0Ð
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Fig[ 0[ Time course for plasma corticosterone in response to novelty and footshock stress in male and female rats[ Open circles\ males ^ closed circles\ females[ First arrow indicates time of transfer to shockbox ^ second arrow indicates application of footshock[ Repeated measures ANOVA\ TIME\ F5\05 20[3\ P ³ 9[990 ^ GENDER ( TIME\ F5\05 4[77\ P ³ 9[91[ Increment in COR in response to novelty stress is signi_cantly greater in females than in males\ P ³ 9[94[ No signi_cant di}erence in the increment of plasma COR at 4 min in response to footshock in the two sexes[
2[ Repeated measures ANOVA revealed a signi_cant e}ect of TIME for all substances measured and signi_cant TIME ( GENDER interactions for EPI\ DHPG\ and COR[ The areas under the curve of plasma COR over time were signi_cantly di}erent in males and females ^ 9[4829[97 and 0[5729[11 "mg:ml:89 min#\ respectively "P ³ 9[990#[ Those for NE were 0[8829[12 "ng:ml:34 min# for males and 1[8029[81 for females\ "NS# and for EPI\ 1[7229[17 for males and 3[5029[31 for females\ "P ³ 9[90#[ Exposure to the shock box\ resulted in a further signi_cant increment in plasma DHPG in males and of DOPA in females[ The increase in DOPAC was signi_cantly higher in females than in males "P ³ 9[94#[ There were signi_cant correlations between the increments in EPI and NE "r 9[649\ P ³ 9[9994#\ EPI and DHPG "r 9[363\ P ³ 9[94# and EPI and DOPAC "r 9[364\ P ³ 9[94# in response to footshock[ The resting levels of COR\ NE or EPI did not predict the magnitude of the response to shock or the area under the curve of plasma concentrations over time[ DISCUSSION The present study found that adult age!matched males had signi_cantly higher MAP than females at rest but lower concentrations of COR and NE[ This is at variance with the _nding of Livezey et al[8 in which there were no di}erences in basal COR and NE in males and females of a similar weight "but presumably of a di}erent age#[ However\ the resting plasma concentration of COR in their males was about three times higher than that in the present study and is in the range usually seen after stress[09\01 On the other hand\ their concentrations of NE and EPI were about half those in the present study[ This may have been due to the method of collection through the jugular vein\ since the values in our study are the same as those in a previous report in SpragueÐDawley rats in which the amines were measured in arterial blood[00 Like Livezey et al[\8 we found a signi_cantly greater variability in the concentrations of NE\ EPI and COR in females than in males[ This also applied to plasma levels of DHPG and DOPA\ which were measured in the present study as indices of NE reuptake and tyrosine hydroxylase activity\ respectively[7 This higher variability remained after excluding the four females that were in diestrous\ which showed higher variances than the majority of the rats which were in estrous[ Since DHPG levels did not di}er in the two sexes at rest\ while those of NE in males were more than double those in females\ it is possible that in the latter\
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Fig[ 1[ Time course for plasma NE and EPI in response to novelty and footshock stress in male and female rats[ Open circles\ males ^ closed circles\ females[ First arrow indicates time of transfer to shockbox ^ second arrow indicates application of footshock[ Repeated measures ANOVA\ NE ] TIME\ F4\05 10[8\ P ³ 9[990 ^ GENDER ( TIME\ F4\05 0[29\ NS[ EPI ] TIME\ F4\05 22[6\ P ³ 9[990 ^ GENDER ( TIME\ F4\05 1[77\ P ³ 9[94[ No signi_cant gender di}erence in plasma NE in response to novelty stress or to footshock[ No signi_cant gender di}erence in plasma EPI in response to novelty stress\ but the increment in EPI in response to footshock is insigni_cantly greater in females than in males\ P ³ 9[94[
leakage of NE from vesicles and:or its neuronal uptake may be lower[ This could be veri_ed by determining the e}ect of desipramine on plasma NE and DHPG levels in females[2 Handling of rats and transfer to a novel environment "the chamber in which they were to receive electric footshock# produced an increase of similar magnitude in NE and EPI within 2 min in rats of both sexes[ The _nding of a greater rise in COR in females than in males after exposure to novelty is in agreement with other reports[6\01 On the other hand\ the increment in DHPG was signi_cantly higher in males\ which suggests that a greater proportion of NE released in response to novelty was recaptured by nerve terminals than in females[ The plasma concentrations of NE and EPI were highly correlated after transfer to the shock box but bore no relationship to that of COR\ indicating a disparate activation of the sympathetic and HPA systems by novelty stress[ Plasma levels of the catechols and COR were still elevated following exposure to the shock box when electric footshock was applied to the rats[ This may be why we did not see clear increases in COR in response to the shock\ since its release is slower than that of the catecholamines and plasma
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Fig[ 2[ Time course for plasma DOPA\ DOPAC and DHPG in response to novelty and footshock stress in male and female rats[ Open circles\ males ^ closed circles\ females[ First arrow indicates time of transfer to shockbox ^ second arrow indicates application of footshock[ Repeated measures ANOVA\ DHPG ] TIME\ F4\05 7[53\ P ³ 9[90 ^ GENDER ( TIME\ F4\05 1[57\ P 9[95[ DOPA ] TIME\ F4\05 6[93\ P ³ 9[94 ^ GENDER×TIME\ F4\05 9[55\ NS[ DOPAC ] TIME\ F4\05 03[36\[ P ³ 9[990 ^ GENDER ( TIME\ F4\05 0[76\ NS[ Signi_cantly greater increment in plasma DHPG in male than in females in response to novelty stress\ P ³ 9[94 ^ no di}erence in response to footshock[ Plasma DOPA levels only increased signi_cantly above after novelty stress and footshock in females[ Plasma DOPAC did not change signi_cantly in either sex in response to novelty stress but increased more in females than in males 4 min after footshock\ P ³ 9[94[
Stress\ gender and sympathoadrenal activity
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levels were still increasing in response to the novel situation 4 min previously[ Thus further increases occurred in some of the rats 4 min\ and in others\ 04 min after shock\ and returned to baseline concentrations in most of them by 89 min[ By contrast\ footshock produced signi_cantly greater increments in EPI and DOPAC than after transfer\ in spite of the fact that their plasma concentrations were already elevated[ These increments in EPI and DOPAC were signi_cantly higher in females than in males[ Footshock resulted in further increments in plasma NE in both sexes that were similar to those seen after exposure to the novel situation[ This study demonstrates that female rats show greater individual variation than males in their concentrations of plasma catechols and COR\ and have higher basal levels of COR and NE[ On the other hand\ they have a lower DHPG:NE ratio\ which suggest that proportionally less NE is taken back into nerve terminals than in males[ In spite of this\ their blood pressures were lower[ The _nding of higher plasma concentrations of EPI and COR in females than in males indicated that the female adrenal gland is more reactive to the stress of a novel environment and footshock[ The higher increments of plasma DOPA and DOPAC produced by these stressors in female rats also supports the suggestion of increased activity of tyrosine hydroxylase in the sympathetic nerve terminals and adrenal medulla[ REFERENCES 0[ Chieuh\ C[ C[ and Kopin\ I[ J[\ Hyperresponsivity of spontaneously hypertensive rat to indirect measurement of blood pressure[ Am[ J[ Physiol[\ 0867\ 123\ H589ÐH584[ 1[ Eisenho}er\ G[\ Goldstein\ D[ S[ and Kopin\ I[ J[\ Plasma dihydroxyphenglycol for estimation of noradrenaline neuronal reuptake in the sympathetic nervous system in vivo[ Clin[ Sci[\ 0878\ 65\ 060Ð071[ 2[ Eisenho}er\ G[\ Pacak\ K[\ Goldstein\ D[ S[ and McCarty\ R[\ Neuronal uptake and sympathoadrenal release of catecholamines in aged rats[ In Stress ] Molecular Genetic and Neurobiolo`ical Advances\ ed R[ McCarty\ G[ Aguilera\ E[ Sabban and R[ Kvetnansky[ Gordon and Breach Science Publishers S[A[\ New York\ 0885\ pp[ 838Ð854[ 3[ Goldstein\ D[ S[\ Udelsman\ R[\ Eisenho}er\ G[\ Stull\ R[\ Keiser\ H[ R[ and Kopin\ I[ J[\ Neuronal sources of plasma dihydroxyphenylalanine[ J[ Clin[ Endocr[ Metab[\ 0876\ 53\ 745Ð750[ 4[ Herskowitz\ M[\ Eliash\ S[ and Cohen\ S[\ The muscarinic cholinergic receptors in the posterior hypothalamus of hypertensive and normotensive rats[ Eur[ J[ Pharmacol[\ 0871\ 75\ 118Ð125[ 5[ Holmes\ C[\ Eisenho}er\ G[ and Goldstein\ D[ S[\ Improved assay for plasma dihydroxphenylacetic acid and other catechols using high performance liquid chromatography with electrochemical detection[ J[ Liquid Chromato`r[\ 0883\ B542\ 020Ð027[ 6[ Kitay\ J[ I[\ Sex di}erences in adrenal cortical secretion in the rat[ Endocrinol[\ 0850\ 57\ 707Ð713[ 7[ Kvetnansky\ R[\ Armando\ I[\ Weise\ V[ K[\ Holmes\ C[\ Fukuhara\ K[\ Deka!Starosta\ A[\ Kopin\ I[ J[ and Goldstein\ D[ S[\ Plasma DOPA responses during stress ] dependence on sympathoneural activity and tyrosine hydroxylation[ J[ Pharmacol[ Exp[ Ther[\ 0881\ 150\ 788Ð898[ 8[ Livezey\ G[ T[\ Miller\ J[ M[ and Vogel\ W[ H[\ Plasma norepinephrine\ epinephrine and corticosterone stress responses to restraint in individual male and female rats\ and their correlations[ Neurosci[ Lett[\ 0874\ 51\ 40Ð45[ 09[ Maccari\ S[\ Piazza\ P[ V[\ Deminiere\ J[ M[\ Lemaire\ V[\ Mormede\ P[\ Simon\ H[\ Angelucci\ L[ and Le Moal\ M[\ Life events!induced decrease of corticosteroid type I receptors is associated with reduced corticosterone feedback and enhanced vulnerability to amphetamine self!administration[ Brain Res[\ 0880\ 436\ 6Ð01[ 00[ McCarty\ R[ and Kopin\ I[ J[\ Sympatho!adrenal medullary activity and behavior during exposure to footshock stress ] a comparison of seven rat strains[ Physiol[ Behav[\ 0867\ 10\ 456Ð461[ 01[ Weinstock\ M[\ Matlina\ E[\ Maor\ G[ I[\ Rosen\ H[ and McEwen\ B[ S[\ Prenatal stress selectively alters the reactivity of the hypothalamic!pituitary adrenal system in the female rat[ Brain res[\ 0881\ 484\ 084Ð199\