Journal ofNeurochemistryRaven Press, Ltd., New York© 1994 International Society for Neurochemistry

Stress-Induced Sensitization of Dopamine and NorepinephrineEfflux in Medial Prefrontal Cortex of the Rat

*Paul J . Gresch, *tAlan F. Sved, *tMichael J . Zigmond, and *Janet M. Finlay

Departments of *Behavioral Neuroscience and tPsychiatry, and Centerfor Neuroscience, University of Pittsburgh,Pittsburgh, Pennsylvania, U.S.A .

Abstract : We examined whether prior exposure tochronic cold (17-28 days, 5°C) alters basal or stress-evoked (30-min tail shock) catecholamine release in me-dial prefrontal cortex, nucleus accumbens, and striatum,using in vivo microdialysis. Basal norepinephrine (NE)concentrations in medial prefrontal cortex did not differbetween chronically cold-exposed rats and naive controlrats (2 .7 ± 0.3 vs . 2.5 ± 0.2 pg/20 pl, respectively). Basaldopamine (DA) efflux in any of the brain regions was notsignificantly different between chronically cold-exposedrats and naive rats . However, atrend for lower basal DAefflux in the cold-exposed relative to naive rats was ob-served in medial prefrontal cortex (1 .5 ± 0.2 vs . 2.2 ± 0 .3pg/20 pl, respectively), nucleus accumbens (3 .7 ± 0.8 vs .5.4 ± 0.9 pg/20 pl, respectively), and striatum (4 .4 ± 0.5vs . 7 .2 ± 1 .5 pg/20 pl, respectively) . In medial prefrontalcortex of rats previously exposed to cold, tail shock elic-ited a greater increase from baseline in both DA and NEefflux relative to that measured in naive rats (DA, 2.3 ± 0.3vs . 1 .2 ± 0.1 pg, respectively ; NE, 3.8 ± 0.4 vs . 1 .4 ± 0 .2pg, respectively) . However, in nucleus accumbens or stri-atum of rats previously exposed to cold, the stress-in-duced increase in DA efflux was not significantly differentfrom that of naive rats (nucleus accumbens, 1 .8 ± 0.7 vs .1 .5 ± 0.3 pg, respectively ; striatum, 1 .9 ± 0.4 vs . 2.6± 0.7 pg, respectively) . Thus, both cortical NE projectionsand cortically projecting DA neurons sensitize afterchronic exposure to cold . In contrast, subcortical DA pro-jections do not sensitize under these conditions . KeyWords: Nucleus accumbens-Striatum-3,4-Dihy-droxyphenylacetic acid-Homovanillic acid-Microdia-lysis-Cold stress .J. Neurochem. 63,575-583 (1994) .

Stress-induced changes in the functioning of centralcatecholaminergic neurons are thought to contribute tothe behavioral response of the organism to that stressor(Anisman et al ., 1985) . In rats, acute exposure to astressor increases catecholamine turnover in tissuefrom various brain regions (Thierry et al ., 1968 ; Korfet al ., 1973 ; Fadda et al ., 1978 ; Dunn and File, 1983 ;Deutch et al ., 1985 ; Irwin et al ., 1986) . In addition,many laboratories have now reported that acute stressincreases extracellular norepinephrine (NE) and dopa-

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mine (DA) concentrations in several brain regions (Ab-ercrombie et al ., 1988, 1989 ; Imperato et al., 1990,1991 ; Rossetti et al ., 1990 ; Sorg and Kalivas, 1991 ;Britton et al ., 1992) .However, animals are also confronted with repeated

or prolonged exposure to stressors, and it is clear thatexposure to chronic stress can alter an animal's re-sponse to subsequent stressors (Anisman et al ., 1985) .For example, rats previously exposed to chronic stressexhibit a greater increase in NE turnover upon reexpo-sure to a stressor, a phenomenon referred to as stress-induced sensitization (Weiss et al ., 1975 ; Anisman andSklar, 1979 ; Cassens et al ., 1980 ; Irwin et al ., 1986 ;Adell et al ., 1988) . Furthermore, it has recently beenshown that chronic exposure to cold stress results inan enhanced efflux of NE in hippocampus and medialprefrontal cortex (mPFC) in response to an acute novelstressor (Finlay and Abercrombie, 1991 ; Nisenbaumet al ., 1991) . In contrast, reports of the effects ofchronic stress on DA turnover and efflux have beencontroversial . Several groups have reported that theacute stress-induced increase in DA metabolism inmPFC is unaltered by subsequent presentations of thestressor (Herman et al ., 1982 ; Deutch et al ., 1985) . Incontrast, others have observed that chronically stressedrats exhibit a larger increase in frontal cortical DAmetabolism upon subsequent exposure to a stressor(Blanc et al ., 1980 ; Kalivas and Duffy, 1989) . Further-more, recent reports suggest that chronic stress resultsin either habituation (Imperato et al ., 1992) or sensiti-zation (Doherty and Gratton, 1992) to the effects ofthe stressor on DA efflux in nucleus accumbens (NAS) .

Using in vivo microdialysis, we have now examinedthe effect of prior exposure to chronic stress on extra-

Received August 30, 1993 ; revised manuscript received December22, 1993 ; accepted January 6, 1994 .

Address correspondence and reprint requests to P. J. Gresch atDepartment of Behavioral Neuroscience, 446 Crawford Hall, Uni-versity of Pittsburgh, Pittsburgh, PA 15260, U.S.A .

Abbreviations used : DA, dopamine ; DOPAC, 3,4-dihydroxyphen-ylacetic acid; HVA, homovanillic acid ; mPFC, medial prefrontalcortex ; NAS, nucleus accumbens; NE, norepinephrine ; STR,striatum .

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cellular DA in response to presentation of an acutenovel stressor . Because the extracellular DA levels innigrostriatal, mesolimbic, and mesocortical dopamin-ergic projection fields respond differently to acutestress (Abercrombie et al ., 1989), we compared theeffects of chronic stress on these dopaminergic sys-tems . Furthermore, because mPFC receives a promi-nent noradrenergic and dopaminergic innervation, wealso compared the impact of chronic stress on NE andDA efflux in this region .

MATERIALS AND METHODS

Animals and chronic cold exposureMale Sprague-Dawley rats (Zivic-Miller Laboratory,

Pittsburgh, PA, U.S.A .) were housed two per cage in wiremesh cages in a colony room (ambient temperature, 22-23°C) . Naive control rats remained in the colony room for-1-2 weeks before probe implantation. During this time,control rats increased their body weight from an initialweight of 175-200 g to a final weight of -310 g . Ratssubjected to chronic cold exposure (chronic cold rats)weighed ^-100-125 g upon arrival . After 1 week in thecolony room, these rats were shaved and placed immediatelyinto a cold room (ambient temperature, 5 °C), where theywere housed singly in wire mesh cages for 17-28 days .While in the cold room, the rats increased their body weightfrom ^-175 g to -340 g. Under both housing conditions,food and water were available ad libitum and lights weremaintained on a 12-h light/dark cycle . All animal use proce-dures were in strict compliance with the National Institutesof Health Guidefor the Care and Use ofLaboratory Animalsand were approved by the Animal Care Committee at theUniversity of Pittsburgh .

Microdialysis probe construction andimplantation

Construction of microdialysis probes of concentric designand the microdialysis procedure were performed as describedpreviously (Abercrombie and Finlay, 1991) . The activelength of the dialysis membrane (molecular weight cutoff,6,000 ; o .d ., 250 pm ; Spectrum Medical Industries, LosAngeles, CA, U.S.A .) was restricted to 2.0 mm for probesimplanted in NAS and 4.0 mm for probes implanted in stria-tum (STR) and mPFC . Naive and chronic cold rats wereremoved from their cages and immediately anesthetized withEquithesin (3 .0 ml/kg i .p .) and placed in a stereotaxic frame .Microdialysis probes were implanted at the following coordi-nates, relative to bregma and dura: STR, AP +0.5, ML ±- 2 .5,DV -7 .0 ; NAS, AP +1 .7, ML _- 1 .2, DV -8.0 ; mPFC, AP+3.2, ML ±1.2, DV -6.0 (Paxinos and Watson, 1982) . Onlyone brain region per rat was examined.

Experimental protocolImmediately after implantation, the dialysis probe was

perfused continuously with an artificial cerebrospinal fluid(145 mM NaCl, 2.7 mM KCI, 1 .0 MM MgC12 , and 1 .2 mMCaC1 2 ) at a rate of 1 .5 pl/min . Approximately 18-24 h afterimplantation of the probe, dialysate collection was initiatedin conscious, freely moving rats . Fifteen-minute collectionperiods were used for probes implanted in NAS and STR,and 30-min intervals for probes located in mPFC. Dialysatewas collected for 1 h to establish baseline concentrations ofneurotransmitter efflux . Afterward, a cuff containing two

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stainless-steel electrodes was positioned on the base of thetail . Intermittent tail shocks were comprised of constant cur-rent pulses of 1 .0 mA intensity delivered for 1 s every 10 sfor a duration of 45 s ; this series was repeated every 5 minfor 30 min . Thus, a total of 30 shocks was delivered . Thecuff was then removed and poststress samples were collectedfor 1 h .

Neurochemical analysis of dialysate samplesFor probes implanted in NAS or STR, 20 pl of dialysate

was assayed for DA, 3,4-dihydroxyphenylacetic acid (DO-PAC), and homovanillic acid (HVA) content. Samples col-lected from probes implanted in mPFC (45 pl) were dividedinto two 20-p1 fractions ; one fraction was assayed for DA,DOPAC, and HVA, and the other for NE concentration .The dialysate was analyzed with reverse-phase HPLC withelectrochemical detection . The HPLC systems each con-sisted of an injector (Rheodyne model 7126, Cotati, CA,U.S .A ., or Waters model U6K, Milford, MA, U.S.A .), adual-piston pump (Waters model 510), an external pulsedampener (Scientific Systems ; model LP-21, State College,PA, U.S.A .), and a C-18 column (100 x 3 .2 mm, 3 pmpacking ; Applied Biosystems, Brownlee Labs, San Jose, CA,U.S .A .) .

Separation of DA, DOPAC, and HVA was achieved usinga mobile phase consisting of 100 mM sodium acetate, 100pM EDTA, 1 .6 mM sodium octyl sulfate, and 9% methanol(vol/vol) ; the mobile phase was adjusted to pH 4.1 withglacial acetic acid . The detector (Waters model 460) wasequipped with a glassy carbon working electrode set at apotential of +600 mV vs . a n Ag/AgCI reference electrode .Chromatograms were recorded and analyzed by a DynamaxHPLC method manager for the Macintosh (Rainin Instru-ments Co., Woburn, MA, U.S .A .) . Peak heights producedby oxidation of DA, DOPAC, and HVA were compared withthose produced by a standard . The limit of detection of theassay was -0.5 pg of DA/20 pl .

Separation of NE was achieved using a mobile phase con-sisting of 60 mM sodium phosphate, 75 p.M EDTA, 1 .36mM sodium octyl sulfate, and 3 .5% methanol (vol/vol) ; themobile phase was adjusted to pH 2.75 with 12 M HCI . NEcontent in the dialysate was measured using a coulometricdetector (model 5100a ; ESA, Inc., Bedford, MA, U.S.A .)configured with three electrodes in series . A model 5021conditioning cell (+260 mV) was placed immediately afterthe column, followed by a model 5011 high-sensitivity ana-lytical cell (first electrode at -210 mV; second electrode at+210 mV) . The signal generated at the second analyticalelectrode was recorded on a chart recorder (Houston Instru-ments, Austin, TX, U.S.A .) . Peak heights produced by oxida-tion of NE were measured and compared with those of astandard . The limit of detection was -0.5 pg of NE/20 pl .

Histological analysisAfter each experiment, the rat was given an overdose of

Equithesin and perfused intracardially with 10% formalin insaline . The brain was removed, sectioned, and stained withLuxol fast blue and Safranin-O . The placement of the dial-ysis probe was determined histologically . If any portion ofthe active membrane was determined to be outside the targetlocation, based on the coordinates from the atlas of Paxinosand Watson (1982), data from that animal were excluded .

Data analysisNeurochemical concentrations in the dialysate (not cor-

rected for in vitro recovery) were expressed as absolute con-

EFFECT OF CHRONIC STRESS ON NE AND DA EFFLUX

FIGA The effect of acute tail shock on extracellular NE in mPFCof naive and chronic cold rats expressed as the absolute concen-tration of NE/20 pl of dialysate (A) or the net change in NE frombaseline (B) . Thirty minutes of intermittent tail shock produceda significantly greater increase in the absolute concentration ofNE in chronic cold rats than naive rats [group x time interaction,F(4,72) = 9.05 ; p < 0.001] . In a similar manner, the stress-in-duced net increase in NE efflux from baseline was significantlygreater in chronic cold rats than naive rats [group x time interac-tion, F(4,72) = 9.05 ; p < 0.001] . Results are expressed as meanSEM ; n = 10/group . 'Significantly different from respective

within-group baseline at 60 min. tSignificantly different from na-ive controls (Tukey's HSD test; p < 0.05) .

centration (pg/20 lal) or as net changefrom baseline (calcu-lated by subtracting the mean of the baseline samples fromeach of the stress and poststress samples) . Comparisons ofbasal values were made by averaging the absolute concentra-tions measured during the final two baseline samples andcomparing the means of the control and chronic cold groupsby independent t test with Bonferroni corrections for multi-ple comparisons (10 comparisons, p < 0.005) (Kirk, 1982).When examining either the absolute concentration or netchange from baseline, the effect of tail shock on the concen-tration of NE, DA, DOPAC, and HVA in naive and chroniccold rats was determined by a two-way ANOVA with re-peated measures . The contribution of individual means to asignificant F ratio was then determined by Tukey's HSD test(p < 0.05) .

RESULTSEffect of cold exposure on NE, DA, DOPAC, andHVA concentrations in mPFC

Basal concentrations of NE in dialysate from mPFCof chronic cold rats were not significantly differentfrom those of naive rats [t(18) = -0.51 ; Fig. IA] .

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Thirty minutes of intermittent tail shock significantlyincreased the absolute concentration of NE in mPFCof both naive and chronic cold rats . The stress-inducedincrease in absolute concentration of NE was signifi-cantly greater in rats previously exposed to chroniccold than in naive rats . In a similar manner, when thedata were analyzed as net change from baseline, tailshock elicited a significantly greater net increase inextracellular NE in chronic cold rats than naive rats(3 .8 ± 0.4 pg vs . 1 .4 -- 0.2 pg above baseline, respec-tively) and this difference was sustained during thetwo poststress samples (Fig . 1B).

Basal concentrations of DA in dialysate from mPFCof chronic cold rats were not significantly differentfrom those of naive rats, although baseline values were31% lower in chronic cold rats [t(18) = 1 .79 ; Fig.2A] . Tail shock significantly increased the absoluteconcentration of DA in both naive and chronic coldrats and the absolute concentrations of DA achieved

FIG. 2. The effect of acute tail shock on extracellular DA in mPFCof naive and chronic cold rats expressed as the absolute concen-tration of DA/20 pl of dialysate (A) or the net change in DA frombaseline (B) . Thirty minutes of intermittent tail shock produceda significant increase in DA efflux in both groups [group x timeinteraction, F(4,72) = 3.74 ; p < 0.05] ; however, pairwise compar-isons revealed that the absolute DA concentration attained wasnot significantly different between the groups . In contrast, whendifferences in basal DA were considered, it was apparent that thestress-induced net increase in DA from baseline was significantlygreater in the chronic cold rats than naive rats [group x timeinteraction, F(4,72) = 3.81 ; p < 0.05] . Results are expressed asmean -! SEM ; n = 10/group . "Significantly different from respec-tive within-group baseline at 60 min . tSignificantly different fromnaive controls (Tukey's HSD test ; p < 0.05) .

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FIG. 3. The effect of acute tail shock on extracellular DOPAC and HVA in mPFC of naive and chronic cold rats expressed as theabsolute concentration of DOPAC/20 pI of dialysate (A), the net change in DOPAC from baseline (B), the absolute concentration ofHVA/20,uI of dialysate (C), or the net change in HVA from baseline (D). Thirty minutes of intermittent tail shock produced a significantincrease in the absolute concentration of extracellular DOPAC [time, F(4,72) = 19.4 ; p < 0.001] and there was not a significant differencebetween the two groups in their response to the stressor [group x time interaction, F(4,72) = 0.50; p > 0.05] . In a similar manner, tailshock produced asignificant net increase in DOPAC above baseline [time, F(4,72) = 19.43; p < 0.001] and there was not a significantdifference between the groups in the net change observed [group x time interaction, F(4,72) = 0.50; p > 0.05] . Because naive andchronic cold rats did not exhibit a difference in their response to stress, the means from the two groups were combined for post hocanalysis . Thirty minutes of intermittent tail shock produced a significant increase in the absolute concentration of HVA [group x timeinteraction, F(4,72) = 5.63; p < 0.01] . However, pairwise comparisons revealed no difference in the absolute concentration achievedby the two groups . In contrast, when the data were presented and analyzed as net change from baseline, it was apparent that ratspreviously exposed to cold exhibited a greater net increase in HVA [group x time interaction, F(4,72) = 5.63; p < 0.01] . Results areexpressed as mean ± SEM; n = 10/group . 'Significantly different from respective within-group baseline at 60 min. tSignificantlydifferent from naive controls (Tukey's HSD test ; p < 0.05) .

were not significantly different between the twogroups . However, when the data were analyzed as netchange from baseline, tail shock elicited a significantlygreater increase in DA concentration in chronic coldrats than in naive rats (2 .3 ± 0.3 pg vs . 1 .2 ± 0.1 pgincrease above baseline, respectively ; Fig . 2B) . Fur-thermore, in chronic cold rats, DA remained elevatedabove baseline during the two poststress samples,whereas in naive rats DA returned to baseline valuesby the second poststress sample .

Basal DOPAC and HVA concentrations in mPFCof chronic cold rats were not significantly differentfrom those of naive rats, although basal DOPAC andHVA values were 20 and 27% lower in chronic coldrats, respectively [t(18) = 0.77 and 1 .99, respectively ;Fig . 3A and C] . Tail shock increased DOPAC concen-trations to the same extent in both groups, regardlessof whether the data were analyzed as absolute concen-tration or net change from baseline (Fig . 3A and B) .

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In a similar manner, tail shock significantly increasedthe absolute concentration of HVA in both naive andchronic cold rats, and the absolute concentration ofHVA attained after tail shock was not significantlydifferent between the two groups (Fig . 3C) . However,when the HVA data were expressed as net changefrom baseline, tail shock elicited a significantly greaterincrease in HVA concentration in mPFC of chroniccold rats than in naive rats (324 ± 25 pg vs . 179 -!- 36pg increase from baseline, respectively ; Fig . 3D) . Thenet increase in HVA in chronic cold rats was signifi-cantly greater than that observed in naive rats duringboth poststress periods .

Effect of cold exposure on DA, DOPAC, andHVA concentrations in NAS and STR

Basal concentrations of DA in NAS and STR ofchronic cold rats were not significantly different fromthose of naive rats, although baseline values of DA in

EFFECT OF CHRONIC STRESS ON NE AND DA EFFLUX

FIG. 4. The effect of acute tail shock on extracellular DA in NASof naiveand chronic cold rats expressed as the absolute concen-tration of DA/20,uI of dialysate (A) or the net change in DA frombaseline (B). Thirty minutes of intermittent tail shock produceda significant increase in the absolute concentration of extracellu-lar DA [time, F(7,98) = 11 .59; p < 0.001] and there was not asignificant difference between the two groups in their responseto the stressor [group x time interaction, F(7,98) = 1 .25; p> 0.05] . In a similar manner, tail shock produced a significantnet increase in extracellular DA above baseline [time, F(7,98)= 11 .63; p < 0.001] and there was not a significant differencebetween the groups in the net change observed [group x timeinteraction, F(7,98) = 1 .25; p > 0.05] . Because naive and chroniccold rats did not exhibit a difference in their response to stress,the means from the two groups were combined for post hoccomparisons. Results expressed as mean ± SEM; n = 8/group.`Significantly different from combined group baseline at 30 min(Tukey's HSD test ; p < 0.05) .

chronic cold rats were 32% less than naive rats in NASand 40% less than naive rats in STR [t(14) = 1 .28 andt(12) = 1 .66, respectively ; Fig . 4A and 5A] . Tail shockincreased the concentration of DA in NAS and STRof both groups and there was no significant differencein the stress-induced efflux of DA in the two groupsregardless of whether the data were analyzed asabsolute concentration or net change (Figs . 4A, B and5A, B) .

Basal DOPAC and HVA concentrations in NAS andSTR of chronic cold rats were not significantly differ-ent from those of naive rats [NAS, t(14) = 2.77 and1 .62, respectively ; STR, t(12) = 0.61 and 0.04, respec-tively ; Figs . 6A, C and 7A, C] . Tail shock increasedextracellular DOPAC and HVA in NAS and STR tothe same extent in both groups regardless of whether

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the data were analyzed as absolute concentration ornet change (Figs . 6A through 7D) .

DISCUSSION

Our data indicate that in rats previously exposed tochronic cold, presentation of an acute novel stressorelicits a greater increase in DA efflux in mPFC. Thesedata are consistent with previous reports indicating thatchronic stress causes sensitization of DA metabolismin frontal cortex . For example, in rats subjected toisolation stress, acute foot shock elicits a greater in-crease in the DOPAC/DA ratio in frontal cortex (Blancet al ., 1980) . In a similar manner, repeated foot shockresults in a sensitized stress-induced increase in tissuelevels of DOPAC and HVA in prefrontal cortex (Kali-

FIG. 5. The effect of acute tail shock on striatal extracellularDA in naive and chronic cold rats expressed as the absoluteconcentration of DA/20 pl of dialysate (A) or the net change inDA from baseline (B). Thirty minutes of intermittent tail shockproduced a significant increase in the absolute concentration ofextracellular DA [time, F(7,84) = 13.59; p < 0.001] and therewas notasignificant difference between the two groups in theirresponse to the stressor [group x time interaction, F(7,84)= 0.76; p > 0.05] . In a similar manner, tail shock produced asignificant net increase in extracellular DA above baseline [time,F(7,84) = 13.62; p < 0.001] and there was not a significantdifference between the groups in the net change observed[group x time interaction, F(7,84) = 0.76; p > 0.05] . Becausenaive and chronic cold rats did not exhibit a difference in theirresponse to stress, the means from the two groups were com-bined for post hoc comparisons. Results are expressed as mean± SEM; n = 7/group. 'Significantly different from combinedgroup baseline at 30 min (Tukey's HSD test ; p < 0.05) .

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FIG. 6. The effect of acute tail shock on extracellular DOPAC and HVA in NAS of naive and chronic cold rats expressed as the absoluteconcentration of DOPAC/20 yI of dialysate (A), the net change in DOPAC from baseline (B), the absolute concentration of HVA/20 plof dialysate (C), or the net change in HVA from baseline (D). Thirty minutes of tail shock produced a significant increase in the absoluteconcentration of extracellular DOPAC [time, F(7,98) = 15.26 ; p < 0.001] and there was not a significant difference between the twogroups in their response to the stressor [group x time interaction, F(7,98) = 0.15 ; p > 0.051 . In a similar manner, tail shock produceda significant net increase in extracellular DOPAC above baseline [time, F(7,98) = 15.26 ; p < 0.001] and there was not a significantdifference between the groups in the net change observed [group x time interaction, F(7,98) = 0.15 ; p > 0.05] . Thirty minutes ofintermittent tail shock produced a significant increase in the absolute concentration of extracellular HVA [time, F(7,98) = 21 .14 ; p< 0.001] and there was not a significant difference between the two groups in their response to the stressor [group x time interaction,F(7,98) = 0.45 ; p > 0.05] . In a similar manner, tail shock produced a significant net increase in extracellular HVA above baseline [time,F(7,98) = 21 .14 ; p < 0.001] and there was not a significant difference between the groups in the net change observed [group x timeinteraction, F(7,98) = 0.45 ; p > 0.05] . Because naive and chronic cold rats did not exhibit a difference in their response to stress, themeans of the two groups were combined for the post hoc comparisons. Results are expressed as mean -- SEM; n = 8/group.*Significantly different from combined group baseline at 30 min (Tukey's HSD test ; p < 0.05) .

vas and Duffy, 1989) . Thus, our data extend theseobservations by demonstrating that stress-induced sen-sitization of DA metabolism in frontal cortex is alsoassociated with a stress-induced sensitization of DAefflux .

In our experiments, sensitization of DA efflux inmPFC was associated with an enhanced stress-in-duced increase in HVA efflux . In contrast, DOPACefflux was not significantly sensitized after chroniccold exposure, although a trend toward an enhancedresponsiveness was observed in the chronic cold rats .Together, these data suggest that extracellular levelsof DA metabolites reflect the stress-induced sensitiza-tion of DA efflux in mPFC.

In contrast, stress-induced sensitization of DA effluxwas not observed in NAS or STR, suggesting thatchronic cold differentially influences subpopulationsof midbrain DA neurons . The absence of an effect ofchronic cold on evoked DA efflux in STR agrees with

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the finding that prior daily foot shock does not alterstriatal DA metabolism in response to subsequent footshocks (Kalivas and Duffy, 1989) . However, our ob-servation that chronic exposure to cold did not affectstress-evoked DA efflux in NAS contrasts with previ-ous reports in which chronic stress results in habitua-tion (Imperato et al ., 1992) or sensitization (Kalivasand Duffy, 1989; Doherty and Gratton, 1992) to theeffects of the stressor on DA metabolism and effluxin NAS. This discrepancy may be due to the nature ofthe stress paradigm . For example, we examined theimpact of a continuous stressor (cold) on the responseelicited by a subsequent presentation of an acute novelstressor (tail shock), whereas previous experiments ex-amined the impact of daily intermittent stress (restraintstress or foot shock) on the response elicited by asubsequent presentation of the same stressor . In con-trast to a continuous stressor, intermittent stress mayelicit discrete responses associated with presentation

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FIG. 7 . The effect of acute tail shock on extracellular DOPAC and HVA in STR of naive and chronic cold rats expressed as the absoluteconcentration of DOPAC/20 pI of dialysate (A), the net change in DOPAC from baseline (B), the absolute concentration of HVA/20 pIof dialysate (C), or the net change in HVA from baseline (D) . Thirty minutes of intermittent tail shock produced a significant increasein the absolute concentration of extracellular DOPAC [time, F(7,84) = 7.47 ; p < 0.01] and there was not a significant difference betweenthe two groups in their response to the stressor [group x time interaction, F(7,84) = 1 .12 ; p > 0.05] . In a similar manner, tail shockproduced a significant net increase in extracellular DOPAC above baseline [time, F(7,84) = 7.47 ; p < 0.01] and there was not asignificant difference between the groups in the net change observed [group x time interaction, F(7,84) = 1 .12 ; p > 0.05] . Thirtyminutes of intermittent tail shock produced a significant increase in the absolute concentration of extracellular HVA [time, F(7,84)= 7 .51 ; p < 0.01] and there was not a significant difference between the groups in their response to the stressor [group x timeinteraction, F(7,84) = 1 .16 ; p > 0.05] . In a similar manner, tail shock produced a significant net increase in extracellular HVA abovebaseline [time, F(7,84) = 7.51 ; p < 0.01] and there was not a significant difference between the groups in the net change observed[group x time interaction, F(7,84) = 1 .16 ; p > 0.05] . Because naive and chronic cold rats did not exhibit a difference in their responseto stress, the means from the two groups were combined for post hoc analysis . Results are expressed as mean ± SEM ; n = 7/group .*Significantly different from combined group baseline at 30 min (Tukey's HSD test ; p < 0.05) .

and subsequent removal of the stressor. Indeed, in re-sponse to repeated immobilization stress, the stress-induced increase in DA efflux in NAS habituates,whereas the increase in DA that occurs after releasefrom the stressor does not (Imperato et al ., 1992) .

In our study, prior chronic exposure to cold resultsin an enhanced NE efflux in mPFC in response to acutetail shock. These data extend the previous observationsfrom this laboratory that cold exposure results in sensi-tization of NE efflux in hippocampus in response toacute tail shock (Nisenbaum et al., 1991) as well asenhanced NE efflux in mPFC in response to acute tailpressure (Finlay and Abercrombie, 1991) . Together,these data suggest that NE-containing projections fromthe locus ceruleus to cortical regions respond in a ho-mogenous manner to chronic stress .The stress-induced sensitization of NE efflux in

mPFC was evident as both a greater increase in theabsolute concentration of NE attained in response to

an acute novel stressor and a greater net increase in NEfrom baseline . In contrast, sensitization of DA efflux inmPFC was apparent only when the data were expressedas a net change from baseline values, in part, becausebasal values of DA in mPFC of chronic cold rats weresomewhat lower than those of naive rats . Determiningthe net change in DA efflux from baseline may be anappropriate index ofthe responsivity of the DA neuron .However, the significance to the postsynaptic cell ofa greater absolute concentration or a greater net changein catecholamine concentration remains to be deter-mined (J . M. Finlay, M. J . Zigmond, and E. D . Aber-crombie, submitted) .

Several hypotheses may be proposed to explain re-gional differences in the response of DA neurons tocold stress . First, the stress-induced sensitization ofDA efflux from mesocortical DA neurons may be aconsequence of the distinct properties of the mesocorti-cal DA projection (Wolf and Roth, 1987b) . For exam-

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ple, relative to DA systems projecting to subcorticalregions, the mesocortical neurons exhibit (1) a higherfiring rate and increased bursting (White and Wang,1983 ; Chiodo et al., 1984), (2) a higher rate of DAturnover in the terminals (Bannon et al ., 1981, 1983),(3) reduced responsiveness to DA agonists and antago-nists (Bannon et al ., 1983), (4) a lack of tolerance torepeated administration of DA antagonists (Bacopou-lous et al ., 1982), and (5) resistance to neuroleptic-induced depolarization block (Chiodo and Bunney,1983 ; White and Wang, 1983 ; for reviews, see Bannonand Roth, 1983 ; Wolf and Roth, 19ß7a) . These uniquecharacteristics of mesocortical DA neurons are thoughtto be, in part, a function of the absence of synthesis-and impulse-modulating autoreceptors on these neu-rons (Wolf and Roth, 19ß7a) . It is possible that theabsence of these regulatory mechanisms also providesthe neurobiological basis for the expression of stress-induced sensitization of DA efflux in mPFC.A second hypothesis is that exposure to cold stress

selectively activates afferents projecting to the cellbodies of mesocortical DA neurons in ventral tegmen-tal area but not to mesolimbic and nigrostriatal DAneurons . Tachykinin peptide-containing neurons havereceived considerable attention for their possible rolein activating ventral tegmental area DA neurons . Forexample, substance P-containing neuronal input mayselectively regulate DA neurons projecting to cortex,whereas substance K-containing neuronal input mayactivate mesolimbic DA neurons (Deutch and Roth,1990) . In addition to tachykinin peptides, other neuro-transmitters, such as opiates and excitatory aminoacids, have been implicated in the stress-induced acti-vation of the prefrontal DA system (see Deutch andRoth, 1990 ; Kalivas, 1993, for reviews) .

Finally, a third hypothesis is that stress-induced sen-sitization of DA efflux in mPFC may be due to thepresence of local interactions that are different fromthose found in the terminal regions of the subcorticalDA projections . We are particularly interested in therole that NE efflux, which is also sensitized in chroniccold animals, may play in the expression of the en-hanced DA efflux in mPFC. Such an interaction mayexplain the regional variation in the development ofsensitization in DA neurons in that both NE and DAare present in similar concentrations in mPFC, whereasthe concentration of DA in subcortical regions is muchgreater than NE (Versteeg et al ., 1976) . Indeed, thereis recent evidence to suggest that NE terminals inmPFC may be involved in regulating extracellular DAin this region (Carboni et al., 1990 ; Gresch et al .,1993) . In a similar manner, local factors may playa role in the expression of sensitized NE efflux . Forexample, in rats previously exposed to chronic cold,local application of 30 mM K+ evokes an enhancedefflux of NE in the hippocampus (Nisenbaum and Ab-ercrombie, 1993) . It is noteworthy that local factorsare also thought to play a role in the expression ofamphetamine-induced sensitization of DA efflux (for

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P. J . GRESCH ET AL.

a review, see Kalivas and Stewart, 1991) . Furthermore,there have been several reports of cross-sensitizationbetween amphetamine and stress on neurotransmitterrelease and behavior (Kalivas and Stewart, 1991), thusimplying that a similar mechanism may be involved.

In summary, our results demonstrate that whereasacute tail shock increases DA efflux in mPFC, NAS,and STR, chronic exposure to cold results in sensitiza-tion of DA efflux only from mesocortical DA neurons .These data suggest that midbrain DA neurons mayrespond in a heterogenous manner to chronic stress .In contrast, this study together with previous workfrom our laboratory suggest that cortically projectingNE neurons respond in a homogenous manner tochronic stress .

Acknowledgment: We thank J.-S. Yen for the histologi-cal preparations and B . Vojta for helpful comments on themanuscript. This work was supported by U.S . Public HealthService grants MH43947 andMH45156 and research grantsfrom the Tourette Syndrome Association and Scottish RiteSchizophrenia Research Program. J. M. F. is a PostdoctoralFellow of the Medical Research Council of Canada . Thesestudies were conducted while A. F. S. was supported by anEstablished Investigator Award from the American HeartAssociation.

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