Brain Research 908 (2001) 197–203www.elsevier.com/ locate /bres

Research report

Delayed stress-induced increase in tissue level of cholecystokinin inrat prefrontal cortex: modulation by microdialysis probe implantation

and systemic ketaminea , b c a* ¨Diana Radu , Ernst Brodin , Gunther Weber , Nils Lindefors

aDepartment of Clinical Neuroscience, Psychiatry Section, Karolinska Institute, SE-171 76 Stockholm, SwedenbDepartment of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden

cCenter for Molecular Medicine, Karolinska Institute, Stockholm, Sweden

Accepted 30 May 2001

Abstract

In the brain, the neuropeptide cholecystokinin (CCK) appears to be involved in the mediation of stress responses. Here we provide newevidence that mild stress induces long-term changes in CCK-like immunoreactivity (CCK-LI) in the prefrontal cortex (PFC). The changesin CCK-LI show a biphasic pattern, with a decrease 20 min after and an increase 8 h after mild stress. These changes seem to be regionspecific. Measurement of CCK mRNA in prefrontal cortex neurons 4 or 8 h after the stress stimulus did not reveal changes in mRNAlevels, suggesting that afferent CCK-containing neuron terminals may be more affected than local cortical CCK-ergic neurons.Furthermore, treatment with the glutamate NMDA receptor antagonist ketamine, led to more pronounced decreases in CCK-LI observedwithin 20 min after mild stress and counteracted the stress induced increase in cortical CCK-LI levels observed at 8 h. Implantation of amicrodialysis probe in the PFC affected the response to mild stress, with no significant decrease in the CCK-LI level 20 min after, andattenuated reactivity to stress 8 h after the saline injection. Our results indicate that a mild stressful stimulus such as an intraperitonealsaline injection may have long-lasting effects on CCK-ergic transmission in the PFC. The use of microdialysis to study stress induced invivo CCK-LI release in awake animals may, however, be significantly compromised by the impact of the microdialysis probe implantationon CCK-ergic mechanisms in the PFC. In addition, we hypothesize that subanesthetic doses of the psychotomimetic drug ketamineinterfere with CCK-ergic mechanisms in the PFC during stress. 2001 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Peptides: anatomy and physiology

Keywords: Brain; Cholecystokinin; Ketamine; Microdialysis; Prefrontal cortex; Stress

1. Introduction behavior and reactions to stress stimuli has been docu-mented in both human and animal studies [6,11]. In

Cholecystokinin (CCK) is a brain-gut neuropeptide humans, systemic bolus injection of a peptide consisting ofdistributed particularly within the cerebral cortex in ani- the C-terminal four to five amino acids of CCK producemals and man [8,18]. CCK or its mRNA is co-localized anxiety in a CCK-B receptor antagonist-sensitive mannerwith e.g. glutamate and GABA in the cerebral cortex [3]. In animals the CCK-ergic system seems to be very[5,14,17] and with dopamine in mesocortical neurons sensitive to experimental handling and mild stress [4,15].projecting from the mesencephalon to the prefrontal cortex The PFC is an important region in the mediation of(PFC) [13]. A role for CCK in adaptive and social reactions to stress and several previous reports deal with

the effects of stressful stimuli on extracellular levels andtissue levels of CCK and CCK mRNA in the PFC.*Corresponding author. Tel.: 146-8-5177-5598; fax: 146-8-5177-However, results reported from animal experiments cor-5550.

E-mail address: diana.radu@ks.se (D. Radu). relating extracellular peptide levels from microdialysis in

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02648-8

198 D. Radu et al. / Brain Research 908 (2001) 197 –203

vivo with peptide levels from tissue extracts and tissue 2.2. Surgical procedure for implantation of microdialysislevels of mRNA in the PFC are not easy to compare. In guide cannulaaddition, use of different stressors reduces the possibilityof comparing results from such experiments. Further, Rats were anesthetized with halothane and placed in aapparently inconsistent results can be caused by differ- stereotaxic frame. The skull was fixed with blunt earbars.ences in preexperimental handling, seasonal variations After exposing the skull, a burr hole (0.8 mm in diameter)[15], age, gender and strain [9]. was drilled at the following co-ordinates: AP 12.7, L 22.3

Microdialysis studies in the PFC show acute increases in [24]. A guide cannula containing a ‘dummy probe’ wasthe release of CCK-like immunoreactivity (CCK-LI) after lowered into the right PFC DV 23.4 at an angle of 308.restraint or exposure to dietylether [21]. Long-lasting The guide cannula was secured in place with two anchorincreases (up to 4 h) are described after systemic or local screws and dental cement. A 1% lidocaine spray wasadministration of the anxiogenic drug yohimbine [21]. administered locally and the wound was closed with oneAdministration of N-methyl-D-aspartate (NMDA) in the stitch of 4.0 Ethicon. Animals were allowed to recover forperfusate also leads to an increase in CCK-LI release 2 days in individual cages. On the 3rd postoperative day,during the first 30 min [19] and pre-treatment with the dummy probe was removed under halothane anesthesiaanxiolytic agents such as benzodiazepine [21] or 5-HT1A and replaced with a microdialysis probe (CMA 12, CMAreceptor agonists [2] attenuate this increase. Microdialys, Solna, Sweden). Animals were allowed to

Rats exposed to foot shock for 30 min show increased recover for 1 or 24 h after probe implantation beforetissue levels of CCK-LI in the PFC [30]. Increased tissue receiving an i.p. saline injection.levels are also found in rats exposed for 15 min to catsmell [23]. Subcutaneous [27] or intraperitoneal (i.p.) 2.3. Animals used for CCK-LI analysessaline injection [25] or isolation [11] have no acute effect(up to 30 min) on tissue levels of CCK in PFC. Adminis- A total of 175 rats were used, grouped as follows withtration of the NMDA receptor antagonist ketamine by i.p. seven rats in each group: groups 1–8: rats administeredinjection has no effect on tissue levels of CCK in PFC saline or ketamine, respectively, and decapitated after 5,when compared to a saline injected group [25]. Long-term 10, 20 or 480 min (S , S , S , S , K , K , K and5 10 20 480 5 10 20

isolation [7] or repeated restraint stress [10] has been K , respectively); group 9: non-injected non-operated480

reported to have little or no effect on CCK mRNA levels controls (C) decapitated during the same time interval asin rat cortex, but leads to increases in CCK mRNA in the animals in groups S , S , S , K , K , and K ;5 10 20 5 10 20

subcortical regions. group 10: non-injected non-operated controls (C ) de-480

Our aims in the present set of experiments were to capitated during the same time interval as the animals inestimate changes in tissue levels of CCK-LI and CCK groups S and K ; groups 11–14: animals having a480 480

mRNA in the PFC at time points following mild stress. We microdialysis guide cannula operated into the right pre-also wanted to evaluate the possibility of using in vivo frontal cortex receiving saline injection 1 h after probemicrodialysis to measure the effect of stress on the release implantation and decapitated at 5, 10, 20 or 480 min afterof CCK-LI in this brain region. In addition, the possible the i.p. saline injection (S1 , S1 , S1 andOP5 OP10 OP20

effects of the glutamate NMDA receptor antagonist S1 ); groups 15–18: animals having a microdialysisOP480

ketamine on stress related CCK-ergic mechanisms were guide cannula operated into the right prefrontal cortexexamined in the PFC. Ketamine effect was considered of receiving saline injection 24 h after probe implantation andrelevance since glutamate appears to modulate CCK-ergic decapitated at 5, 10, 20 or 480 min after the i.p. salinemechanisms in this part of the brain [1,19]. injection (S24 , S24 , S24 and S24 ); groupOP5 OP10 OP20 OP480

19: control animals (C1 ) having a microdialysis guideOP

cannula operated into the right prefrontal cortex decapi-tated during the same time interval as animals in groups

2. Methods S1 , S1 and S1 ; group 20: control animalsOP5 OP10 OP20

(C24 ) having a microdialysis guide cannula operatedOP

2.1. Animals into the right prefrontal cortex decapitated during the sametime interval as animals in groups S24 , S24 andOP5 OP10

Male Sprague–Dawley rats (ALAB Sweden) weighing S24 ; group 21: control animals (C1 ) having aOP20 OP480

250–300 g at delivery were housed five per cage at microdialysis guide cannula operated into the right pre-constant room temperature of 218C with unlimited access frontal cortex decapitated during the same time interval asto food and water and a 12-h light–dark cycle. Animals animals in group S1 ; group 22: control animalsOP480

were allowed to accommodate for 5 days before the (C24 ) having a microdialysis guide cannula operatedOP480

experiments. The experiments were approved by the local into the right prefrontal cortex decapitated during the sameethical committee, ethical approval: Dnr N188/98, date time interval as animals in group S24 ; groups 23–25:OP480

8 /27 /98. non-injected non-operated controls (C, C1 , and C24 )480 480

D. Radu et al. / Brain Research 908 (2001) 197 –203 199

35for groups C1 and C24 C1 , and C24 (Pharmacia) to obtain a [ S]UTP (Amersham) labeledOP OP, OP480 OP480

respectively. riboprobe complementary to mRNA coding for the ratAnimals were anesthetized for 2 min in a CO chamber CCK peptide.2

prior to decapitation. The studied brain regions were Sections were fixed in 4% formaldehyde for 5 min,microdissected on ice using a brain blocker to acquire deproteinated for 15 min in 0.2 M HCl, treated in 0.25%1-mm slices in a standardized fashion. A circular micro acetic anhydride solution for 20 min and dehydrated in apunch was used to dissect nucleus accumbens and series of ethanol including a 5-min chloroform step prior toamygdala, and a scalpel for the prefrontal cortex, caudatus- hybridization. The sections were incubated at 558C for 16putamen, hippocampus and entorhinal cortex, according to h with a hybridization buffer containing 50% formamide,Paxinos and Watson brain atlas [24] and rapidly frozen in 20% 503 dextran sulphate, 6.7% 5 M sodium chloride, 4%pre-weighed 2-ml Eppendorf tubes on dry ice. 5 M DTT, 2% 1 M Tris–HCl pH 7.6, 2% 503 Denhardt’s

solution, 2% yeast tRNA (25 mg/ml), 13.3 ml H O262.4. Animals used for in situ hybridization containing 10310 cpm probe per ml hybridization buffer.

After hybridization, the sections were washed in 43 SSC,A total of 30 rats were used, grouped as follows with six treated with RNase 10 mg/ml at 378C for 30 min and

rats in each group. Rats were decapitated 4, or 8 h after i.p. washed in 23 SSC, 13 SSC and 0.53 SSC for 10 minsaline or ketamine administration. Rats receiving no treat- each, 0.13 SSC at 608C, and 0.13 SSC at room tempera-ment served as controls. Brains were immediately removed ture for 5 min. The sections were dehydrated in gradedand frozen on dry ice. series of ethanol, air-dried and exposed to X-ray film

(B-Max, Amersham). The developed films were scanned2.5. Tissue extracts using a flat bed scanner with transparency adapter, ScanJet

6100 C/T (HP) and analyzed using Scion Image forTissue extraction was first performed in 10 vol. of Windows, Beta 4.02 version (Scion).

distilled water. Samples were homogenized using a Poly- One-way ANOVA followed by Tukey’s H.S.D. test wastron PT 1200, incubated at 1008C for 10 min and cen- used for the statistical analysis of data (Statistica 5.5,trifuged at 3000 rpm. The pellets were then resuspended in StatSoft Scandinavia, Uppsala, Sweden).1 M acetic acid at 1008C. The supernatants from thedistilled water and acid extraction were pooled and dried ina Speed-Vac, resuspended in 1 ml 0.15 M NaCl containing0.2% BSA and used for radioimmunoassay, diluted 1:10. 3. Results

2.6. Radioimmunoassay 3.1. Effects of i.p. saline and ketamine injection in non-operated animals

Samples were analyzed using antiserum 2609 [26],125[ I]]gastrin as radioligand and synthetic CCK8 as stan- Intraperitoneal saline injection led to a significant de-

dard. The total incubation volume was 1.1 ml and the crease in the PFC tissue levels of CCK-LI at 20 min aftersample volume was 0.1 ml. The radioligand and diluted administration (F 54.4752, P50.041239, Tukey’s6, 42

antiserum were added to the samples in 0.5 ml barbital H.S.D. test) and a significant increase at 8 h after adminis-buffer (0.02 M, pH 8.6) containing 0.8% BSA. Samples tration (F 511.060, P50.000829, Tukey’s H.S.D. test)2, 18

were incubated at 48C for 72 h and separated by adding 0.5 in PFC CCK-LI. Levels at 5 and 10 min after salineml of sheep anti-rabbit IgG coupled to Sepharose solution injection were not different from non-injected controls.(decanting suspension NR 3, Pharmacia), followed by Ketamine treated rats displayed significant decreases inincubation at 48C for 30 min and centrifugation (10003g, CCK-LI at 5 and 20 min after injection (F 54.4752,6, 42

10 min, 48C). The supernatants were discarded and pellets P50.042174 and P50.001340, respectively, Tukey’swere counted 10 min each in a gamma counter. H.S.D. test) as compared to the non-injected control group.

The group decapitated at 10 min after ketamine injection2.7. In situ hybridization did not reach significance as compared to the non-injected

controls. Animals treated with ketamine showed no signifi-Frozen brains were cut at 2148C in 14-mm coronal cant difference to saline injected animals. Rats adminis-

sections using a Frigocut 2800 E (Leica, Germany) tered ketamine had significantly lower levels of CCK-LI incryostat at bregma 12.70 mm according to Paxinos and PFC as compared to saline injected animals at 480 minWatson brain atlas [24] and thawed onto slides pre-treated after injection (F 511.060, P50.009471, Tukey’s2, 17

with 2% 3-aminopropyl-triethoxy-silane. A 293-basepairs H.S.D. test), but no significant change was seen as(bp) cDNA (bp 263–535, acc. no. X01032) was cloned compared to the non-injected controls (Fig. 1). Theinto an pGEM-T easy vector (Promega), linearised with increase in CCK-LI observed in the PFC at 480 min afterPstI (Pharmacia) and transcribed with T7 polymerase saline administration was not observed in the nucleus

200 D. Radu et al. / Brain Research 908 (2001) 197 –203

Fig. 1. Effects of saline (S) and (K) ketamine i.p. injections on tissue levels of CCK-LI in rat PFC; subscripts indicate minutes after injection.Means6S.E.M. as % of non-injected non-operated control group, control levels similar to absolute levels in PFC (Fig. 2). Line indicates mean values fornon-injected non-operated control groups. Means6S.E.M. are 10064.25 for controls (C), 100612.60 for 480-min controls (C ); *P,0.05, **P,0.01480

Tukey’s H.S.D. test.

accumbens, caudatus-putamen, amygdala, hippocampus administration, respectively, as measured in the prefrontaland entorhinal cortex (Fig. 2). cortex region. Levels of CCK mRNA in PFC in non-

injected animals served as controls (Table 1).3.2. Effects of i.p. saline and ketamine injection in non-operated animals on CCK mRNA in the PFC 3.3. Effects of i.p. saline injection in animals having a

microdialysis probe implant in the right PFCThere was no significant change in levels of mRNA

encoding for CCK 4 or 8 h after either saline or ketamine Animals having a microdialysis probe cannula in the

Fig. 2. Effect of saline and ketamine i.p. injections on tissue levels of CCK-LI 480 min after injection in selected regions of the rat brain: AC, nucleusaccumbens; AMG, amygdala; CP, caudatus-putamen; EC, entorhinal cortex; HIPP, hippocampus; PFC, prefrontal cortex; ***P,0.001 as compared tonon-injected controls, Tukey’s H.S.D. test.

D. Radu et al. / Brain Research 908 (2001) 197 –203 201

Table 1Results from measurement of CCK mRNA levels in rat prefrontal cortex on autoradiogram from in situ hybridization histochemistry

Control Saline 240 min Saline 480 min Ketamine 240 min Ketamine 480 min

330.14626.57 352.65628.01 333.70624.90 312.34611.34 297.20618.23

Mean values6S.E.M. of CCK mRNA in rat prefrontal cortex (optical density, arbitrary units).

right medial prefrontal cortex did not show any significantchange in CCK-LI at 5, 10 and 20 min following salineadministration as compared to non-injected operated con-trols and non-injected non-operated control animals bothanalyzed 1 h after probe implantation (Fig. 3) and animalsanalyzed 24 h after probe implantation (Fig. 4). Thenon-injected group studied 480 min after probe implanta-tion displayed a significant increase in CCK-LI (F 52, 17

4.1287, P50.041257, Tukey’s H.S.D. test) in studies done1 h following probe implantation (Fig. 3). Saline injectedanimals analyzed at 24 h after probe implantation hadsignificantly lower levels of CCK-LI than control animals,(F 56.9532, P50.006609, Tukey’s H.S.D. test) (Fig.2, 16

4). There was no significant effect at 8 h after salineadministration (S1 , and S24 ) in operated animalsOP480 OP480

as compared to non-injected operated controls (C1 ,OP480

and C24 respectively) (Figs. 3 and 4).OP480Fig. 3. Effect of i.p. saline injection on tissue levels of CCK-LI in ratPFC. Experiments were done 1 h following probe implantation. C1 ,OP

non-injected operated controls; S1 , saline injected animals; subscriptsOP 4. Discussionindicate minutes after i.p. saline injection. Means6S.E.M., % of non-injected non-operated control group. Line indicates mean values for

The present results provide further support for a CCK-non-injected non-operated control groups. Control levels similar toabsolute levels in PFC (Fig. 2). Means6S.E.M. are 100612.68 for ergic involvement in stress. As a novel finding we putcontrols (C), 100610.58 for 480-min controls (C1 ). *P,0.05, Tukey’s480 forward the biphasic change in tissue response to mildH.S.D. test. stress, with significant decrease in tissue level of CCK-LI

in PFC after 20 min and increase following 8 h after a mildstress stimulus. The decrease at 20 min after injectioncould be the result of increased CCK release, as suggestedby microdialysis experiments [21], or possibly a decreasein synthesis. The observed lack of change in CCK-LI inPFC 5 and 10 min after injection is in accordance with thefindings of Rosen and collaborators [27]. The observationof a significant CCK-LI increase as late as 8 h after theinjection-induced stress could be the result of a compensat-ory activation of synthesis. This finding is most original asearlier studies have focused on time intervals much shorterthan 8 h following stress-inducing stimuli.

4.1. CCK in prefrontal cortex

While we found significant changes in PFC tissue levelsof CCK-LI 8 h after the mild stress stimulus, no corre-sponding changes in CCK mRNA in this part of the brainFig. 4. Effect of i.p. saline injection on tissue levels of CCK-LI in ratwere detected either 4 or 8 h following the stress, in bothPFC. Experiments were done 24 h following probe implantation. C24 ,OP

non-injected operated controls; S24 , saline injected animals; subscripts saline and ketamine injected animals. Upregulation ofOP

indicate minutes after i.p. saline injection. Means6S.E.M., % of non- CCK mRNA in subcortical regions, with only smallinjected non-operated control group. Line indicates mean values for increases in cortex, have been reported using other stressnon-injected non-operated control groups. Control levels similar to

models, such as restraint or isolation stress [7,10]. In thisabsolute levels in PFC (Fig. 2). Means6S.E.M. are 100612.68 forstudy, the changes in CCK-LI levels could not be linked tocontrols (C), 10066.04 for 480-min controls (C24 ). *P,0.05, Tukey’s480

H.S.D. test. changes in CCK gene-expression in adjacent cortical

202 D. Radu et al. / Brain Research 908 (2001) 197 –203

neurons, but could possibly be due to posttranslational tent ketamine administration on dopaminergic andprocessing of neuronal prepro-peptide pools, gene upregu- serotonergic transmission in rat PFC [16].lation in subsets of cells in the prefrontal cortex and/or inafferent CCK-ergic systems. The observed increase in 4.3. Microdialysis probe implantationpeptide levels could be the result of local processing ofCCK precursors into immunoreactive material, alone or in Different stressors such as restraint, ether and yohimbinecombination with upregulation of CCK gene expression administration lead to increase in CCK release in the PFCand peptide synthesis in afferent neurons with terminals in starting within the first 30 min and lasting up to 4 h asthe PFC, projecting from other parts of the cerebral cortex, assessed by microdialysis studies [2,21]. It seems ap-the hippocampal formation, the amygdala or projecting pealing to use in vivo microdialysis in awake animals tofrom neurons originating in or close to the ventral tegmen- study the role of CCK-ergic transmission in the response total area [13,28]. CCK mRNA in the cerebral cortex stress. In an attempt to examine this possibility we firstappears to be expressed in GABAergic interneurones [12] addressed the issue of effects on tissue levels of CCK-LIbut also in a majority of other cortical neurons. CCK-LI is by microdialysis probe implantation in itself. Thus weexpected to be synthesized in CCK mRNA expressing cell examined the tissue level of CCK-LI adjacent to the probebodies and is present in local glutamatergic neuronal cell and found evidence for an operation-induced influence onbodies [14] and dopaminergic [13,29] afferents. the tissue response with respect to CCK-LI content. Tissue

levels of CCK-LI decreased by up to 20–30% up to 32 h4.2. Effects of ketamine in non-injected animals. In addition, the stress-induced

effects were also attenuated (Figs. 3 and 4). SignificantAn i.p. injection containing ketamine resulted in signifi- methodological development seems necessary before mi-

cant decreases in CCK-LI in PFC at 5 min in addition to crodialysis detection of CCK-LI release in the cerebralthe 20-min time point following injection. It is not possible cortex may be used in mild stress paradigms. This cautionat present to conclude whether this is due to increased may possibly be relevant to the study of other corticalCCK release or other mechanisms. neurotransmitters that may be affected by CCK.

The glutamate agonist N-methyl-D-aspartate stimulatesCCK release in rat PFC in a dose-dependent fashion [22].The inclusion of the NMDA blocking drug ketamine in 5. Conclusionsuch doses as used in our study may induce increases inglutamatergic neurotransmission in PFC at non-NMDA Experimental handling of animals might trigger long-sites [1,20]. term (several hours) changes in CCK-ergic mechanisms in

The observed lack of difference in cortical CCK-LI the PFC of rat, possibly due to the effects of mild stress.between saline injected and ketamine injected animals NMDA antagonism by subanesthetic doses of ketamine5–20 min after injections is consistent with data from seems to interfere with CCK-ergic mechanisms as ob-Pongdhana and collaborators [25], where no difference in served by a more pronounced decrease within 20 min andlevels of CCK-LI in frontal cortex was reported after an elimination of the increase in CCK peptide level 8 hadministration of ketamine as compared to saline injected after an i.p. saline injection. In addition, it appears as ifcontrols, non-injected controls not being included in the factors associated with the presence of a microdialysisstudy. The results are however not completely comparable probe in the PFC tissue interfere with the CCK-ergicsince in that report only the response 30 min following response to mild stress.injections was reported and the doses of ketamine usedwere four times higher than the doses used in the presentstudy. Ketamine seems to have a biphasic dose dependent

Acknowledgementsaction on glutamate release, with low subanesthetic dosesleading to increased and anesthetic doses to decreased

This work has been supported by the Swedish Medicalglutamate outflow in rat PFC [20]. CCK has been shown to

Research Council, grant nr 8653 and by the Swedishincrease glutamate and aspartate release in rat cortex via

Cancer Foundation.both CCK and CCK receptor subtypes [31].A B

The observation of unchanged tissue levels of CCK-LIat 8 h after ketamine administration is more difficult to

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