increased sensitivity to cocaine, and over-responding during cocaine self-administration in tpa...

Ž .Brain Research 826 1999 117–127

Research report

Increased sensitivity to cocaine, and over-responding during cocaineself-administration in tPA knockout mice

Tamzin L. Ripley a, Beatriz A. Rocha b,c, Michael W. Oglesby b, David N. Stephens a,)

a Laboratory of Experimental Psychology, UniÕersity of Sussex, Falmer, Brighton, BN1 9QG, UKb Department of Pharmacology, UNTHSCrFW, Fort Worth, TX, 76107, USA

c NIDAr IRP, BehaÕioural Neuroscience, Intermural Program, 5500 Nathan Shock DriÕe, Baltimore, MD, 21224, USA

Accepted 16 February 1999

Abstract

Tissue plasminogen activator, tPA, is induced in the brain by electrical activity leading to synaptic remodeling. It is also induced in theŽ .prefrontal cortex PFC by acute cocaine. We investigated cocaine-induced locomotor activity, the development of sensitisation to cocaine

Ž .and cocaine self-administration in mice lacking the gene encoding tPA. Mice lacking tPA tPA knockout mice, tPAyry showedŽ .normal spontaneous activity, exhibited cocaine-induced locomotor activity at lower doses than wild-type WT control mice and showed a

greater degree of cocaine-induced locomotor activity following repeated administration. tPAyry and WT mice did not differŽ .significantly in the time to acquire self-administration of cocaine 20 mgri.v. infusion under an FR2 schedule. Following acquisition of

this behavior, these groups also did not differ significantly in the rate of cocaine self-administration across the next three sessions.However, WT mice decreased responses on the active lever during signaled periods when reinforcer was not available; in contrast,tPAyry mice did not. The emission of non-reinforced responses was most marked at the beginning of each 90 min daily session. Thispattern of responding was not seen in tPAyry mice pressing for food under an FR2 schedule of reinforcement. These results suggestthat tPA may play a specific role either in retention of information between sessions or in behavioural inhibition in cocaineself-administration. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cocaine; I.V. self-administration; Tissue plasminogen activator; Synaptic plasticity; Prefrontal cortex; Transgenic mice

1. Introduction

Neuronal adaptation following repeated drug adminis-tration leading to addiction is believed to involve processessimilar to those that underlie other forms of synaptic

w x Ž .plasticity 22,10 . Tissue plasminogen activator tPA is aserine protease that has been suggested to play a role in theplasticity of neural connections. Electrical activation ofneurones initiates a cascade of intracellular events leading

w xto induction of immediate early genes, including tPA 16 .In the adult brain, tPA is expressed in Purkinje cells of the

w xcerebellum and in the hippocampus 17,20,25 , two areaswhich undergo synaptic plasticity following high levels of

w xneuronal activity 8,13 . tPA is expressed in the brain withdistinct spatial patterns by events including convulsive

) Corresponding author. Fax: q44-1273-678611; E-mail:[email protected]

seizures and stimulation of the perforant path leading toŽeither kindling or long-term potentiation LTP; Refs.

w x.2,16 . Induction of tPA in dentate gyrus granule cellsduring LTP is prevented by the NMDA receptor antagonistdizocilpine, suggesting that tPA may play a role in NMDAreceptor-mediated neuronal plasticity underlying kindlingand LTP. In particular, tPA may be induced during thecascade of events leading to late-phase LTP, subsequent toactivation of cAMP response element binding proteinŽ . w xCREB 16 ; correspondingly, tPA knockout mice show aselective interference with late-phase LTP in the Schaffercollateral-CA1 pathway, a phase thought to involve anNMDA receptor-dependent modification of GABA trans-

w xmission in the CA1 region 7,3 .In keeping with behavioural sensitisation to psychostim-

ulants depending on events analogous to those underlyingw xLTP 22,10 , tPA mRNA is expressed in several brain

areas, including PFC, following psychostimulant adminis-tration. The majority of PFC tPA-expressing neurones

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01280-9

( )T.L. Ripley et al.rBrain Research 826 1999 117–127118

w xproject to the medial striatum 5 , an area intimately impli-w xcated in reward processes 11,26 . Expression of tPA

mRNA is first detectable 1 h following amphetamineadministration, and reaches a maximum at 3 h, consistentwith a role in late plastic events following drug administra-tion. Amphetamine-induced expression of tPA in PFC is ofparticular interest since this brain area has been implicatedin both behavioural sensitisation to psychostimulants, and

w xin drug self-administration 23 .Therefore, it may be speculated that tPA is involved in

mediating behaviours associated with repeated administra-tion of psychostimulants through an action of tPA in thePFC. To explore this possibility, we have examined loco-motor activity following acute and repeated administrationof cocaine in mice that lack the gene that encodes tPA.Lack of sensitisation in these mice would indicate that tPAmay be involved in the cascade of cellular events leadingto behavioural sensitisation. However, a potentiated re-sponse to cocaine or a more rapid development of sensiti-sation would indicate an involvement of tPA in inhibitoryresponses controlling the expression of behavioural sensiti-sation. This latter effect would resemble behavioural pro-files seen when normal neuronal functioning in the PFC is

w x w xmanipulated by lesions 21 or electrical kindling 23 .Since prior behavioural sensitisation to cocaine facilitates

w xacquisition of cocaine self-administration 6 , changes inthe rate of behavioural sensitisation might predict alteredacquisition of drug self-administration; cocaine self-admin-istration was therefore also studied in these animals.

2. Materials and methods

2.1. Animals

Ž .A colony of tPA null mutant knockout; tPAyryŽ .mice and wild-type WT controls were raised from origi-

nal homozygous breeding pairs obtained from D. Collenand P. Carmeliet, University of Leuven, Belgium. Theanimals were genetically manipulated from SV129 stem

w xcells implanted into a C57rBl parent strain 1 . OffspringŽfrom pure bred lines of homozygous knockout mice male

. Ž .and female yry or WT male and female qrq didnot differ in body weight, body length or size or frequencyof litter. Animals were born and maintained in a 12 hlightrdark cycle with lights on at 7 AM. Male mice wereindividually housed at 6 weeks of age.

2.2. Genetic status

Genetic status was verified in the Sussex colony usingŽ .polymerase chain reaction PCR methodology to amplify

any remaining signal from the tPA gene. Genomic DNAwas isolated from samples of mouse blood from both thetPAyry and WT colonies. A PCR fragment of mouse

tPA was produced and run on a 1.4% agarose gel produc-ing a band at 410 bp. The null mutation was confirmed bya lack of this band.

2.3. Locomotor actiÕity

Male mice at 7 weeks of age were placed in circularŽ .runways 24 cm diameter containing 8 infrared light

beams at 458 separation and forward locomotor activitywas recorded as adjacent beam breaks. Activity was sam-pled for 30 s every 4 min throughout the experiment.tPAyry mice and WT controls, naive to the apparatusand drug treatment, were habituated to the apparatus for 1h over which time spontaneous forward locomotor activity

Žwas recorded. Animals were injected with cocaine 3, 10.or 30 mgrkg, 10 mlrkg, i.p. or saline and locomotor

activity was recorded for a further 1 h period.To study the development of behavioural sensitisation,

Ž .a single dose of cocaine was selected 10 mgrkg andanimals were treated for 10 sessions over a 4 week periodŽtreatment days over each 2 week period were: M, W, F,

.Tu, Th . On each test day animals were habituated to theŽapparatus for 1 h, injected with either cocaine 10 mgrkg,

.10 mlrkg, i.p. or saline and locomotor activity wasrecorded for a further 1 h period. tPAyry and WT micewere divided into 3 treatment groups. The first group

Žreceived saline treatment on each of the 10 sessions con-.trol group , the second group received saline treatment for

the first 9 sessions and cocaine on the final session and thethird group received repeated cocaine treatment on each ofthe 10 sessions.

ŽData were analysed by 2-way ANOVA factors geno-.type and dose of drug for cumulative forward locomotor

activity over the acute 1 h test period, and 3-way ANOVAŽfactors genotype, drug dose, and time bin as a repeated

.measure for the 4-min time bins, for the pattern oflocomotor activity. Post-hoc analyses consisted of one-wayanalysis of variance comparing tPAyry and WT miceat individual doses of cocaine or at individual time bins.Effect of increasing doses of cocaine within each mousetype was also analysed using one-way analysis of variance.

For the behavioural sensitisation experiment, cumula-tive forward locomotor activity over the 1 h test period onthe 10th treatment session was analysed by two-way

Ž .ANOVA factors genotype and treatment . Post hoc analy-ses consisted of one-way analysis of variance comparingtPAyry and WT mice for each treatment and the effectof treatment within each mouse type.

2.4. Operant behaÕiour

2.4.1. OÕernight food shapingOne week before the start of operant training male mice

were food restricted to 80% of their ad libitum body

( )T.L. Ripley et al.rBrain Research 826 1999 117–127 119

weight. Animals were trained to press a lever for a milkreinforcer in two 16 h sessions run during the dark phase,

Žin mouse operant chambers model ENV-307; MedAssoci-.ates, Georgia, VT, USA , constructed of clear perspex, and

measuring 18=18=15 cm, and contained in sound- andlight-attenuating cubicles. A single house light was locatedin the wall opposite the levers. The front wall of the boxes

Ž .were fitted with a liquid dipper model ENV-202A , lo-Žcated between 2 ultrasensitive mouse levers model ENV-

. Ž .310A . Activation of one of the levers the active leverraised the liquid dipper for 5 s, giving access to a 0.01 ml

Žcup containing 30% solution of condensed milk the rein-. Ž .forcer Fussell’s, York, UK . Activation of the other lever

Ž .inactive lever had no programmed consequences. Freereinforcers were delivered every 2 min until the animalbegan lever pressing. The response requirement to obtain areinforcer was then increased progressively; a single acti-vation of the lever was required for each of the first twenty

Ž .reinforcers fixed ratio 1; FR1 , after which two leverŽ .presses were required Fixed ratio 2; FR2 for each of the

Ž .next 10 reinforcers, and four FR4 for subsequent rein-forcers, until a minimum of 30 reinforcers were obtainedand for each reinforcer, 75% of responses were on theactive lever. To reduce lever bias, the inactive lever nowbecame the active lever and the schedule returned to FR1.

Ž .Such reversals cycles occurred throughout the trainingsession. Animals that reached criterion on the second

Žtraining session, a minimum of 100 earned reinforcers and.2 completed cycles , progressed to the next part of the

study. tPAyry and WT mice were randomly assignedto either continued operant training for food reinforcementor to the cocaine self-administration procedure.

2.4.2. Food reinforcementAnimals remained on a restricted diet and were placed

in the operant boxes for 90 min daily sessions for a total of30 sessions during which pressing the active lever resultedin the presentation under an FR2 schedule of a reinforcerconsisting of 0.01 ml 30% condensed milk solution, theactive lever being randomly assigned. The dipper was upand reinforcer therefore available for 20 s, followed by a60-s time-out period signaled by the houselight going off.This procedure provided a similar schedule of reinforce-ment to that used in the cocaine self-administration study.The animals received a single ‘priming’ presentation of thedipper at the start of each session. The maximum numberof reinforcers that could be obtained in a single sessionwas 66.

2.4.3. Cocaine self-administrationAnimals that were to be included in the cocaine self-ad-

ministration study were given access to ad libitum food fora minimum of 24 h before surgery. Animals were then

Žanaesthetised with a ketaminerxylazine mixture induction

dose: 260 mgrkg ketamine, 2 mgrkg xylazine; Sigma,.UK . Additional anaesthetic was administered as necessary

throughout the surgery to maintain anaesthesia. A silasticŽcatheter o.d.s0.025 in.; i.d.s0.012 in.; 35 mm long;

.Altec Products, Hants, UK was inserted into the rightexternal jugular vein under a dissection microscope, ad-vanced towards the atrium, and secured by sutures andsurgical glue, following the procedure described by Rocha

w xet al. 19 . The distal end of the catheter was attached to a15 mm length of 23 g hypodermic needle tubing usingsilicone sealant and was guided subcutaneously behind theear and secured to an exposed region of the skull using

Žacrylic cement Geristore Dual Cure; Grafton International,.Staffordshire, UK . The open end of the hypodermic tub-

ing was sealed with a silicone cap. The dead space of thecatheter assembly was approximately 5 ml. Animals wereallowed to recover from surgery for 24 h before cocaineself-administration sessions began. The animals were againfood restricted and the cannula was flushed daily with 0.02ml of 30 urml heparin in physiological saline followingself-administration sessions.

ŽAnimals were placed in the operant boxes with the.liquid dipper disabled for daily 90 min sessions during

which activation of the active lever resulted in infusion ofŽcocaine 20 mgr20 ml infusion; approximately 0.8 mg

y1 y1 .kg infusion ; Sigma, UK under an FR2 schedule, theactive lever being randomly assigned. Each session beganwith a priming infusion of cocaine. Each cocaine infusionlasted 1.8 s and was followed by a 60-s time-out period,signaled by extinguishing the houselight, during whichperiod further lever pressing had no programmed conse-quences. The time-out was incorporated to prevent rapidsuccessive infusions leading to overdose.

Behavioural data are divided into 2 sections: acquisitionand stable performance. Acquisition refers to the sessionsduring which animals were learning the cocaine self-ad-ministration paradigm. Completion of acquisition was de-fined as having occurred on the first of three days overwhich the animals showed stable performance. Stable per-

Ž .formance or maintenance of self-administration was de-fined as three consecutive days over which the number ofcocaine reinforcers taken did not vary by more than 20%w x19 .

2.5. Data analysis

Data were analysed using analysis of variance for re-peated measures, the between subject factor being the

Ž .mouse genotype tPAyry vs. WT and the within sub-ject factors being either consecutive time bins or consecu-tive daily sessions. The dependent measures were takenfrom data of self-administration performance and includednumber of reinforcers, number of responses on the activeand inactive levers. Post hoc analyses consisted of one-wayanalysis of variance comparing tPAyry and WT mice


at either individual time bins or within individual sessions,and paired Student’s t-tests for comparing either tPAyryor WT mice across sessions. The slope of the sigmoidalcurves fitted to the acquisition data were analysed usingStudent’s t-test on the mean and standard error value.Non-parametric analysis was used to compare the numberof reinforcers taken during the food acquisition study,since the introduction of a cut off on the maximum numberof reinforcers available, led to a skewed distribution.

3. Results

3.1. Locomotor actiÕity

No difference between tPAyry and WT mice wasseen in spontaneous forward locomotor activity measured

Žover the habituation period forward locomotor activitymeasured as adjacent infra-red light beam breaks: tPAy

. Ž Ž . .ry: 116"12; WT: 105"9 F 1,39 s0.13, p)0.5 .Acute cocaine dose-dependently increased activityŽ Ž . .F 3,39 s28.63, p-0.001 , a significant interaction be-tween genotype and cocaine dose indicating that tPAyrymice showed higher cocaine-induced locomotor activity

Ž Ž . .than WT controls F 3,39 s2.78, ps0.05 leading to aŽ Ž .significant main effect of genotype F 1,39 s7.39, p-

.0.01; Fig. 1a . Post-hoc analysis using one-way analysis ofvariance revealed no differences between saline treatedtPAyry and WT mice. Cocaine had a stimulatory effect

Ž .in tPAyry mice at the lowest dose tested 3 mgrkgwhen compared with saline-treated animals of the same

Ž Ž . .mouse type F 1,9 s8.19, p-0.05 but a higher doseŽ .10 mgrkg was required to produce a similar stimulatory

Ž Ž . .effect in WT mice F 1,10 s12.07, p-0.01 . At theŽ .lowest dose of cocaine tested 3 mgrkg tPAyry mice

Ž Ž .were significantly more active than WT controls F 1,9.s9.76, p-0.05 . There was no difference in activity

between the tPAyry and the WT mice at either the 10Ž Ž .mgrkg or 30 mgrkg dose of cocaine 10 mgrkg: F 1,10Ž .s0.202, ps0.66; 30 mgrkg: F 1,10 s4.29, ps

.0.065 . The pattern of locomotor activity over time follow-ing drug treatment was not significantly different between

Ž .the tPAyry and WT mice data not shown .Repeated cocaine treatment led to an increase in loco-

motor activity in both types of mouse, however this effectŽ .was potentiated in the tPAyry group Fig. 1b . ThereŽ Ž .was a genotype by treatment interaction F 2.77 s3.75,

. Ž Ž .p-0.05 , and a main effect of both genotype F 2,77 s. Ž Ž .7.13; p-0.01 and treatment F 2,77 s47.46, p-

.0.0001 . Post-hoc analysis showed that locomotor activitywas higher in the tPAyry mice than in the WT mice

Ž Ž . .after both acute cocaine F 1,23 s5.57; p-0.05 andŽ Ž . .repeated cocaine F 1,28 s5.66; p-0.05 although there

Ž Ž .was no difference in saline treated animals F 1,26 s1.45;

ŽFig. 1. Forward locomotor activity measured as the number of adjacentŽinfrared beam breaks, time sampled over a 60 min period 30 s sample

..time every 4 min in tPAyry and WT mice following acute orŽ .repeated cocaine. a Forward locomotor activity during habituation to the

Ž .apparatus spontaneous activity and following acute cocaine administra-Ž . Žtion 0, 3, 10 or 30 mgrkg i.p. in naive tPAyry and WT mice dark

symbol, tPAyry mice ns23 habituation, ns5–6rcocaine dose;.white symbol, WT mice ns24 habituation, ns6rcocaine dose . Post-

hoc analysis showed that the dose of cocaine that had no effect on WTŽmice, significantly increased locomotor activity in tPAyry mice 3

mgrkg cocaine tPAyry vs. WT mice p-0.05; tPAyry mice 0 vs..3 mgrkg p-0.05 ANOVA . There was no significant difference in

Ž .spontaneous activity seen in tPAyry or WT mice. b Forward locomo-Žtor activity following acute and repeated cocaine treatment 10 mgrkg

. Ži.p. or saline treated controls in tPAyry and WT mice dark bars,.tPAyry mice ns13–15; white bars, WT mice ns12–15 . Post-hoc

analysis showed that tPAyry mice were more sensitive to both theŽacute and repeated effect of cocaine tPAyry vs. WT acute or repeated

.cocaine p-0.05 ANOVA and that the acute dose of cocaine enhancedŽlocomotor activity in the tPAyry mice only control vs. acute cocaine:

.tPAyry mice p-0.005; WT mice ps0.08 ANOVA . There was nosignificant difference in saline-treated tPAyry or WT mice.

.ps0.2 . Acute cocaine significantly enhanced locomotorŽ Ž .activity in the tPAyry mice F 1,25 s10.44; p-

. Ž Ž . .0.005 but not in the WT mice F 1,24 s3.45; ps0.07when activity was compared with saline-treated animals ofthe same genotype. Repeated cocaine treatment lead to asignificant increase in locomotor activity in both mousegenotypes when compared with either saline or acute


Žcocaine treated animals tPA y ry mice: cf salineŽ . Ž .F 1,27 s40.19; p-0.0001; cf acute cocaine F 1,26 s

Ž .29.81; p-0.0001; WT mice: cf saline F 1,27 s17.14;Ž . .p-0.0005; cf acute cocaine F 1,25 s11.68; p-0.005 .

3.2. Operant behaÕiour

3.2.1. OÕernight food shapingThe tPAyry mice showed a significantly shorter

latency to completion of the first cycle when comparedŽ Ž . .with WT controls F 1,31 s9.07; p-0.01; Fig. 2a .

Post-hoc analysis showed that this effect was due toa shorter latency of the tPAyry mice to complete the

Žfirst cycle in the first overnight session only tPAyry:. Ž Ž .203.8"40.8 min; WT: 455.9"73.0 min F 1,33 s9.87;

.p-0.01 . Both tPAyry and WT mice showed a signi-ficant decrease in latency to complete the first cycle from

Žthe first to the second session paired Student’s t-test;Ž .tPAyry mice ts3.19, d.f.s18; p-0.01 ; WT mice

Fig. 2. Behavioural parameters measured during 2 overnight sessionsshowing acquisition of lever pressing for a condensed milk reward on a

Žprogressive ratio schedule in tPAyry and WT mice dark bars, tPAy. Ž .ry mice ns19; white bars, WT mice ns16 . a Time required to

reach criterion for completing the first cycle of acquisition of lever-Ž .pressing for milk reinforcement. b Number of cycles of repeated

acquisition of lever-pressing on alternatingly active levers during two16-h sessions.

Ž . Ž .Fig. 3. Time course of reinforcers taken during the a first and b secondŽovernight training sessions in tPAyry mice and WT controls closed.circles, tPAyry mice ns19; open triangles, WT mice ns16 . Post-

hoc analysis showed that the tPAyry mice took a larger number ofreinforcers than the WT controls in the first overnight training session in

Žthe first, second, third and sixth hours of the test session p-0.05;.ANOVA .

Ž ..ts3.10, d.f.s15; p-0.01 . The faster onset of leverpressing behaviour is also reflected by the tPAyryanimals completing a significantly greater number of cy-cles as shown by a genotype by session interactionŽ Ž . . .F 1,31 s5.25 ; p-0.05; Fig. 2b . Post-hoc analysisshowed that this effect was due to the tPAyry micecompleting a greater number of cycles in the first overnight

Ž .session only tPA y ry: 6.9 " 0.9; WT: 3.9 " 0.7Ž Ž . .F 1,33 s7.03; p-0.05 . Both tPAyry and WT miceshowed a significant increase in the number of completed

Žcycles from the first to the second session paired Student’sŽ .t-test; tPAyry mice tsy2.68, d.f.s18; p-0.05 ;

Ž ..WT mice tsy9.21, d.f.s15; p-0.01 .Finally, when the time course of reinforcer administra-

tion was studied, the tPAyry mice took significantlymore reinforcers on the first overnight session than the WT

Ž Ž . .animals F 1,31 s5.72; p-0.05; Fig. 3 with this effectbeing concentrated over the first 6 h of the session. Therewas no difference in the pattern of reinforcer delivery onthe second overnight session.


3.2.2. Food reinforcementThere was no significant difference between tPAyry

and WT mice when the number of reinforcers taken onŽeach of the 30 sessions was compared Mann–Whitney U

.test; p)0.05 . For both groups of animals, there was a

significant increase in the number of reinforcers takenŽ . Žacross the first 3 sessions Wilcoxon test; p-0.05 Fig.

.4a .No differences were seen between the two genotypes in

the number or pattern of responses emitted on either the

ŽFig. 4. Behavioural parameters measured during daily food acquisition sessions in tPAyry and WT mice closed circles, tPAyry mice ns8; open. Ž . Ž .triangles, WT mice ns8 . a Number of food reinforcers taken in each session, b number of responses on the active lever contributing towards

Ž . Ž .obtaining a reinforcer; c number of responses the active lever made during the time-out period; d number of responses on the inactive lever during theŽ .period when the reinforcer was available and e number of responses on the inactive lever made during the time-out period. There was no difference in

either the number of reinforcers taken by tPAyry and WT mice or in the number or pattern of responses made on the active or inactive levers.


Žactive or inactive lever rewarded responses on the activeŽ .lever: F 1,13 s0.73; ps0.407; non-rewarded responses

Ž .on the active lever: F 1,14 s0.00; ps0.990; responsesŽ .on the inactive lever during the FR2 period: F 1,13 s

0.51; ps0.489; responses on the inactive lever during theŽ . .time-out period: F 1,14 s0.13; ps0.719: Fig. 4b–e . In

contrast to the pattern of responding for cocaine reinforce-ment, analysis of the patterns of lever pressing duringsession 1, 10, 20 and 30 did not suggest that the tPAyrymice over-responded more than the wild-type at the begin-

Ž . Ž .ning of each daily session p)0.05 data not shown .

3.2.3. Cocaine self-administration

3.2.3.1. Acquisition. The mean number of sessions re-quired to reach the first stable performance day did not

Ždiffer between tPAyry and WT mice 3.6"0.3 and.3.3"0.8 days, respectively . However, when the data

were expressed as the percentage of animals reachingstable performance against session number, the slope ofthe curve fitted to this data was significantly steeper in the

ŽtPAyry mice when compared with WT controls Stu-.dent’s t-test: ts3.994, d.f.s22; p-0.01 , indicating

that there is more variation in performance in the popula-Ž .tion of WT mice than in the tPA knockouts Fig. 5 .

(3.2.3.2. Stable performance maintenance of cocaine self-)administration . The number of cocaine reinforcers ob-

tained over the three stable performance days did not differŽ .between the tPAyry and WT groups Fig. 6a . How-

ever, the pattern of non-reinforced responses, attributableboth to responses on the inactive lever and to responses onthe active lever during cocaine infusion and time-out peri-ods varied between the two groups. In WT mice there wasa decrease in non-reinforced lever presses on the active

Fig. 5. Acquisition of intravenous cocaine self-administration in tPAyryand WT mice shown as the percentage of animals reaching acquisition.The slope of a sigmoidal curve fitted to the WT data was significantlyless than that fitted to the tPAyry data showing that there was more

Žvariability in the WT group closed circles, tPAyry mice ns14; open.triangles, WT mice ns11 .

Žlever from session 1 to session 3 ts4.55, d.f.s10;.p-0.001; Fig. 6c , but this decrease was not seen in the

tPAyry animals, resulting in a significant main effectŽ Ž . .of mouse genotype F 1,23 s5.16; p-0.05 . Post-hoc

analyses showed that there was a significant differencebetween the tPAyry and WT mice by the third stable

Ž Ž . .session F 1,23 s8.18; p-0.01 . Although there was atrend towards a decrease in responses on the inactive leverin the WT animals this failed to reach significance acrossthe 3 sessions due to large individual animal variation.

ŽThis trend was not seen in the tPAyry animals Fig..6d,e .

Data for non-reinforced lever-pressing from each ses-Ž .sion was further analysed for its three components, A

data from periods during which the reinforcer was avail-Žable, and houselight on i.e., responses on inactive lever

. Ž .while cocaine was available on the active lever ; B dataŽ .from the periods 1.8 s during which cocaine infusion wasŽ . Žtaking place, and C data from time-out periods each of

.60 s , during which the houselight was extinguished, andneither lever gave rise to reinforcement. The WT miceshowed a significant decline in non-reinforced responding

Ž .on the active lever during the time-out period C on theŽsecond and third days session 1 vs. 2: ts2.74, d.f.s10;

.p-0.05; session 1 vs. 3: ts4.41, d.f.s10; p-0.001 .This decline was not seen in the tPAyry animals,

Ž Ž .resulting in a main effect of mouse genotype F 1,23 s.6.04; p-0.05 . Post-hoc analysis revealed that responding

by the WT animals was significantly lower than that of thetPAyry mice on the second and third stable perfor-

Ž Ž .mance days session 2: F 1,23 s5.49; p-0.05; sessionŽ . . Ž .3 F 1,23 s7.56; p-0.05 Fig. 6c . Again there was a

trend towards a decrease in inactive lever presses duringŽ .the time when cocaine was available component A and

Ž .during time-out component C in WT animals. However,due to large individual animal variation, this failed to reachsignificance. tPAyry animals did not show this trendŽ .Fig. 6d,e .

The pattern of responding for cocaine was further exam-ined in 15 min time bins for each stable performance day.tPAyry and WT animals did not vary in the pattern inwhich the reinforcers were taken. Reinforcers were spreadevenly across each session and this did not vary across

Ž .sessions Fig. 7a . When non-reinforced responses on theinactive lever and active lever during time-out were exam-ined in this way it was seen that on the first stableperformance day, when an equal number of non-reinforcedresponses were made by the tPAyry and WT mice,these responses were equally distributed across the sessionfor both sets of animals and across both levers. Bythe third stable performance day, the WT control animalswere making very few non-reinforced responses and thesewere equally spread across the session. In contrast, thetPAyry animals made significantly more non-rein-forced responses in the first 15 min time bin, when com-pared with either the WT animals at the same time point


Fig. 6. Behavioural parameters measured during i.v. cocaine self-administration in tPAyry and WT mice when the animals were displaying stableŽ . Žperformance defined as three consecutive days when the number of reinforcers taken by an individual mouse did not vary by more than 20% closed

. Ž . Ž . Ž .circles, tPAyry mice ns14; open triangles, WT mice ns11 . a Number of cocaine reinforcers 20 mgrinfusion taken in each session; b numberŽ . Ž .of responses on the active lever contributing towards obtaining a reinforcer; c number of responses the active lever made during the time-out period; d

Ž .number of responses on the inactive lever during the period when the reinforcer was available and e number of responses the inactive lever made duringthe time-out period. There was no difference in the number of reinforcers taken by tPAyry and WT mice. However, tPAyry mice displayed asignificantly greater number of non-reinforced lever presses on the active lever on the third stable performance day when compared with WT controlsŽ .p-0.01; ANOVA .

Ž Ž . .F 1,22 s5.164; p-0.05 or compared with tPAyryŽ .animals at any other time point p-0.05 . Further analy-

sis of the time course data showed that the non-reinforcedlever presses shown by the tPAyry mice were equally


Ž . Ž .Fig. 7. Time course of a cocaine reinforcers taken and b non-rewardedŽ .responses on the active and c inactive levers during the first, second and

Žthird stable performance day in tPAyry mice and WT controls closed.circles, tPAyry mice ns14; open triangles, WT mice ns11 . There

was no significant differences in the number or pattern of cocainereinforcers taken between the 2 groups or across the time bins. However,on the third stable performance day the tPAyry animals showed asignificantly greater number of non-rewarded lever presses in the first 15min time bin when compared to subsequent time bins and when com-

Ž .pared to WT animals in the same time bin p-0.05; ANOVA . Thiseffect was distributed across both levers.

distributed across the inactive lever and the active leverŽ .during time-out periods Fig. 7b,c .

4. Discussion

In the present experiments, tPAyry mice showed anincreased sensitivity to the locomotor stimulant propertiesof cocaine, compared with WT control mice, withoutshowing alteration in spontaneous activity in a novel envi-ronment. The reason for the increased sensitivity is notknown, but, considering that tPA is induced in prefrontal

Ž .cortex PFC as a result of psychostimulant administrationw x5 , it is of interest that a similar sensitivity to the stimulanteffects of cocaine is also seen in rats in which the normalfunctioning of the PFC is altered by either 6-hydroxy-

Ž . w xdopamine lesions 6-OHDA 21 or electrical kindlingw x23 . 6-OHDA lesions of the PFC reduce DA levels in this

w xarea 21 and it is speculated that a decrease in DAtransmission may occur in the PFC of the tPAyry mice.

This hypothesis is supported by the enhancement ofbehavioural sensitisation seen in tPAyry mice whencompared with WT mice as it has been shown that adecrease in DA transmission is also seen following be-havioural sensitisation to repeated cocaine administrationw x24 . Low DA transmission in the PFC could result in theanimals being in a ‘pre-sensitised’ state rendering themsensitive to the stimulant effects of cocaine. We havepreviously shown that the tPAyry mice show a morerapid development of behavioural sensitisation to cocaineat a dose of cocaine where there is no difference in the

w xacute sensitivity of the tPAyry and WT mice 18 .Dopamine levels in the PFC influence the direction ofchanges following increased neuronal activity, low levelsof dopamine supporting LTP induction, while high levels

w xbias to LTD 12 . To date, activity-dependent plasticchanges in the PFC have not been studied in tPAyrymice. In the hippocampus, tPAyry mice show dis-rupted late-phase LTP due to an enhancement of GABAer-

w xgic transmission 3 and a similar disruption in the PFCcould result in a functional down regulation of dopaminer-gic transmission.

6-OHDA lesions, which deplete PFC dopamine, facili-w xtate self-administration of cocaine by rats 21 , which is

consistent with the enhanced sensitivity of such animals tococaine. Nevertheless, and contrary to our predictions,tPAyry mice did not show facilitation of acquisition ofcocaine self-administration. Although there was greatervariation among the WT mice than the knockouts in thenumber of training sessions required to achieve the crite-rion for stable performance, no differences were observedin the number of sessions required to reach criterion, nor inthe number of reinforcers obtained at stable performance.These data are not what we expected based both upon theenhanced rate of behavioural sensitisation to cocaine in the

w xtPAyry mice 18,6 and upon increased cocaine sensi-w xtivity following PFC lesions 21 . However, cocaine self-

administration was acquired very rapidly in this experi-ment; thus, the failure to find an increase in the rate ofacquisition may reflect inadequate sensitivity of the


method. The variation seen in the WT mice could possiblyreflect a difference in genetic background. However, this isunlikely as a carefully controlled animal breeding proce-dure was enforced to minimise behavioural traits seen inselective breeding programs.

Although tPAyry mice self-administered similaramounts of cocaine, the pattern of responding differedbetween the knockout and control mice. WT mice showedan improvement in efficiency of performance over testdays, as shown by a decrease in the number of non-rein-

Ž .forced responses on the active lever during time-out ,while the knockout animals did not show this increasedefficiency across sessions. The tPAyry mice continuedto respond during time-out, and they emitted more than50% non-reinforced responses. They thus failed to acquire

Ž .a conditional discrimination press only when light on toobtain cocaine. Analysis of the time course of the re-sponses indicated that the tPAyry mice made the ma-jority of their non-reinforced lever responses over the first

Ž .15 min of each 90 min daily session Fig. 7b,c , and thatthe tendency to respond inefficiently declined during thecourse of each session.

The inefficient response pattern did not reflect an inabil-ity of the knockouts to learn a simple operant response,lever pressing. Indeed, during the first overnight trainingsession of the acquisition of lever pressing for food, thetPAyry mice were superior to the WT mice when thenumber of food rewards taken or the number of reversalscompleted were considered. On session 2, as the WTanimals became more practised at the task, there was asignificant increase in the number of reinforcers taken,which matched the level of the tPAyry mice.

During responding for cocaine reinforcers, the higherror rate of the tPAyry mice was particularly pro-nounced at the start of each day’s session. A possibleexplanation of this pattern is that while the knockout micewere able to acquire the discrimination during the courseof a self-administration session, and although they retainedthe lever-pressing response overnight, they did not retainthe discrimination. Such a pattern would be consistent withtPA being necessary only for longer term storage of certainkinds of information, in keeping with its proposed role inlate-phase LTP. Although tPAyry mice show disrup-

w xtion of late-phase LTP 3,7 , which many think to beinvolved in learning and memory formation, behaviouraldeficits in appropriate memory tasks have not previouslybeen documented in the tPAyry mice. For instance,Huang et al. 1996 reported that tPAyry mice were notimpaired in their ability to learn a contextual fear response,or spatial navigation of either a Morris Water Maze, or a

ŽBarnes maze, behaviours thought to require LTP review:w x.Ref. 15 .

Since rates of lever pressing are strongly influenced bypsychomotor stimulants like cocaine, and tPAyry miceshow greater sensitivity to cocaine, it was important toinvestigate whether the pattern of errors displayed during

cocaine self-administration was also seen during operantperformance for a conventional reinforcer. The reinforcerchosen was condensed milk, as in the initial training phase,but the experimental contingencies pertaining were chosento maximise the likelihood of the kinds of errors seenduring cocaine self-administration—responding during re-inforcer delivery, and time-out, and responding on theinactive lever. Under this schedule no differences betweenknockout and wild-type mice were seen across 30 sessionsin terms of the numbers of reinforcers received or in thenumber or pattern of responses emitted on the active orinactive levers. In contrast to the self-administration ofcocaine, there was no evidence of the mutants improvingperformance within sessions, only to relapse to high errorrates at the beginning of the next day’s session, as wasseen when the tPAyry mice were responding for co-caine. An interpretation of our results in terms of impairedconsolidation of discrimination learning must therefore betreated with caution, and it may be more appropriate toview the behaviour of the tPAyry mice as uncontrolledresponding during the early part of each day’s session,which, since the knockouts were more sensitive than WTmice to low doses of cocaine, was exacerbated by cocaine.

The behaviour of the tPAyry mice can thus besummarised as showing enhanced sensitivity to the loco-motor stimulant effects of acute cocaine which may repre-sent a form of ‘pre-sensitisation’ in these animals. Re-peated exposure to cocaine further emphasised this en-hanced sensitivity as the tPAyry mice showed height-ened behavioural sensitisation. However, we found no

Ž .changes in acquiring an operant response lever pressingto obtain either an intravenous cocaine injection or thepresentation of food reinforcers. Nevertheless, tPAyrymice did show a deficit in their ability to increase theefficiency with which they obtained cocaine but not foodreinforcers, by suppressing responding either on a non-re-inforced lever, or on the active lever at times when rein-forcement was signaled to be unavailable. The failure tofind changes in cocaine administration cannot be taken asdefinitive in determining whether tPA is important incocaine self-administration in normal animals, since com-pensatory changes during development may have counter-acted the effect of the knockout manipulation. More impor-tantly, these experiments are provocative in suggesting thatdeletion of the tPA gene may lead to a deficit in ability tosuppress unrewarded responding. In the present experi-ments, there was no deleterious consequence for the micein failing to suppress these kinds of responses, and thepresent work was not designed to explore the nature ofthese deficits in depth. Nevertheless, the inability of themutant mice to suppress unrewarded behaviour, whilemaintaining the ability to learn a lever-pressing response,is reminiscent of rodents with either PFC or hippocampal

Ž w x.damage for reviews, see Refs. 4,9,14 , and the implica-tion of tPA in hippocampal plasticity would be consistent

.with this idea .


Acknowledgements

We are indebted to P. Carmeliet and D. Collen forproviding us with breeding pairs of tPAyry and wild-type mice. This work was supported by MRC ProjectGrant G9510898 to DNS.

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