Brain Research 971 (2003) 40–46www.elsevier.com/ locate/brainres

Research report

O lfactory discrimination learning deficit in heterozygous reeler mice*John Larson , Jeffrey S. Hoffman, Alessandro Guidotti, Erminio Costa

Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois, 1601 W. Taylor St., Chicago, IL 60612,USA

Accepted 11 December 2002

Abstract

Adult mice heterozygous for the ‘reeler’ mutation (HR mice) have been found not to have a reeler phenotype but to express a numberof abnormal traits including reduced sensorimotor gating, lower density of dendritic spines in frontoparietal cortex and hippocampus, andselectively decreased expression of one form of glutamic acid decarboxylase (GAD67). Since reelin is expressed in olfactory areas of theadult mouse brain, we have tested for olfactory functions in HR mice that express only half of the reelin found in wild-type (WT) mice.HR and WT mice were trained to criterion performance of 90% correct in a block of 20 trials on eight distinct simultaneous-cue, two-odordiscriminations. HR mice required significantly more training sessions and made more errors than did WT mice in acquiring the firstolfactory discrimination. Subsequent discriminations were learned equally rapidly by both HR and WT mice. Memory retention for thefinal discrimination was tested 1 week after training and was equally good for both groups. Both HR and WT mice showed equivalentsensitivity in discriminating low concentrations of either ethyl acetate or butanol from non-odorized air. Whether or not the olfactorylearning deficit observed in HR mice is related to the low dendritic spine density or GAD67 reduction observed in hippocampus andcortex are currently under investigation. 2003 Elsevier Science B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Learning and memory: systems and functions

Keywords: Reelin; Odor discrimination; Acquisition; Memory; Learning set; Schizophrenia

1 . Introduction down-regulation in psychosis has been seen in everytelencephalic structure examined [9,11].

Adult mice heterozygous for the ‘reeler’ mutation have Adult heterozygous reeler (HR) mice do not exhibit thebeen proposed as animal models relevant for studies of the severe behavioral or anatomical traits of reeler (null) micemolecular neuropathology associated with schizophrenia but are haploinsufficient for the reelin gene and express[4,13,22]. The gene affected in reeler mice encodes an about half of the reelin mRNA and protein as wild-typeextracellular matrix protein, reelin, that is synthesized by (WT) control mice [13,22], replicating the expressionGABAergic neurons throughout the telencephalon [16,17]. pattern in psychotic patients. HR mice also exhibit severalMeasurements of Reelin mRNA and protein in post other characteristics reminiscent of schizophrenia, includ-mortem brains indicate that the expression of theReelin ing a decrease in dendritic spine density on pyramidalgene is down-regulated by about 50% in schizophrenics neurons in frontal cortex and hippocampus, increasedcompared to non-psychiatric patient controls [9,11]. Simi- packing density of frontal cortical pyramidal cells, down-lar findings in patients with bipolar disorder with psychosis regulation of one form of glutamic acid decarboxylasebut not in patients with unipolar depression suggest that (GAD67), a synthetic enzyme forg-aminobutyric acidthe Reelin deficiency is related to psychosis or vulnerabili- (GABA), and a significant impairment in prepulse inhibi-ty to psychosis [9]. An important finding is that Reelin tion of the auditory startle reflex [13,22].

The localization of reelin near dendritic spines and itshigh-affinity binding to integrin receptors suggest that*Corresponding author. Tel.:11-312-413-4572; fax:11-312-413-reelin may participate in synaptic plasticity associated with4569.

E-mail address: jrlarson@uic.edu(J. Larson). cortical memory functions. Reelin binds with high affinity

0006-8993/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0006-8993(03)02353-9

41J. Larson et al. / Brain Research 971 (2003) 40–46

to integrin receptors in postsynaptic densities of dendritic for photobeam detection of nose pokes. Odor and waterspines [6,17] and likely activates a focal tyrosine kinase delivery were controlled by electrically-driven, Teflon-that participates in the activation of translation of dendritic body solenoid valves (General Valve Co., Fairfield, NJ); aresident mRNAs [5]. Integrin receptors are known to be microcomputer (PC) detected infrared photobeam breaksnecessary for stabilization or ‘consolidation’ of long-term and activated the valves under custom software control.potentiation (LTP) [25], a memory mechanism that may The whole chamber was enclosed and the ceiling wasinvolve dendritic protein synthesis and modifications or equipped with an exhaust fan to remove odorants.genesis of spine synapses. An air dilution system described previously [12] was

It has been suggested that olfactory learning paradigms used to generate odorants. Bottles (30 ml capacity) con-may be particularly useful for studying the cellular and taining odorants (1–4 ml, undiluted) were located down-synaptic basis for cortical memory formation [7,12,15,21]. stream of computer-operated control valves and flowmetersIt is also significant that schizophrenia is often accom- in order to minimize odorant contamination of thesepanied by alterations in olfactory perception, most likely of elements. The clean air supply (bottled zero air, AGA Gascentral origin [3,10,14,24]. The present study was under- Co., Lansing, IL) in each channel was run at 1.8 l /min;taken to determine whether olfactory functions, including odorized air was injected into this stream at 0.2 l /min for alearning and memory, are altered in mice with a reelin dilution of 10%. The bottles and all common tubing in thedeficiency. This hypothesis was tested by comparing the system were made of teflon to facilitate cleaning. Theabilities of heterozygous reeler mice and wild-type mice odorant bottles and the tubing that was exposed to theto: (i) acquire a series of simultaneous-cue, two-odor odorants were replaced as a unit when odor pairs werediscrimination problems, (ii) maintain long-term memory changed. Odorants used in these experiments were selectedfor an odor-reward association, and (iii) detect low con- from a large stock of chemicals obtained from Internation-centrations of two different odors. al Flavors and Fragrances (Union Beach, NJ), Aldrich

Chemical Co. (Milwaukee, WI), and McCormick and Co.(Baltimore, MD). The same odorants were always paired

2 . Materials and methods together. The odor pairs and valences were as follows:strawberry (S1) and banana (S2), propionic acid (S1)

2 .1. Subjects and hexyl octanoate (S2), ethyl lactate (S1) and methylsalicylate (S2), terpinyl acetate (S1) and anisole (S2),

Eight male, wild-type (WT) and nine male, hetero- pineapple (S1) and cherry (S2), maple (S1) and cycla-zygous reeler (HR) mice (3–4 months old) were trained in prop (S2), dihydrojasmone (S1) and cis-3-hexen-1-olan automated olfactory discrimination chamber using (S2), walnut (S1) and butter (S2). In a previous study,procedures reported previously [12]. Both types of mice these odor discriminations were acquired with comparablewere littermates obtained from HR–HR matings in our facility by mice [12].mouse colony and were genotyped as described previously[22]. They were housed individually on a normal 14:10 hlight:dark cycle and maintained on a water deprivation 2 .3. Procedureschedule with access to 1.5–2.5 ml water once per day forat least 5 days prior to and throughout training. Thisschedule reduced body weight by about 20% in the first 2 .3.1. Shapingfew days but maintained the mice at a stable weight A shaping procedure was used to familiarize the mice tothroughout the study. All testing was done during the light the training procedures and to reinforce nose poke re-phase. sponses prior to the introduction of odor cues. This

proceeded in four stages. The first stage consisted of2 .2. Apparatus 20-trial, daily sessions in which the mice were reinforced

with a small drop of water (12.5ml) for a nose poke inThe testing chamber was made of black acrylic and either sniff port at either end of the alley. Each trial was a

consisted of a straight alley 60 cm long and 10 cm wide. maximum of 120 s long and was followed by a 10 sThe two side (long) walls sloped upward and outward at inter-trial interval (ITI). After each reinforced response,an angle of 158 off vertical and were 30 cm high. The end the next reinforcement was contingent on the mousewalls were vertical. At each end (‘East’ and ‘West’) of the making a nose poke in one of the sniff ports at the end ofalley were two circular ‘sniff ports’ (1.5 cm, I.D.) for nose the alley opposite the last correct response, i.e., the mousepoke responses (2 cm from the floor and centered 5 cm was required to traverse the alley repeatedly. If a correctapart) and a single small cup in the floor for water response did not occur within the 120 s period, that trialdelivery. The two sniff ports at the West end of the alley was scored as incorrect and the identical contingency waswere connected to individual air-dilution olfactometers for in effect on the following trial. A lamp at each end of theodor stimulus delivery; all of the sniff ports were equipped alley was lit during the ITI and was extinguished over the

J. Larson et al. / Brain Research 971 (2003) 40–4642

ports at which reinforcement was available during the However, no water was given on either correct or incorrecttrials. Each mouse was trained in this way until it had trials and the ITI after each trial was 30 s.made 90% reinforced responses in one 20-trial session.The second stage of shaping was identical to the first 2 .3.4. Olfactory sensitivity testsexcept that each session had 40 trials and mice were Eight HR and eight WT mice were also trained in antrained to criterion of 90% reinforced trials in one 40-trial odor detection task. Two experiments were conducted, thesession. The third stage of shaping consisted of 20-trial first using ethyl acetate as the test odorant and the secondsessions in which each trial began with the extinguishing using butanol. The training procedure was similar to theof the lamp at the East end of the alley. A nose poke in simultaneous-cue, two-odor discrimination problems de-either of the East sniff ports (a ‘trial initiation’ nose poke) scribed above except that the S1 was the test odorant andwithin 120 s of the trial onset was reinforced with a drop the S2 had no odor. In the first experiment, mice were firstof water, turned on the lamp, and extinguished the lamp at trained to respond to an S1 consisting of the vapor of athe West end of the alley. No response within the 120 s 0.1% aqueous solution (5 ml in a 20 ml bottle) of ethylperiod terminated the trial and was followed by a 10 s ITI. acetate, diluted 10-fold in air. We refer to this as aA nose poke at either West sniff port within 60 s after the concentration of 0.01%. The S2 was a vapor of deionizedtrial initiation nose poke was rewarded with water and water (5 ml in a 20 ml bottle), diluted 10-fold in air. Lowerfollowed by the ITI. Mice were trained to a criterion of concentrations were made by diluting the ethyl acetate90% of trials rewarded at both ends in one 20-trial session. further in deionized water. Mice were trained at the 0.01%The fourth stage of shaping was identical to stage three concentration until all had reached a performance criterionexcept that trial initiation nose pokes were not rewarded of 90% correct in a 20-trial session. Thereafter, mice werewith water, each session was 40 trials, and mice were tested in one or two 20-trial sessions per day using lowertrained to a criterion of three sessions in which 90% of concentrations of the test odorant. Each concentration wastrials were rewarded. run on two consecutive days with the olfactometer chan-

nels containing S1 and S2 reversed on the second day.Tests using a concentration of 0.001% ethyl acetate

2 .3.2. Olfactory discrimination (prepared as 0.01% aqueous solution diluted 10 times inOlfactory discrimination training used a simultaneous- air) were interleaved between tests at lower concentrations

cue, two-odor, forced-choice paradigm. The trial proce- (0.0003%, 0.0001%, 0.00003%, and 0.00001%) in order todures and timing were similar to those of shaping stage maintain performance. The second sensitivity experimentfour except that a trial initiation nose poke at the East end was run in the same way except that the test odorant wasalso activated the delivery of the two discriminative odors butanol. The percentage of correct responses was averagedto the West sniff ports. The spatial position of the two for all test sessions at a given concentration for eachodors (S1 and S2) on any given trial was randomly mouse.determined except that no more than three identical trialscould occur in succession. Nose pokes in the port con-taining the S1 stimulus were rewarded with a drop of 3 . Resultswater; nose pokes in the S2 port were not rewarded. TheITI after a correct response trial was 10 s, that after an Each of the mice reached criterion performance levels atincorrect (S2) or no-response trial was 30 s. Each mouse each of the stages of shaping. There were no apparentwas given a single session of 40 trials per day. Olfactory differences between WT and HR mice in the number ofdiscrimination training continued until criterion perform- sessions required to reach criterion at each stage. Measure-ance of 90% correct responses or better in the last 20 trials ments of response latency at different stages of shapingof a session was reached. The mice were trained succes- also did not reveal any differences between HR and WTsively to criterion on eight different two-odor discrimina- mice. Statistical analysis (two-way, repeated measures,tion problems. analysis of variance by genotype and stage) confirmed the

lack of genotype effect on either of these variables(sessions to criterion:F 50.19,P.0.05; latency:F 51,15 1,15

2 .3.3. Memory tests 0.33, P.0.05) and the absence of interaction betweenAfter each mouse reached criterion performance on the genotype and stage (sessions:F 50.44, P.0.05;3,45

eighth (final) discrimination, it was not tested for a 1 week latency:F 50.27, P.0.05).5,75

period. After this delay, it was given a series of 10 probe Wild-type mice reached criterion performance on thetrials which were identical to the training trials for the final first two-odor discrimination after two to five trainingdiscrimination problem except that no differential rein- sessions (mean53.7560.45) whereas heterozygous reelerforcement was provided. Responses at the port containing mice required 5–13 sessions (mean57.1160.89) to reachthe odor trained as S1 were scored as correct and criterion on the first discrimination (Fig. 1a). This differ-responses to the trained S2 were scored as incorrect. ence was statistically significant (t 53.24, P,0.01). The15

43J. Larson et al. / Brain Research 971 (2003) 40–46

Fig. 1. Acquisition and retention of two-element, simultaneous-cue, olfactory discriminations in heterozygous reeler (HR) and wild-type (WT) mice. (a)Mean (6S.E.M.) number of training sessions required to reach criterion performance on the first discrimination problem. HR mice took longer to acquirecriterion performance that did WT mice. (b) Mean (6S.E.M.) number of errors made before reaching performance criterion for the first discrimination inthe two groups of mice. HR mice committed significantly more errors. (c) Mean (6S.E.M.) errors made before criterion was reached on a series of eighttwo-odor discrimination problems in WT (open circles) and HR (filled circles) mice. (d) Memory scores after a 1-week retention interval. Histogram barsshow the mean (6S.E.M.) percentage of correct responses on unreinforced probe trials given 1 week after acquisition of the final olfactory discriminationproblem. (*P,0.05; **P,0.01).

number of errors made before criterion was reached (Fig. [8,20,21]. The number of discriminations acquired within1b) was also significantly higher in the HR mice than in one session ranged from two to four for WT mice (mean5

the WT mice (t 52.54, P,0.05). 3.0060.27) and from zero to four for HR mice (mean515

Mean errors to criterion for all of the two-odor discrimi- 2.7860.46), a difference devoid of statistical significancenation problems for HR and WT mice are summarized in (t 50.40, P.0.5). There were few instances of truly15

Fig. 1c. A two-way, repeated measures analysis of variance ‘errorless’ learning: three different mice each acquired oneindicated significant main effects of genotype (F 54.84, of the discrimination problems without making any errors1,15

P,0.05) and discrimination problem number (F 5 after the first trial (all three were WT). However, if we7,105

14.30, P,0.0001) but the interaction was not significant define ‘near errorless’ learning [19] as 90% correct per-(F 51.78, P.0.05). Multiple comparisons tests indi- formance on both blocks of 20 trials in the first session of7,105

cated that HR mice made significantly more errors than a problem (excluding trial[1), then WT and HR mice metWT mice only for the first discrimination of the series of this standard on 1.75 (60.25) and 0.89 (60.31), respec-eight (Newman–Keuls test,P,0.01). tively, of the eight discrimination problems. This differ-

As noted above, none of the mice learned the first odor ence only approached statistical significance (t 52.13,15

discrimination in fewer than two training sessions. How- P50.05).ever, all but one of the mice learned at least one sub- The mean latency from the ‘trial-initiate’ nose poke tosequent discrimination within one 40-trial session. Rapid the ‘odor-choice’ nose poke was calculated for the correctlearning is one indication that rodents adopt a ‘win stay– trials in the last block of 20 trials for each odor discrimina-lose shift’ strategy or develop a learning set for odors tion problem for each mouse. Since this was the criterion

J. Larson et al. / Brain Research 971 (2003) 40–4644

run for each problem, performance accuracy was very high(.90% correct) in these trials. The latency includes thetime needed to run the length of the alley, sample theodors, and make a choice and thus could be sensitive todifferences in these ‘performance’ variables. Analysis ofvariance indicated that there was no main effect ofgenotype on response latency (F 51.73,P.0.05). There1,15

was a highly significant effect of discrimination problemnumber (F 511.25, P,0.0001); mice in both groups7,105

became faster with training. However there was no inter-action between genotype and problem number (F 57,105

1.94, P.0.05).Since the only reliable learning-related difference be-

tween WT and HR mice was found in the speed ofacquisition of the first odor discrimination problem, thelatency data for the initial trials of these discriminationswere also examined. The first training session was dividedinto two blocks of 20 trials and latencies for correct andincorrect trials were calculated and analyzed separately.For correct response trials, the first block latencies were8.42 s (61.45) for WT and 9.56 s (60.87) for HR; secondblock latencies for WT were 5.92 s (60.76) and for HRwere 5.81 s (60.76). ANOVA indicated that the decreasein latencies from block one to block two was significant(F 515.55, P,0.01) but the genotype effect was in-1,13

significant (F 50.18, P.0.5) as was the interaction1,13

(F 50.62, P.0.4). Results for incorrect trials were1,13

indistinguishable from those for correct trials.Long-term memory for the eighth (final) olfactory

discrimination problem was tested 1 week after the lasttraining session for that problem. The results are shown inFig. 1d. There were no differences in memory scoresbetween the HR and WT mice (t 50.03, P.0.5). Both15

Fig. 2. Accuracy of HR and WT mice in odor detection. (a) Percentgroups accurately selected the previously-rewarded odor incorrect responses (mean6S.E.M.) for WT (open circles) and HR (filled

the probe trials since scores were significantly above circles) mice in tests of detecting ethyl acetate (S1) from clean air (S2)chance (one-samplet-tests for WT:t 53.46, P,0.05; for at indicated concentrations of the odorant. Dashed line indicates level of7

HR: t 56.18, P,0.001). performance if responses are random. (b) Same as (a) except that test8odorant was butanol.Eight of the WT and eight of the HR mice were tested

for olfactory sensitivity to ethyl acetate and butanol usingthe odor detection task. The results for ethyl acetate areshown in Fig. 2a. Performance accuracy was very high athigh concentrations of the odorant and decreased with centration on detection scores (F 521.72, P,0.0001).4,56

concentration in both groups. Analysis of variance re- The effect of genotype was not significant (F 52.24,1,14

vealed significant main effects of both genotype (F 5 P.0.05), nor was the interaction between genotype and1,14

5.45, P,0.05) and odorant concentration (F 525.02, concentration (F 50.12, P.0.5).4,56 4,56

P,0.0001) on accuracy, but no interaction between thetwo factors (F 50.52, P.0.5). Pair-wise comparisons4,56

(Newman–Keuls tests) did not detect significant differ- 4 . Discussionences in accuracy between HR and WT mice at anyspecific concentration. The main finding of the present experiments was that

The results for butanol are shown in Fig. 2b. The pattern HR mice were slower to learn the first two-odor discrimi-was very similar to that obtained with ethyl acetate: high nation problem. The learning deficit is not global since weaccuracy at high concentrations and a decline in accuracy could detect no difference between HR and control mice inwith decreasing concentration of the odorant. Analysis of acquiring the nose poke task prior to introduction of odorsvariance indicated only a significant main effect of con- and the HR mice also learned subsequent discriminations

45J. Larson et al. / Brain Research 971 (2003) 40–46

as fast as controls. In fact, both groups of mice showed memory in rodents [7,15,18]. The present study supportsstrong evidence of learning set acquisition, with large the relevance of the HR mouse model to psychosis sincesavings in acquiring later problems and some instances of we found that the mutant mice have an olfactory learning‘near errorless’ learning. deficit and olfactory identification deficits are common in

The defect in HR mice relative to controls does appear schizophrenic patients. It will be of interest to determine ifto involve learning since the animals showed high levels of olfactory cortex of HR mice displays the neuropil changesdiscrimination performance once the first discrimination seen previously in frontal cortex and hippocampus.problem was acquired and exhibited good performance onall subsequent discriminations. Tests of odor sensitivityusing two very different odorants failed to reveal differ-

A cknowledgementsences between the two sets of mice making it unlikely thatthe HR mice have a generalized olfactory disturbance.

This research was supported by NIH grants MH062188Finally, comparison of response latencies did not indicateand MH062090. We thank Ms Dagmara Sieprawska forthat the HR mice were impaired significantly in motortechnical assistance.aspects of the task, since they were equivalent to WT mice

in the time required to run the alley, sample the odors, andexecute the nose poke response. The response latencies ofHR mice were no different from those of WT mice even in R eferencesthe initial trials of the first odor discrimination problem.This also suggests that purely sensory or motor factors [1] S. Alcantara, M. Ruiz, G. D’Arcangelo, F. Ezan, L. de Lecea, T.

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