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Habituation to a test apparatus during associative learning is sufficient to enhance muscarinicacetylcholine receptor-immunoreactivity in rat suprachiasmatic nucleusvan der Zee, Eelke; Biemans, BAM; Gerkema, Menno P.; Daan, S

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DOI:10.1002/jnr.20300

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Citation for published version (APA):Van der Zee, E. A., Biemans, B. A. M., Gerkema, M. P., & Daan, S. (2004). Habituation to a test apparatusduring associative learning is sufficient to enhance muscarinic acetylcholine receptor-immunoreactivity inrat suprachiasmatic nucleus. Journal of Neuroscience Research, 78(4), 508-519. DOI: 10.1002/jnr.20300

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Habituation to a Test Apparatus DuringAssociative Learning Is Sufficient ToEnhance Muscarinic AcetylcholineReceptor-Immunoreactivity in RatSuprachiasmatic Nucleus

Eddy A. Van der Zee,1,2* Barbara A.M. Biemans,1 Menno P. Gerkema,1 andSerge Daan1

1Department of Animal Behaviour, University of Groningen, Haren, The Netherlands2Department of Molecular Neurobiology, University of Groningen, Haren, The Netherlands

The suprachiasmatic nucleus (SCN) is engaged in modula-tion of memory retention after (fear) conditioning, but it isunknown which pathways and neurotransmitter system(s)play a role in this action. Here we examine immunocyto-chemically whether muscarinic acetylcholine receptors(mAChRs), mediating cholinergic signal transduction in theSCN, are involved. For this purpose, mAChR immunoreac-tivity (mAChR-ir) was studied in the SCN after variousstages of passive shock avoidance (PSA) and active shockavoidance (ASA) training and, for ASA, at various posttrain-ing time points. mAChR-ir was significantly enhanced inSCN neurons as a result of the training procedure, and thenumber of mAChR-positive glial cells in the SCN increasedsignificantly. The increase in mAChR-ir as a result of PSAand ASA training was not due to fear conditioning or thenumber of correct avoidances (in case of ASA training) butrather to behavioral arousal as a consequence of (brief)exposure to a novel environment (the test apparatus). Thisfinding was confirmed by a cage-change experiment inwhich the rats were allowed to stay in a novel cage for15 min or 24 hr. Only the brief exposure to the fresh cagetriggered alterations for SCN mAChRs 24 hr later. Theseresults shed new light on a possible function of the cholin-ergic system in the SCN mediated by mAChRs in relation tomodulation of memory processes and demonstrate thatbehavioral arousal during (the habituation stage of) a learn-ing task is sufficient to alter the mAChR system in the SCN.© 2004 Wiley-Liss, Inc.

Key words: active shock avoidance; cholinergic signaltransduction; glial cells; novelty; passive shock avoid-ance; cage change

The suprachiasmatic nucleus (SCN) of the anteriorhypothalamus is the master circadian clock (biologicalclock) in mammals (Rusak and Zucker, 1979), controllingand synchronizing many biochemical, physiological, andbehavioral processes. Studies in the 1970s suggested that it

plays a role in learning and memory processes; these stud-ies used passive shock avoidance (PSA) and active shockavoidance (ASA) as associative learning tasks (Hollowayand Wansley, 1973a, b; Wansley and Holloway, 1976;Stephan and Kovacevic, 1978). The pathways and neuro-transmitter systems through which the SCN is engaged inmemory processes are not clear, but the cholinergic systemis a possible candidate.

The cholinergic basal forebrain system, acting viamuscarinic acetylcholine receptors (mAChRs), is involvedin various aspects of PSA and ASA learning (Lo Conteet al., 1982; Dekker et al., 1991; Van der Zee and Luiten,1999). The major function of the cholinergic system is toevaluate sensory stimuli for their importance (Sarter andBruno, 1994; Blokland, 1996). Cholinergic neurons areactivated by behaviorally salient and arousing stimuli andplay a key role in behavioral arousal and attention towardstimuli crucial for (associative) learning (Hasselmo, 1995;Acquas et al., 1996; Wenk, 1997). Bina and coworkers(1993) demonstrated that cholinergic neurons of the me-dial septum, nucleus basalis, and diagonal band project tothe SCN. These cholinergic projections consist of fiberscontaining large numbers of varicosities and make axo-somatic and axodendritic synaptic contact with SCN neu-rons (Kiss and Halasy, 1996).

Acetylcholine (ACh) levels in the SCN do not showan endogenous circadian fluctuation but these levels canbe enhanced fivefold within 30 min by arousing an animal

Contract grant sponsor: NWO (Netherlands Organization for ScientificResearch); Contract grant number: GpD 970-10-025; Contract grant spon-sor: Vernieuwingsimpuls; Contract grant number: 016.021.017.

*Correspondence to: Eddy A. Van der Zee, Department of MolecularNeurobiology, University of Groningen Biological Center, Kerklaan 30,9751 NN Haren, The Netherlands. E-mail: e.a.van.der.zee@biol.rug.nl

Received 20 July 2003; Revised 21 July 2004; Accepted 13 August 2004

Published online 5 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.20300

Journal of Neuroscience Research 78:508–519 (2004)

© 2004 Wiley-Liss, Inc.

(Murakami et al., 1984). Cholinergic agonists can phaseshift in a phase-dependent manner the locomotor rhythmsin rats and hamsters (Earnest and Turek, 1985; Meijeret al., 1988; Wee and Turek, 1989; Bina and Rusak, 1996)and neuronal firing activity in rat SCN slices (Liu andGillette, 1996). These actions are predominantly mediatedby mAChRs. Furthermore, cholinergic depletion of theSCN by nucleus basalis lesioning reduces the synthesis andexpression of SCN neuropeptides, indicating a role in theregulation of metabolic activity of SCN neurons (Madeiraet al., 2004), and alters the responses of the SCN to phaseshifting effects of light (Erhardt et al., 2004).

Localization of mAChRs in the SCN has been dem-onstrated by ligand binding (Kobayashi et al., 1978; Rotteret al., 1979; Bina et al., 1998). In contrast to the case formany other neuroactive substances, the expression ofmAChRs in the SCN (like ACh levels) does not revealdaily fluctuations in rats and hamsters (Van der Zee et al.,1991; Bina et al., 1998). The distribution of mAChRs inthe rat SCN has been described in detail by using themonoclonal antibody M35, which binds to all fivemAChR subtypes (Van der Zee et al., 1991; Carsi-Gabrenas et al., 1997). Using subtype-selective antibodies,we reported that SCN neurons express at least m1, but notm2, mAChRs, although both subtypes are present onaxon terminals within the SCN (Van der Zee et al., 1999).

The aim of the current study was to examine 1)whether the SCN is cholinergically engaged by a learningtask via its muscarinic receptors, 2) which aspects of thelearning task are critical, and 3) whether the neurochem-ical changes correlate with learning and memory perfor-mance. Behaviorally induced alterations in the immuno-cytochemical expression of mAChRs in learning andmemory-related brain regions (e.g., hippocampus, neo-cortex, and amygdala) have been described and examinedin detail in rats following PSA and ASA learning, but theSCN was not studied (for review see Van der Zee andLuiten, 1999). Here we analyzed whether associativelearning changes mAChR expression in the SCN. For thispurpose, we examined mAChR-ir in the SCN after var-ious stages of PSA and ASA training (experiment 1) andfor ASA at 10 different posttraining time points (experi-ment 2) in order to determine the time course of thechanges observed in experiment 1. Experiment 3, inwhich rats were placed in a novel cage, served to deter-mine the impact of a brief (15 min) or prolonged (24 hr)exposure to a novel environment on mAChR-ir in theSCN.

MATERIALS AND METHODS

Animals and Assignment to Experimental Groups

Male Wistar rats (body weight �300 g; 121 in total) wereobtained from the breeding colony at our institute (Departmentof Animal Physiology, University of Groningen). Animals weregroup housed (5–7 individuals per cage). Food and water wereavailable ad libitum in a temperature-controlled environment of21°C � 1°C on a 12:12 hr light/dark cycle, with lights on from8:30 to 20:30 hr. PSA and cage changes were performed be-

tween 10:30 and 12:30 [Zeitgeber Time (ZT) 2 and 4, and ASAwas performed between 10:30 and 16:30 hr; ZT 2 and 8]. Theexperiments were approved by the Animal Experimental Com-mittee of the University of Groningen and the animals havebeen acquired and cared for in accordance with the guidelinespublished in the NIH Guide for the Care and Use of LaboratoryAnimals.

In experiment 1, 48 rats were used. For PSA, rats werehabituated to the test apparatus (habituated group HPSA; n � 6);habituated and shocked twice by a mild foot shock (trainedgroup TPSA; n � 10); or habituated, shocked twice by a mildfoot shock, followed by a retention trial (tested group TEPSA;n � 8). Experimentally naıve rats served as controls (naıve groupNPSA; n � 6). For ASA, rats were habituated to the shuttle box(habituated group HASA; n � 6) or habituated and trained byreceiving 60 trials in the shuttle box (trained group TASA; n �6). Experimentally naıve rats served as controls (naıve groupNASA; n � 6). All experimental rats were killed 24 hr after theirlast exposure to the test apparatus.

In experiment 2, 55 rats were used. Fifty rats weretrained for ASA and killed 2, 8, 16, 20, 22, 24, 32, 40, or 48 hror 13 days (13 � 24 hr) after training [trained groupsTASA2, -8, -16, -20, -22, -24, -40, -48 (n � 5); TASA32 (n � 6);TASA13days (n � 4)]. Five rats served as naıve cage controls.

In experiment 3, 18 rats were used. Twelve rats wereplaced in a novel cage in the beginning of the light period eitherfor 15 min (followed by a return to the home cage for 23.5 hr;n � 6) or for 24 hr (n � 6). All rats were killed 24 hr followingthe start of the experiment, and 6 rats served as control rats thatdid not receive a cage change. For cage changing, the animalswere provided with a novel cage, containing fresh sawdust,food, and water. All 18 rats were kept in their home cage for14 days prior to the experiment.

Behavioral Procedures for PSA and ASA

A step-through-type passive avoidance apparatus was usedto investigate one-trial associative learning. Briefly, the apparatusconsisted of a dark compartment (40 � 40 � 40 cm) connectedvia a small opening to an elevated, well-lit platform. Access fromthe platform to the dark compartment was regulated by a slidingdoor in the opening. The floor of the dark compartment wasmade of stainless steel bars, through which a scrambled footshock could be delivered.

Two habituation trials were given on 2 consecutive days,24 hr apart. The first habituation trial (day 1) started with a5-min adaptation to the dark compartment. Immediately after-ward, the rat was placed on the illuminated platform and al-lowed to enter the dark compartment. The sliding door wasthen closed, and the rat stayed for another 3 min in the dark.During the second habituation trial (day 2), the rat was placeddirectly on the illuminated platform. After entering the darkcompartment the sliding door was closed. The animal wasallowed to stay in the dark for 3 min once more. Twenty-fourhours later (day 3), the training trial was performed. The pre-shock entry latency was measured, and the rat received twoscrambled electric foot shocks (0.6 mA a.c. for 3 sec) 30 secapart, shortly after entering the dark compartment with thesliding door closed. The rat was removed from the dark com-partment 30 sec after the last foot shock. The HPSA group was

Novelty and Cholinergic Signaling in SCN 509

also placed in the dark compartment for 30 sec but did notreceive a foot shock. Twenty-four hours (day 4) later, HPSA andTPSA groups were killed as described below, whereas the TEPSAgroup was placed on the platform to measure the postshockentry latency to the dark compartment with a cutoff latency of5 min. Twenty-four hours later (day 5), these animals werekilled. As such, HPSA and TPSA animals were killed 72 hr andTEPSA animals 96 hr after their first exposure to the test appa-ratus, but all experimental subjects were killed 24 hr after theirlast exposure.

Active shock avoidance conditioning, a multitrial associa-tive learning task, was performed in a two-way shuttle box(50 � 26 � 17 cm). All experimental animals were habituatedto the shuttle box for 5 min. The sound of a buzzer served as theconditioned stimulus (CS), presented for 5 sec prior to the onsetof the unconditioned stimulus (US) of a scrambled electric footshock (0.3 mA a.c.) delivered through the grid floor of theshuttle box for 3 sec. When the rats crossed the barrier within5 sec after the onset of the CS presentation, the CS was termi-nated immediately, no foot shock was applied, and the responsewas recorded as an avoidance. If the rat did not make anavoidance, it either received the foot shock for 3 sec or escapedfrom it during the 3-sec delivery by crossing to the other side ofthe shuttle box. Eight seconds after the onset of the CS, both theCS and the US were terminated when a rat failed to make theresponse within that time. The animals received 60 trials in onedaily session, with an intertrial interval varying between 10 and30 sec (20 sec on average).

Immunocytochemical Procedure

Rats were deeply anesthetized with 6% sodium pentobarbitaland transcardially perfused with 100 ml heparinized saline, followedby 300 ml fixative composed of 2.5% paraformaldehyde, 0.05%glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer (PB;pH 7.4). The brains were removed from the skull and cryopro-tected by overnight storage in 30% sucrose in 0.1 M PB at 4°C.Brains were cut coronally on a cryostat into 25-�m sections. (Thebrain of one animal from the TASA32 group was discarded becauseof a failed perfusion.) Thereafter, immunostaining was carried outon the free-floating sections for mAChRs by using the monoclonalmouse anti-mAChR IgM M35 raised against purified bovinemAChR protein as described in detail elsewhere (Van der Zeeet al., 1989). M35 does not discriminate between the five mAChRsubtypes (Carsi-Gabrenas et al., 1997; Van der Zee and Luiten,1999). In short, tissue sections were preincubated for 15 min in0.1% H2O2 in phosphate-buffered saline (PBS) and immersed in5% normal rabbit serum (NRS) in PBS for 30 min to reduceaspecific binding in the following incubation step. Next, the sec-tions were incubated with the first antibody (M35), diluted 1:200 in1% NRS overnight at 4°C under gentle movement of the incu-bation medium. After the primary incubation, sections were rinsedin PBS and again preincubated with 5% NRS for 30 min before thesecondary incubation step in biotinylated rabbit IgG anti-mouse-IgM [mu-chain directed, F(ab�) fraction; Zymed, South San Fran-cisco, CA], diluted 1:200 in PBS for 2 hr at room temperature.Thereafter, the sections were thoroughly rinsed in PBS and incu-bated in streptavidin-horseradish peroxidase (HRP; Zymed) di-luted 1:200 in PBS for 2 hr at room temperature. Finally, afterrinsing in PBS and 0.05 M Tris buffer, the sections were processed

by the diaminobenzidine (DAB)-H2O2 reaction (30 mg DAB and0.003% H2O2/100 ml 0.05 M Tris buffer, pH 7.4), guided by avisual check. Control experiments were performed by the omissionof the primary antibody, yielding immunonegative results. Cresylviolet background staining was performed on M35-processed tissuesections of experimentally naıve animals (NPSA and NASA, TPSA andTASA of experiment 1) to estimate the number of principal SCNcells.

For fluorescent double labeling, sections were labeled forglial fibrillary acidic protein (GFAP; 1:100; Amersham, Arling-ton Heights, IL) with incubation steps as described above, andwith phycoerrythrin-conjugated goat anti-mouse IgG (1:50;Tago, Burlingame, CA) as secondary antibody. After comple-tion of GFAP staining, the sections were immunoprocessed formAChRs as described above, with fluorescein isothiocyanate(FITC)-conjugated streptavidin (1:50; Zymed) instead of HRPconjugate.

Data Analysis and Quantification

The optical density (OD) of M35-ir was determined inthe SCN of all animals (experiments 1 and 2). Five levels alongthe rostrocaudal axis of the SCN were distinguished, with levels1 and 5 corresponding to bregma levels –0.92 and –1.50 mm,respectively, according to the brain atlas of Paxinos and Watson(1986). Rostrocaudal levels 2, 3, and 4 were then equallydistanced from each other between levels 1 and 5. Brain sectionscontaining the SCN were then assigned to these levels. SCNM35-ir OD was determined in brain sections corresponding tothe five levels of all animals from experiment 1 (one brainsection per level per animal; the obtained values of the left andright SCN were averaged). Based on the outcome of this ros-trocaudal analysis, we decided to perform all further OD mea-sures on brain sections corresponding to level 3 (the middleportion of the SCN; two brain sections per animal were ana-lyzed and the values of the left and right SCN were averaged).In addition to the SCN, the OD of M35-ir was also determinedof the medial preoptic area (mPOA), the retrochiasmatic area(RChA), and the lateral hypothalamus (LH) of animals fromexperiment 1. The mPOA was analyzed at bregma level–0.80 mm, and the RChA and LH at bregma level –1.80 mm.The OD was expressed in arbitrary units corresponding to graylevels with a Quantimet 600 image-analysis system. The value ofbackground staining was measured in the corpus callosum,which was relatively devoid of M35-ir. The relative ODwas calculated by the formula [(ODarea – ODbackground)/ODbackground], thus correcting for variability in backgroundstaining between sections.

The number of mAChR-positive SCN neurons andmAChR-positive astrocytes was determined in both the left andthe right SCN of one brain section (rostrocaudal level 3, rep-resenting the mid-SCN region), and the counts were summedper individual [all experimentally naıve (NASA and NPSA; n �12); TPSA (n � 10); TASA (n � 6) rats from experiment 1 wereanalyzed]. Neurons and astrocytes were counted only when thenucleus was present in the section (the longest diameter of theSCN neurons ranged from 7.5 �m to 23.0 �m). The samplearea was delineated by an ocular grid containing a square of250 � 250 �m. Total number of SCN neurons in this sample

510 Van der Zee et al.

area was based on cresyl violet background staining as men-tioned above.

Statistical Evaluation

A Wilcoxon signed rank test (WSR), a one-way ANOVAfor effect of treatment followed by post hoc pair wise compar-ison (Dunn’s test), or a two-way ANOVA followed by post hocmultiple comparison (Bonferroni) was used for statistical analysiswhen appropriate. P � 0.05 was used as an index of statisticalsignificance in all cases. All tests were applied two-tailed, and alldata are presented as mean � SEM.

RESULTSmAChR-ir in Experimentally NaıveRats (Experiment 1)

Loosely scattered mAChR-positive cells with asso-ciated dendritic profiles were found within the SCN

(Fig. 1a,d) as well as in the mPOA and, to a lesser degree,in the RChA and LH, as described earlier (Van der Zee etal., 1989, 1991). The mAChR-positive cells belonged to asubpopulation of relatively large, often somewhat elon-gated, monopolar or bipolar neurons. No apparent differ-ences in distribution or morphological appearance of thesecells were found in the dorsoventral axis of the SCN.mAChR immunostaining, as determined by OD, differedlittle along the rostrocaudal axis in experimentally naıverats (see Fig. 3). No differences in any of these measureswere obtained between the NPSA and the NASA groups.Some immunolabeled neurons were present in the opticchiasm. Few mAChR-ir glial cells were found in theSCN, but those observed often were associated with thelarge blood vessels or found at the border of the opticchiasm.

Fig. 1. Photomicrographs of muscarinic acetylcholine receptor immunoreactivity in the SCN ofbehaviorally naıve rats (NPSA; a,d) 24 hr after passive shock avoidance retention test (TEPSA; b,e)and 24 hr after active shock avoidance training (TASA; c,f); d, e, and f are enlargements of theSCN area shown in a, b, and c, respectively. 3V, third ventricle; OC, optic chiasm. Scale bars �48 �m in a, b, and c, 12 �m in d, e, and f.

Novelty and Cholinergic Signaling in SCN 511

Behavior and Alterations in mAChR-ir inExperimentally Tested Rats (Experiment 1)

PSA: behavior. All rats displayed exploratory be-havior during the habituation period to the test apparatus.PSA preshock entry latency did not differ among thevarious groups (Fig. 2A; HPSA 5.2 � 1.4 sec; TPSA 3.5 �0.8 sec; TEPSA: 3.3 � 0.7 sec; one-way ANOVA: P �0.05). The memory retention test of the eight TEPSA

animals revealed that two rats did not enter the darkcompartment before the 5 min cutoff time, whereas theremaining six animals stayed on the platform for 156.4 �16.6 sec on average (Fig. 2A). Postshock entry latency wassignificantly increased compared with preshock entry la-tency (P � 0.01; WSR test), indicating that the animalsassociated the dark compartment with the foot shockdelivery.

PSA: immunostaining. Enhanced mAChR-irwas found in SCN cell soma and their dendritic profiles inHPSA, TPSA, and TEPSA rats (a TEPSA rat is shown inFig. 1b,e). HPSA, TPSA, and TEPSA groups revealed sig-nificantly enhanced mAChR-ir compared with the be-haviorally naıve group (Fig. 3A; one-way ANOVA effectof experimental treatment: P � 0.01). Although the ODseems to increase further in the trained and tested groups(TPSA and TEPSA) compared with the habituated group(HPSA), post hoc testing revealed that all groups differedsignificantly from the naıve group (HPSA P � 0.01; TPSAand TEPSA P � 0.05) but that no significant differenceswere present among the HPSA, TPSA, and TEPSA groups.Densely stained cells were homogeneously distributedthroughout the dorsoventral axis of the SCN. The numberof mAChR-ir neurons in TEPSA rats increased signifi-cantly compared with that in naıve rats (Table I). Thestaining intensity for mAChRs was more pronounced atthe anterior than at the posterior part of the SCN (two-way ANOVA main effect of SCN level: P � 0.05; and ofexperimental treatment: P � 0.001), as revealed by ODmeasures (analyzed for the TEPSA group; Fig. 4A), but therostrocaudal “gradient” was present only in the TEPSAgroup (interaction effect of SCN level � treatment: P �0.05). Post hoc testing revealed that mAChR staining inTEPSA rats differed significantly from that in naıve rats atall levels. No correlation was found between the postshockentry latency as a measure of memory retention (rangingfrom 83 to 300 sec) and the OD values for mAChR-ir inthe SCN (Spearman rank correlation: r � 0.13; P � 0.68).

ASA: behavior. All animals exposed to the two-way shuttle box displayed exploratory behavior. DuringASA training, a gradual increase in number of correctavoidances occurred over time that varied considerablyamong individuals. On average, rats reached 41.51 � 3.22correct avoidances (69.2%) within 60 trials (data notshown).

ASA: immunostaining. An increase in mAChR-ir was found in the SCN in all HASA and TASA rats, andthis was comparable in distribution and appearance to thatfound for PSA (a TASA rat is shown in Fig. 1c,f). Nodifferences in ir were obtained with respect to time oftraining, which varied from ZT2 to ZT8. The number ofmAChR-ir neurons in TASA rats increased significantlycompared with that in naıve rats (Table I). The impact of5 min of habituation to the shuttle box was examined inthe HASA group and of an additional ASA training sessionin de TASA group. Figure 3B shows that the exposure tothe test apparatus, and further training, induced a signifi-cant increase (one-way ANOVA: P � 0.01) in mAChR-

Fig. 2. Performance in behavioral tasks. A: Passive shock avoidancetest. Preshock latencies (black bars) to enter the dark compartment aredisplayed for the three experimental groups (HPSA: habituated rats;TPSA: trained rats; TEPSA: rats receiving training and test). Postshocklatency (gray bar) is displayed for the TEPSA group receiving the testtrial 24 hr later. The dots at 300 sec represent the two rats that did notenter the dark compartment before the cutoff criterion. **P � 0.01.B: Active shock avoidance test. Number of correct avoidances isplotted per block of 10 trials. The performance of all rats of experiment2 together (n � 50) is displayed as well as the performance after splittingthe group into responders and nonresponders (rats making three orfewer correct avoidances in the last block of 10 trials).

512 Van der Zee et al.

ir compared with NASA animals. Post hoc testing, how-ever, revealed that HASA rats did not differ from TASA rats(one-way ANOVA: P � 0.05). A significant increase inimmunostaining in TASA rats as measured by OD was seenthroughout the rostrocaudal axis of the SCN (Fig. 4B;two-way ANOVA effect of experimental treatment: P �0.001; no effect of SCN level) compared with NASA rats.The distribution along the rostrocaudal axis was similar inboth groups (no interaction effect of SCN level � exper-imental group). This increase in mAChR-ir in TASA ratswas less pronounced and showed less variation along therostrocaudal axis than the increase seen in PSA-testedanimals, although it did not differ significantly from that ofTEPSA for all SCN levels. No correlation was foundbetween the number of correct avoidances and the ODvalues in the SCN (Spearman rank correlation: r � 0.41;P � 0.50).

Alterations in mAChR-ir in the Medial Preopticand Retrochiasmatic Area and Other BrainRegions (Experiment 1)

In addition to changes in mAChR-ir in the SCN,other brain regions showed changes as well. Within thehypothalamus in the near vicinity of the SCN, changeswere observed in the mPOA, RChA, and LH of TEPSAand TASA animals compared with NPSA animals. ODmeasures revealed a significant increase for both learningtasks in the mPOA and RChA (one-way ANOVA effectof experimental procedure: P � 0.01 for both regions),but not in the LH (Fig. 3C). The increase in the RChAwas less pronounced and more variable among individualsthan that of the mPOA (RChA: TEPSA and TASA vs.NPSA: P � 0.01 and P � 0.05, respectively; mPOA:TEPSA and TASA vs. NPSA: P � 0.001). Though beyondthe scope of the current study, distinct patterns of thechanges in mAChR-ir were present in the forebrain afterPSA and ASA performance. Enhanced mAChR-ir wasseen in the SCN in both paradigms, but characteristic andlocalized changes were also found elsewhere after eachtask. In PSA-tested rats, changes were present in neocor-tex (in a columnar fashion; Van der Zee et al., 1994; Vander Zee and Luiten, 1999), piriform cortex, and thalamicregions (e.g., the ventrolateral thalamic nucleus and ven-tral posteromedial thalamic nucleus), whereas no changeswere found in the paraventricular nucleus or (dorsal) hip-pocampus or various other brain regions. In ASA-testedrats, changes were seen in neocortex (but to a lesser extentthan after PSA), amygdala (decrease in the central nucleus

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Fig. 3. Relative optical density of mAChR-ir at rostrocaudal level 3 ofthe SCN (A,B). A: PSA. Habituated, trained, and retention-tested ratsare compared with the values for naıve rats. B: ASA. Habituated andtrained rats are compared with the values for naıve rats. C: Relativeoptical density of mAChR-ir in mPOA, RChA, and LH of naıve andPSA- or ASA-trained rats. Significant increases compared with exper-imentally naıve rats are indicated by asterisks: *P � 0.05, **P � 0.01,***P � 0.001.

Novelty and Cholinergic Signaling in SCN 513

and increase in corticomedial nucleus; Roozendaal et al.,1997; Van der Zee and Luiten, 1999), paraventricularnucleus, dentate gyrus of the dorsal hippocampus, andthalamic regions (e.g., the ventrolateral thalamic nucleusand ventral posteromedial thalamic nucleus), whereas nochanges were found in various other brain regions.

The mPOA and RChA in habituated rats (HPSA andHASA) revealed changes in mAChR-ir similar to thoseseen in animals that were used for associative learning(TEPSA and TASA). In contrast, habituated rats revealed noapparent changes in the other brain regions describedabove, which revealed associative-learning-characteristicalterations in mAChR-ir (Van der Zee and Luiten, 1999).

Time Course of Enhanced mAChR-ir in the SCNof ASA-Trained Rats (Experiment 2)

Behavior. The number of avoidances varied con-siderably among animals, ranging from 0% correct avoid-ances (two animals; one of the TASA20 and one of TASA32group) to 90% correct avoidances. A distinction was madebetween slow and fast learners (respectively referred to asnonresponders and responders) on the basis of the numberof avoidances in the last block of 10 trials (Fig. 2B). Withall groups of experiment 2 taken together, rats reached34.06 � 12.18 correct avoidances (56.8%) on averagewithin 60 trials (Fig. 2B).

Immunostaining. Brain sections were studied atrostrocaudal level 3 for mAChR-ir at 2, 8, 16, 20, 22, 32, 40,and 48 hr after training as well as 13 days (13 � 24 hr)posttraining (Fig. 5A). OD values of SCN mAChR-ir in theNASA and TASA24 rats in this experiment (experiment 2)were comparable to those of naıve and ASA-trained rats ofexperiment 1, replicating the previous findings. A signifi-cant effect of time point was found (one-way ANOVA:P � 0.001). Post hoc tests showed that TASA2, TASA8, andTASA16 groups did not differ from NASA animals and thatTASA20 was significantly higher (P � 0.05) than at allprevious time points. Furthermore, TASA24, TASA32, andTASA48 animals differed significantly from all time pointsbefore 24 hr (P � 0.01 in all cases), but not from eachother. Although TASA13days had an intermediate level ofrelative OD, this time point did not differ from either

naıve or TASA24 animals (P � 0.1). It should be noted,however, that, in the four animals of the 13-day group,two had a high relative OD (4.96 and 6.46) resemblingTASA24, TASA32, TASA40, and TASA48, whereas the othertwo were low (0.92 and 0.95), resembling naıve animals.

In addition to OD measures, the numbers ofmAChR-ir astrocytes were counted at rostrocaudal level 3(Fig. 5B). Immunoreactive astrocytes were clearly discern-ible from neurons because of higher staining intensity andcharacteristic morphology (this study; see also Van der Zee

Fig. 4. Relative optical density of mAChR-ir of five equidistant ros-trocaudal levels through the SCN of PSA-tested (A) and ASA-trained(B) rats compared with experimentally naıve rats. In case of PSA, asignificant increase in mAChR-ir was found at levels 1–4, representingthe most caudal part of the SCN. In case of ASA, a significant increasein mAChR-ir was found at all levels. Significant increases comparedwith experimentally naıve rats, as revealed with post hoc testing, areindicated by asterisks: *P � 0.05, **P � 0.01, ***P � 0.001.

TABLE I. Number of mAChR-ir Neurons in the Midregion ofthe SCN (Rostrocaudal Level 3) and the Estimated Percentage ofTotal SCN Neurons That These Cells Represent†

mAChR-positive neurons

Number

Percentageof totalneurons

Naıve rats (NASA NPSA; n � 12) 16.6 � 5.0 8PSA-trained rats (TPSA; n � 10) 110.2 � 17.6*** 51ASA-trained rats (TASA; n � 6) 84.6 � 16.1** 39†Number of mAChR-ir cells was significantly enhanced after PSA and ASAtraining (Experiment 1) compared with naıve rats.**P � 0.01.***P � 0.001.

514 Van der Zee et al.

et al., 1991, 1993). Expression of mAChRs in SCN as-trocytes is demonstrated in Figure 6 by way of fluorescentdouble labeling. Enhanced numbers of immunopositiveastroglial cells (one-way ANOVA main effect of timepoint: P � 0.001) were encountered in all TASA groupscompared with NASA animals (P � 0.05 in all cases), withthe exception of TASA13days. In the latter group, the twoanimals with high mAChR-ir OD also revealed highnumbers of mAChR-positive astrocytes (202 and 184),whereas the two individuals resembling naıve animals forOD also had low numbers of astrocytes (43 and 39). Theastroglial changes were primarily limited to the SCN andwere not obviously present in the mPOA and RChA (datanot shown).

The variation in number of correct avoidances en-abled us to analyze the potential relationship between theOD and the rate of acquisition (expressed as number ofcorrect avoidances). Because alterations in mAChR-irwere fully expressed at 24 hr posttraining and later timepoints, whereas, at 20 hr posttraining and earlier timepoints, no changes had occurred yet, correlations werestudied in two clusters of animals. The first cluster con-sisted of TASA2, TASA8, TASA16, and TASA20 and thesecond cluster of TASA24, TASA32, TASA40, and TASA48. Inboth cases, no significant correlation was found (cluster 1:r � 0.2648; P � 0.258; cluster 2: r � 0.1729; P � 0.447),indicating that neither the initial nor the enhanced level ofmAChR-ir relates to acquisition.

mAChR-ir and a Novel Cage (Experiment 3)Cage changes were performed during a brief period

(15 min) for comparison with the habituation periods in atest apparatus during a learning task or for 24 hr forcomparison with classic cage change experiments andnonphotic effects on circadian rhythms (Mrosovsky,1988). The OD and the number of astrocytes formAChR-ir were determined in the mid-SCN region of

Fig. 5. A: Relative optical density of mAChR-ir at rostrocaudal level3 of the SCN of naıve rats and ASA-trained rats killed at differentposttraining time points. B: Number of mAChR-positive astrocytes inthe SCN at rostrocaudal level 3 of the same rats as shown in A.Significant increases compared with naıve animals are indicated byasterisks: *P � 0.05, **P � 0.01.

Fig. 6. Fluorescent double labeling of mAChRs and GFAP in theSCN. Expression of mAChRs in astrocytes is demonstrated by colo-calization of GFAP and mAChRs (large arrows). Small arrows point tomAChR-positive neurons.

Novelty and Cholinergic Signaling in SCN 515

all animals (Table II). A cage change significantly en-hanced both mAChR-OD and the number of mAChR-positive astrocytes in the 15-min group (P � 0.01) but notin the 24-hr group.

DISCUSSIONThe results of the present study demonstrate that the

cholinergic system in SCN neurons and astroglial cells isaffected as a result of the training procedure for passive andactive shock avoidance learning, two forms of associativelearning. The essential findings of the present study are asfollows. 1) Enhanced mAChR-ir takes place in SCNneurons and glial cells after associative learning. 2) Theincrease in mAChR-ir following PSA or ASA training isnot due to fear conditioning per se (i.e., the association ofthe foot shock with the place of delivery in PSA, or thecoupling of a tone and a foot shock in ASA) but rather toexposure to a novel environment (the test apparatus). 3) Abrief but not a prolonged exposure to a novel environ-ment (a cage change) does affect SCN mAChR-ir in a waysimilar to a brief exposure to a test apparatus. 4) Theincrease in mAChR density is not related to performancemeasures of learning and memory, insofar as no correla-tions were found in any of the experiments. 5) Theincrease in SCN mAChR-ir is differentiated in time forglial cells and neurons; glial cell changes are seen 2 hr afterthe last training trial and neuronal changes not before the22 hr posttraining time point.

Anatomical Aspects of Enhanced mAChR-irImmunocytochemical examination of the distribu-

tion of the m1 subtype of mAChRs has shown that nearlyall SCN cells express mAChRs in experimentally naıverats (Van der Zee et al., 1999). In contrast, only a smallsubset of relatively large SCN cells is strongly M35 posi-tive, representing approximately 8% of the SCN cell pop-ulation. M35 binds to internalized mAChRs, with noselectivity to mAChR subtype (Raposo et al., 1987; Carsi-Gabrenas et al., 1997; Van der Zee and Luiten, 1999). Thissuggests that most SCN cells have few internalizedmAChRs (experimentally naıve home-cage control situ-

ation) and that this number increases significantly after (abrief) exposure to a novel environment. After training forPSA or ASA, the proportion of M35-positive cells in-creased to about 40–50%. This finding implies that thechanges in the SCN are massive but presumably not yetmaximal; approximately 50–60% of the SCN cells seemto be unaffected by the training procedure.

The induction of mAChR changes does not seem todepend on time of day of training or perfusion within thelight period. In the ASA experiments, the time of day oftraining and perfusion varied between ZT2 and ZT8.Although the physiology of the SCN might be changedwithin this period, no differences in mAChR-ir wereobserved in relation to ZT. This is in line with previousobservations that circadian variation in mAChR immu-nostaining when using the M35 antibody was not presentin the SCN (Van der Zee et al., 1991; unpublished ob-servations). Similarly, Bina and coworkers (1998) showedthe absence of daily fluctuations in mAChRs in Syrianhamster SCN. It remains to be tested, however, whethertraining in the dark period induces similar changes in theSCN.

A strong increase in the number of mAChR-positiveglial cells is found after ASA training (and PSA trainingalike; data not shown). This change was induced within2 hr after training, suggesting that it is a direct conse-quence of the behavioral arousal of the animals in the testapparatus. As with the neuronal changes seen after 20–24 hr, high numbers of immunostained glial cells remainpresent up to (at least) posttraining time 48 hr. The declineof this enhanced immunostaining seems to occur concur-rently in glial cells and neurons, as seen in two of the fouranimals studied 13 days after training. Glial cells are sup-posed to be part of a synchronizing mechanism in theSCN, possibly through intercellular Ca2 waves (Van denPol et al., 1992; Welsh and Reppert, 1996), and/or mayregulate SCN glutamate content (Lavaille and Serviere,1995). Our current knowledge of neuron–glia interac-tions in SCN function is limited (Morin et al., 1989;Prosser et al., 1994; Tamada et al., 1998), but the currentresults potentially point to a role of astroglia in regulatingSCN function in relation to novelty and arousal. It istempting to speculate that these affected glial cells inducethe neuronal changes seen almost one circadian cycle later,because these neuronal alterations are initiated intrinsicallyin the SCN, without further external stimuli. Evidently,the relation between the astroglial and neuronal changes inthis context awaits further investigation.

Aspects of Fear Conditioning Causing EnhancedmAChR-ir in the SCN

The results of the different aspects of PSA trainingshow that the changes of mAChRs in the SCN are duenot to the conditioning aspect but rather to the initialnovelty aspects of the learning task. Habituation of theanimals to the PSA test apparatus enhanced mAChR-ir tothe same extent as seen in animals receiving the additionalfoot shock or this foot shock in combination with aretention trial 24 hr later. Similarly, the results of ASA

TABLE II. Optical Density of mAChR-ir and Number ofmAChR-Positive Astrocytes in the Midregion of the SCN of RatsThat Received a Novel Cage for 15 Minutes or 24 Hours andTheir Home-Cage Controls†

mAChR OD

Number ofmAChRpositive

astrocytes

Home-cage controls (n � 6) 1.29 � 0.16 27.0 � 2.9815 Minutes novel cage (n � 6) 4.41 � 0.91** 126.3 � 13.9**24 Hours novel cage (n � 6) 1.36 � 0.21 31.6 � 3.18†Optical density of mAChR-ir and number of mAChR-positive cells in theSCN are significantly changed as a consequence of placement in a novelcage for a brief period.**P � 0.01.

516 Van der Zee et al.

show that habituation to the shuttle box triggers thechanges. Transferring a rat from its home cage to a freshcage for 15 min (but not 24 hr) also induced mAChRalterations. Apparently, brief exposures to a novel envi-ronment are sufficient to set in motion the mAChRchanges, whereas prolonged exposure to a novel environ-ment prohibits or counteracts this change. Interestingly, ashort-lasting, significant increase in c-fos mRNA expres-sion in the SCN is induced within 30 min following thebrief exposure to a novel environment (Emmert and Her-man, 1999), showing SCN sensitivity for novelty throughthe expression of immediate early genes.

There was no correlation between learning andmemory performance and mAChR-ir in the SCN. Onemight argue that, although the SCN may be permissive for(time-dependent) memory retention (Stephan and Ko-vacevic, 1978), this nucleus most likely does not play a keyrole in acquisition processes or consolidation of memoryfor conditioning. The SCN may modulate memory re-tention by either facilitating memory retention at 24-hrintervals following training or inhibiting memory reten-tion at other time points (Stephan and Kovacevic, 1978),possibly by an output signal generated in the mAChR-positive cells (Biemans, 2003). In contrast to the SCN,however, changes in mAChR-ir in the amygdala afterASA and PSA training are associated with number of footshocks, and the temporal dynamics of these alterations(neuronal changes occur within 2 hr in the hippocampusand within 8 hr in the amygdala) differ from those seen inthe SCN (Van der Zee et al., 1997; Roozendaal et al.,1997; Van der Zee and Luiten, 1999). This indicates thatmAChRs in brain regions other than the SCN are moreclosely involved in conditioning aspects and learning andmemory processes.

Fear conditioning experiments in mice revealed acircadian modulation of context-dependent but not tone-cued fear conditioning (Valentinuzzi et al., 2001), alsosuggesting a link between SCN functioning and environ-mental cues. Circadian modulation of the expression offear memory persists for several day, even under constantlight conditions, demonstrating the endogenous nature ofthe rhythms (Chaudhury and Colwell, 2002). These stud-ies clearly indicate modulation of fear memory by thecircadian system in mice, although the expression of theunderlying rhythm differs from that in rats (Holloway andWansley, 1973a, b; Wansley and Holloway; 1976).

M35 Binding Characteristics and PossibleFunctional Consequences of Enhanced mAChR-irin the SCN

Enhanced mAChR-ir detected by M35 binding haspreviously been explained by internalization of themAChRs and the subsequent exposure of the epitope forthe monoclonal antibody M35 (for review see Van derZee and Luiten, 1999, and references therein). Hence, themonoclonal antibody M35 detects neurons affected by theexperimental treatment of the animal, in this study bybriefly exposing a rat to either the PSA or the ASA testapparatus. The responsible mechanisms and pathways in

the SCN are unknown, but mAChRs are internalizedwhen phosphorylated. Stimulation of mAChRs by AChresults in such phosphorylation through the activation ofdifferent kinases, pointing to cholinergic signal transduc-tion as the prime candidate. In this context, it is relevant tonote that, in old rats with a deteriorated cholinergic sys-tem, no mAChR changes could be induced in the SCNby ASA training (Biemans et al., 2003). However, itshould be noted that other neurotransmitters (e.g., gluta-mate) acting via the same types of kinases can also lead tochanges in the phosphorylation state of mAChRs (Van derZee and Luiten, 1999).

Previously, we described the functional impact ofenhanced mAChR-ir in the hippocampus, neocortex, andamygdala (Van der Zee and Luiten, 1999). InternalizedmAChRs make cholinoceptive cells less sensitive to AChand, hence, less sensitive for behavioral arousal accompa-nied by enhanced levels of ACh. Enhanced mAChR-irdetected by M35 binding also reflects enhanced intrinsicinformation processing among the affected neurons withina brain region (Hasselmo and Bower, 1993; Hasselmo,1995; Van der Zee and Luiten, 1999). In case of the SCN,it is of interest to consider the affected SCN cells as aneuronal substrate for time stamping. The time of eventinitiates, through cholinergic signaling, a circadian outputrhythm in the SCN neurons phase locked to the time ofevent instead of phase locked to the light-dark cycle,employing, for example, one or more clock genes and apeptide as output. The internalized mAChRs block fur-ther cholinergic input and, therefore, protect these cellsfrom additional input by which the initiated rhythm couldbe disrupted. Indeed, different phase relationships betweenSCN neurons have been recorded (Herzog et al., 1998;Schaap et al., 2003; Quintero et al., 2003). However,further experiments are required to begin to test such ahypothesis.

It is unlikely that the mAChR changes are related to(nonphotic) phase shifts. PSA training performed underfree-running conditions did cause SCN mAChR changes,but no phase shifts in locomotor activity were observed(Biemans et al., unpublished observations). Cage changes(those of 24 hr) can induce nonphotic phase shifts (Mros-ovsky, 1988) but do not lead to SCN mAChR changes.

Functionally, mAChR changes in the SCN mayindicate that familiarization with a novel environment in ashort and restricted moment in time is placed in a tem-poral, circadian context. This mechanism may be impor-tant for time–place association and incorporation of dailyhabits in the behavioral repertoire of an animal (Daan,1981). One may speculate that mAChR-positive neuronsof the SCN link incoming environmental information totime of day. Individual cholinergic neurons of the basalforebrain system innervate both memory-related brain re-gions (e.g., hippocampus and neocortex) and the SCN(Bina et al., 1993). Interestingly, SCN-lesioned rats arebehaviorally aroused for much longer in a novel environ-ment than intact rats (Buijs et al., 1997), suggesting that

Novelty and Cholinergic Signaling in SCN 517

SCN processing of environmental novelty has adaptivevalues.

The present results indicate a new functional aspectof cholinergic signal transduction in the SCN. Additionalresearch, however, is needed to shed more light on therelevance and mechanisms of this finding.

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