Behavioural Brain Research 116 (2000) 89–98

Research report

Differential participation of the NBM in the acquisition andretrieval of conditioned taste aversion and Morris water maze

Claudia L.R. Gonzalez, Marıa Isabel Miranda, Humberto Gutierrez,Christopher Ormsby, Federico Bermudez-Rattoni *

Instituto de Fisiologıa Celular, Uni6ersidad Nacional Autonoma de Mexico, Apartado Postal 70-253, 04510 Mexico D.F., Mexico

Received 7 February 2000; received in revised form 5 June 2000; accepted 5 June 2000

Abstract

Deficits in both learning and memory after lesions of the cholinergic basal forebrain, in particular the nucleus basalismagnocellularis (NBM), have been widely reported. However, the participation of the cholinergic system in either acquisition orretrieval of memory process is still unclear. In this study, we tested the possibility that excitotoxic lesions of the NBM affect eitheracquisition or retrieval of two tasks. In the first experiment, animals were trained for two conditioned taste aversion tasks usingdifferent flavors, saccharine and saline. The acquisition of the first task was before NBM lesions (to test retrieval) and theacquisition of the second task was after the lesions (to test acquisition). Accordingly, in the first part of the second experiment,animals were trained in the Morris water maze (MWM), lesioned and finally tested. In the final part of this experiment, anotherset of animals was lesioned, then trained in the MWM and finally tested. All animals were able to retrieve conditioned tasteaversion (CTA) and MWM when learned before NBM lesions; however, lesions disrupted the acquisition of CTA and MWM.The results suggest that the NBM and cholinergic system may play an important role in acquisition but not during retrieval ofaversive memories. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Conditioned taste aversion; Morris water maize; Lesions; Acetylcholine

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1. Introduction

In recent years a significant amount of informationhas suggested an important role of the cholinergicsystem in a variety of cognitive processes, in particularlearning and memory [11,17,25,30,36,37,54]. The nu-cleus basalis magnocellularis (NBM) provides the pri-mary cholinergic projection to the cerebral cortex[3,52,53,60] and extensive evidence shows that lesions tothis nucleus impair memory formation in a number oflearning tasks [1,2,4,11,12,16,19,25,34,60]. Additionalevidence has shown that NBM lesions in the neonatal

rat can induce permanent cholinergic hypofunction inadulthood, as well as severe cognitive dysfunction [52].Furthermore, other findings have shown that fetal basalforebrain tissue implanted in the rat neocortex after acholinergic deprivation induced by NBM lesions, pro-duces an important cholinergic reinnervation [18] andpromotes behavioral recovery in different tasks[1,10,26,41,59]. Nevertheless, many researchers have re-ported that the magnitude of decrease in ChAT follow-ing excitotoxic lesions of NBM is unrelated to thedegree of cognitive impairment [4,12,16] and some havereported no effects in conditioned taste aversion [17,28].

Conditioned taste aversion (CTA) is a very robustand widely used model in the study of learning andmemory processes [7,20]. In this behavioral model, ananimal acquires aversion to a taste when it is followedby digestive malaise. The anatomical substrate of CTA

* Corresponding author. Tel.: +525-622-5626; fax: +525-622-5607.

E-mail address: fbermude@ifisiol.unam.mx (F. Bermudez-Rattoni).

0166-4328/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 6 -4328 (00 )00250 -3

C.L.R. Gonzalez et al. / Beha6ioural Brain Research 116 (2000) 89–9890

has been well-described [29] and includes the gustatoryportion of the insular cortex (IC) as a higher projectionsite for taste–visceral information. However, IC lesionsdo not produce obvious deficits in gustatory or gas-trointestinal sensitivity, or taste perception. These pro-cesses seem to take place in the pontine areas of thebrainstem [5,45,61]. Previous experiments in our labora-tory have shown that the adult IC possesses cholineacetyltransferase (ChAT) and cholinesterase activities[32]. Moreover, excitotoxic lesions of the NBM disruptthe ability to learn CTA [34,38], inhibitory avoidancetask in rats [23,34] and spatial maze [27].

Recent studies have shown an increase in cholinergicactivity in the cortex during the acquisition of differentlearning tasks and a gradual decrease in cholinergicactivity after consecutive learning trials [14,38,58]. Thisdata showed that the duration of post-test cholinergicactivation in the cortical NBM-pathway decreased pro-gressively as a function of repeated daily acquisitionsessions. In this regard, evidence from our laboratory[38] has demonstrated an increase in acetylcholine (ACh)release in the IC during acquisition, but not duringretrieval of CTA in free-moving animals. This re-flects the involvement and dependence of the corticalcholinergic system, in the formation of aversive memo-ries. Thus, we decided to explore more extensivelythe role of cholinergic system integrity during the acqui-sition and retrieval of CTA and Morris water maze(MWM).

This report is divided into two experiments. In Exper-iment 1, we tested the effects of N-methyl-D-aspartic(NMDA)-induced lesions in the NBM on acquisitionand retrieval of CTA. Experiment 2 contains the resultsof the effects of NBM lesions on spatial learning(MWM) and the results of NBM lesions on long lastingspatial memory.

2. Method and procedure

2.1. Experiment 1 — effects of NBM lesions onmemory and learning of CTA

In this experiment, we study the biochemical andbehavioral effects of NMDA-induced lesions in theNBM on acquisition and recall of CTA. To test retrieval,intact animals were first trained in conditioned tasteaversion (CTA-1: saccharine aversion) and then lesionedbilaterally in the NBM; 15 days later they were tested forCTA. Ten days later, the same animals were exposed toa second novel taste during a new CTA acquisition trial(CTA-2: saline aversion) and then tested for this flavorby the corresponding retention test (Fig. 1).

2.1.1. SubjectsThirty-two male Wistar rats, weighing 290–310 g at

the beginning of the procedures, were used for thisexperiment. Animals were housed individually with wa-ter and food ad libitum, except during the CTA proce-dure. All behavioral procedures were carried out duringthe light cycle.

2.1.2. CTA-1 acquisitionAnimals were deprived of water for 24 h and habitu-

ated for 7 days to get their daily water intake twice a day(baseline). Distilled water was given to the rat in gradedtest tubes for 10 min and the consumption was mea-sured. On the acquisition day, 0.1% saccharin solutionwas given instead of water as a novel taste (Fig. 1;CTA-1, Day 0). After drinking (20 min), animals wereinjected i.p. with 0.4 M LiCl (127 mg/kg of body weight)to induce gastric malaise, thus completing the acquisi-tion phase of the CTA. After this, water and food wereavailable again ad libitum.

Fig. 1. Schematic representation of the experimental procedures. Surg, surgery; ACQ, acquisition; CTA, conditioned taste aversion; MWM,Morris water maze.

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2.1.3. Surgical proceduresFive days after the acquisition of CTA-1 (Fig. 1; Day

5), animals were randomly divided into three groups:(1) intact control group (CON, n=8); (2) the groupthat received bilateral NMDA-induced lesions in theNBM (NBM, n=16); and (3) a vehicle treated group(SHAM, n=8) which received the same surgical proce-dure with bilateral injections of phosphate buffer 0.1 MpH 7.4 (PBS). The animals were anesthetized withsodium pentobarbital (70 mg/kg). The tips of stainlesssteel dental needles were stereotaxically placed in theNBM using the following coordinates: DV, −7.0 mm;L, + −2.8 mm; AP, −0.8 mm from Bregma; accord-ing to Paxinos and Watson [48]. Each needle wasconnected through a polyethylene tube to a 10-mlHamilton micro-syringe mounted on an automated mi-cro-injector. The needle, tube and syringe were filledback with either a 10 mg/ml solution of NMDA (NBMgroup) or the corresponding vehicle (PBS). Once theneedle was in place, 0.5 ml of the solution was infusedover a 3-min period and the needle was kept in placefor an additional 3 min to allow diffusion. The needlewas then removed and the animals were sutured andallowed to recover.

2.1.4. CTA-1 retention testTen days after the lesion, all animals were again

water-deprived for 24 h and a baseline of water con-sumption was taken for the next 5 days. In the follow-ing morning session (Fig. 1; Day 20), saccharin solutionwas used instead of water and consumption was mea-sured, thereby completing the retention test for theCTA.

2.1.5. CTA-2 acquisitionTen days after the CTA-1 retention test (Fig. 1; Day

30), all animals were trained up for CTA using saline asthe novel flavor (0.15 M NaCl), following the sameprocedure described above.

2.1.6. CTA-2 retention testAnimals were tested 20 days after the acquisition trial

(Fig. 1; Day 50) for CTA-2, as described above.

2.1.7. Histology and biochemistryAfter the behavioral tests were finished, each group

was divided into two groups, the first was subjected tothe diisopropyl-fluorophosphate (DFP)-pharmacohys-tochemical regime [21] (CON, n=5; SHAM, n=5;NBM, n=11). For this procedure, we administered 1.8mg/kg of DFP intramuscularly 2 h before the perfu-sion. For the perfusion, all animals were given anoverdose of sodium pentobarbital and perfused throughthe left ventricle of the heart and the ascending aortawith physiological saline, followed by a 4%paraformaldehyde solution in phosphate buffer. The

brains were then removed and kept in a 30% sucrosesolution for 3 days, after which they were cut in serialcoronal slices (40 mm thick), mounted and then stainedfor acetylcholinesterase histochemistry using a modifiedprotocol from Paxinos and Watson [48]. The slideswere incubated overnight in 50 mM sodium acetatebuffer (pH 5.0), 4 mM copper sulfate, 16 mM glycine,4 mM acetylthiocholine iodide and 0.1 mM etho-propazine. After incubation, the slides were immersedin a developing solution (1% sodium sulfide, pH 7.5)for 10 min and then cover-slipped.

The remaining animals were subjected to ChAT ac-tivity analysis (CON, n=3; SHAM, n=3; NBM, n=5). In this case, animals were decapitated and the brainsrapidly removed onto ice cooled plates. Previous works[21,22,34], showed the existence of a cholinergic path-way arising from the NBM to the IC, so we decided totake samples from this region (IC) for further ChATactivity analysis. Samples of the IC were dissected andChAT activity was measured by high-performance liq-uid chromatography (HPLC), using a modified proto-col from Nitta [44]. Briefly, each tissue was sonicatedfor 30 s in 12.5 ml of cold 25 mM phosphate buffer, pH7.4 containing 0.5% Triton X-100, per gram of wetweight. The homogenate was centrifuged at 12.5 rpmfor 15 min. ChAT activity in the supernatant wasassayed. In the incubation, 50 ml of substrate solutioncontained 10 mM choline chloride, 0.4 mM acetylcoenzyme-A, 0.2 mM neostigmine, 0.3 M sodium chlo-ride and 20 mM EDTA in 0.1 M sodium phosphatebuffer, pH 7.4 and the incubation was carried out at37°C for 20 min. Adding 50 ml of 1 M perhydrochloridein ice stopped the enzyme reaction. After 10 min, 6 mlof 1.0 mM isopropylhomocholine as an internal stan-dard was added and the mixture was centrifuged at 12.5rpm for 10 min at 4°C. An aliquot (150 ml) of the clearsupernatant was taken and passed through a Milliporefilter (0.45 mm). The sample (20 ml) was then injectedinto the HPLC system. The HPLC system consisted ofa polymeric reversed phase column (BAS, ACh-Cholineassay kit) and an enzymatic post column reactor con-taining acetylcholinesterase and choline oxidase (BAS).ACh was converted in the reactor to hydrogen perox-ide, which was then detected electrochemically with aplatinum electrode at 450 mV. A choline oxidase/cata-lase reactor (BAS) was added in order to avoid cholinedetection in the substrate solution.

2.2. Results

2.2.1. Histology and biochemistryFig. 2A,B shows the acetylcholinesterase histochem-

istry results. Lesion sites were characterized by anabsence of magnocellular cell bodies and intense glioticreaction, in contrast to control sites where darklystained cells of the NBM were readily identified.SHAM lesions were characterized by gliosis around the

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Fig. 2. Acetylcholinesterase histochemistry results (A) Control brainat a level containing the NBM; and (B) section of a lesioned animal.On the right, a magnification of the same area. Injections of NMDAproduced a significant loss of magnocellular cells. Results of ChATimmunohystochemistry. (C) Control brain at a level containing theNBM; and (D) at the same level a section of a lesioned animal. Theexcitotoxic lesions produced a significant loss of magnocellularChAT-immunoreactive neurons. Left, scale bar 400 mm; right, scalebar 200 mm.

taste aversion was detected in any of the tested groups.The ANOVA showed that there were no significantdifferences among groups.

2.2.3. Acquisition effects (CTA-2)We expressed the intake of the test for the CTA-2

stimulus as percentages of the baseline of water. Fig. 3Bshows the results for the retention test for the saline. TheANOVA showed that significant differences existedamong groups (F(2,29)=6.97, PB0.01). Post hoc anal-ysis with Fisher test showed that the lesioned group(NBM) presented a significant decrease in aversion tosaline, differing significantly from the CON group (PB0.01). As expected, the CON and the SHAM groupsshowed a marked aversion to saline expressed by the lowpercentage of consumption with respect to the baseline.

2.3. Experiment 2 — effects of NBM lesions on memoryand learning of spatial water Morris task

This experiment was designed to test the role of NBMduring both retention and acquisition of WMM. To thisend, a group of animals was first trained in the MWMand then lesioned bilaterally in the NBM; several daysafter the lesion they were tested for this task.

In order to test NBM lesions upon learning of MWM,a second group of animals was first lesioned and subse-quently trained (14 days post lesion) in the MWM andthen tested for this task (Fig. 1; Experiment 2).

2.3.1. SubjectsThirty-two male Wistar rats, weighing 280–315 g at

the beginning of the procedures, were used for thisexperiment. Animals were housed as in Experiment 1.

2.3.2. MWM acquisitionAll animals were trained on the MWM (Fig. 1, Day

0). A circular pool was used, the inside of which waspainted black and filled to a height of 25 cm with waterat :21°C. A clear Plexiglas platform (10×10 cm) waspresent inside the pool; its top surface was 1 cm beneaththe surface. A trial consisted of placing a rat into thewater at any one out of ten random starting locationswhile the platform remained in the same location (seeRef. [43]).

The behavioral training was conducted on 2 consecu-tive days, each rat receiving ten trials per day. If, on aparticular trial, a rat found the platform, it was permit-ted to remain on the platform for 10 s. The trial wasfinished if a rat failed to find the platform after 45 s, inwhich case the animal was guided to the platform andallow remain there for 10 s. At the end of a trial, the ratwas returned to a holding cage and :40 s elapsed beforebeginning the next trial. The latency to find the platform(escape latency) was recorded. The swimming path foreach rat on every trial was recorded via a video camera

Table 1Results of ChAT activity of the IC

Ach/prot 9S.E.M.Group

Control 4449.54 1181.42Sham 3086.41 388.38Lesion 167.251627.61*

* PB0.05

cannula track, but not by loss of magnocellularneurons. Table 1 shows the results for the analysis ofthe ChAT activity of the IC. A significant main effectof group (F(2,8)=6.212, PB0.03) was found.

2.2.2. Retention effects (CTA-1)To correct for individual baseline drinking, we ex-

pressed the total intake of the novel taste stimulus aspercentages of the baseline of water [7], taking thearithmetical mean of the previous two morning sessionsas 100%. Fig. 3A shows the results for the retention testfor the CTA-1 with saccharin. All the groups showedreduced consumption, indicating that no disruption of

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mounted above the tank. A target scanning system wasable to track the white head of the rat from the blackbackground of the pool. After the ten trials, the animalswere returned to their home cages and the same proce-dure was repeated next day. By the end of the secondday, all animals were placed in their home cages.

2.3.3. Post-training lesioned groupsSixteen animals were trained in the MWM, as de-

scribed above. Three days after the second acquisition ofthe MWM, all animals were randomly divided into threegroups: an intact control group (CON, n=5), an NBM-lesioned group that received NMDA induced lesionsperformed bilaterally (NBM, n=6) and a SHAM group(n=5).

2.3.4. MWM retention testTwo weeks after the surgery, all animals were tested

in the MWM (Day 20). The platform was removed fromthe pool and the animals were allowed to swim for 30s while the swimming path was recorded. After the timeelapsed, they were returned to their home cages.

2.3.5. HistologyAfter the behavioral tests were finished, all animals

were sacrificed and their brains prepared for acetyl-cholinesterase histochemistry using the same protocol asExperiment 1.

2.3.6. Pre-training lesioned groupsAnimals were randomly divided into three groups: an

intact control group (CON, n=5), a SHAM group(n=5) and a lesioned group (NBM, n=6) that receivedNMDA-induced lesions performed as in the previousexperiments.

2.3.7. MWM acquisitionTwo weeks after NBM-lesion (Fig. 1; Day 0), all

animals were trained in the MWM as described in theprevious section.

2.3.8. MWM retention testAfter 20 days of the MWM acquisition (Fig. 1; Day

20), all animals were tested in the MWM retention test.

2.3.9. HistologyAfter the behavioral tests were finished, all animals

were perfused and their brains analyzed for ChATimmunohystochemistry using a modified protocol fromPlaschke and the standard avidin-biotin ABC procedure[22,49].

2.4. Results

2.4.1. HistologyFig. 2C,D, shows the ChAT immunohistochemistry

results. ChAT-immunopositive neurons were foundthroughout the entire basal forebrain, including themedial septal area, vertical and horizontal limbs of thediagonal band of Broca, ventral pallidum and substantiainnominata region that includes the NBM. Injections ofNMDA produced an almost total loss of ChAT-positivecells (Fig. 2D).

2.4.2. Post training lesions: effects on MWM retentionAll the animals learned to find the platform and to

swim directly toward it when released from any startinglocation, which was expected since all animals were notlesioned at the time of MWT acquisition. By the secondday of acquisition, all animals swam directly to theplatform. This improvement was reflected in the declineof latencies to find the platform (Fig. 4A,B). Analysis

Fig. 3. Experiment 1. (A) Retention tests for the conditioned taste aversion (CTA) to saccharine taste acquire before the lesion. (B) Retention testfor the new CTA to saline taste acquired after the lesion. The vertical axis shows the mean percentage of solution intake. **PB0.01.

C.L.R. Gonzalez et al. / Beha6ioural Brain Research 116 (2000) 89–9894

Fig. 4. Summary of the Morris water maze performance. Effects of MWM retention, (A) mean latency over the first ten trial blocks for thecontrol, sham and lesion groups, blocks of two trials; and (B) mean latency over the second ten trial blocks (Day 2). No differences were foundamong the three groups when animals were trained in the task before the NBM lesions. (C) Summary of the time that it took the animals to goto the correct quadrant (escape latency) the day of the probe (20th day after training). No differences were found among the three groups. (D)Summary of swim time in the previously correct quadrant (Dwell) during the probe trial. All animals showed preference for the quadrant wherethe platform used to be.

of variance (ANOVA) on the escape latency revealedno significant differences among the three groups.When the platform was removed on the MWM reten-tion test, all animals swam around the previous correctlocation before heading off to swim in other directions.Two criteria were statistically considered: (1) the timethe animals took to go to the place where the platformwas located right after placing them in the pool (escapelatency) (Fig. 4C); and (2) the time the animals spent inthe quadrant where the platform used to be (dwell)(Fig. 4D). No differences were found in any of thegroups for any of the two criteria.

2.4.3. Pre-training lesions: effects on MWM learningFig. 5A shows that during the first day of the MWM

training, the lesioned group significantly differed fromthe CON group in the seven to eight trial block

(F(2,13)=4.57, PB0.05). The CON and SHAMgroups quickly learned to find the platform and swimdirectly toward it when released from any startinglocation. The ANOVA of the escape latency from thesecond day revealed a significant group main effect intrial blocks one to two, five to six and nine to ten,(F(2,13)=4.79, PB0.05; F(2,13)=5.72, PB0.02;F(2,13)=7.14, PB0.02) (Fig. 5B). Follow-up tests onthe group main effect showed that the CON group wassignificantly better than the NBM group (PB0.05). Nodifferences were found among CON and SHAMgroups. When the platform was removed from the poolon the MWM retention test, the NBM rats showedrandom swimming patterns with high latencies in get-ting to the place where the platform used to be (Fig.5C) and short swimming time in the target regionaround the platform location (Fig. 5D). An ANOVA of

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both criteria (escape latency and dwell time) revealed asignificant group main effect (F(2,13)=2.95, PB0.05;F(2,13)=2.97, PB0.05). In contrast, CON andSHAM groups displayed focused searching for the plat-form and low latencies in arriving at the correct loca-tion. No differences were found among CON andSHAM groups.

3. Discussion

Results of CTA- and WM-memory experiments, inwhich the animals received the lesions after the behav-ioral training, showed that the three different groups(CON, SHAM and NBM) had a significant saccharinaversion, regardless of the treatment received before thetest trial. Similarly, all animals quickly learned to findthe platform in the MWT and during the test, all

animals swam around the place were the platform usedto be. In this way, animals were able to recall bothtasks despite the cholinergic deprivation. Conversely,the learning experiments results, where animals werelesioned before the behavioral training, showed thatNBM-excitotoxic lesions disrupted taste aversion sincetheir saline consumption were similar to the day of thetraining, when the stimuli was presented for the firsttime. The control and SHAM groups showed a strongaversion to saline. Analogous effects were shown in theMWT, where we found that animals with NBM lesionshad trouble in learning the platform location and there-fore, showed equal preference for swimming in all fourdifferent quadrants on the test day.

Our results demonstrate that animals with excitotoxiclesions of the NBM before training in CTA and Morriswater task are unable to perform the tasks when tested,but not when the lesion is made after the acquisition of

Fig. 5. Summary of the Morris water maze performance. Effects on MWM learning, (A) mean latency over the first ten trial blocks. (B) Meanlatency over the second day of training. *PB0.01. The NBM group was significantly impaired when animals were trained in the task after thelesion. (C) Summary of the escape latency 20 days after training. *PB0.01. When lesioned before acquiring the task, NBM animals took almostsix times longer before heading in the correct direction. (D) Summary of Dwell when animals were trained before the surgical procedures.*PB0.05. NBM animals performed at chance levels spending less time in the quadrant than the other two groups.

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the tasks. These results suggest that an intact choliner-gic system is required to acquire but not to recallinformation already learned. Moreover, our biochemi-cal and histological results showed that excitotoxiclesions of the NBM significantly decrease the levels ofChAT in the insular cortex and are in accord withprevious works [21,34], supporting the idea of the exis-tence of a cholinergic pathway from the NBM to theIC. Moreover, it has been demonstrated that NMDA-induced lesions in the NBM reduced the ChAT levels inthe amygdala, another important structure involved inCTA [9,23]. In this regard, we have demonstrated aredundant basal forebrain modulation that implies thatlearning deficits associated to excitotoxic lesions of theNBM are the result of the simultaneous destruction ofthe corticopetal and basoamygdaloid interaction[22,23]. The results reported here also coincide withdifferent studies that have shown a significant decreaseof cholinergic activity in different regions of the cortexafter lesions of the NBM [6,8,31,35,40,52], like theparietal cortex which plays a role in spatial navigation[28]. Nevertheless, the discrepancy between our obser-vations of NBM lesions on taste aversion learning andothers who did not find effects [13,23,28], can be ex-plained for the kind of the excitotoxin used and theactual lesioned area (see Ref. [16]).

It is important to mention that the experiments werecarefully planned in terms of timing (Fig. 1), so wecould make sure that the effects observed were directlyrelated to manipulations of the cholinergic basalo-corti-cal connections and not a matter of forgetfulness. Wehad evidence that animals can recall the MWM up to 2weeks and the CTA for up to 3 weeks [46]. Our resultsshowed that the aversive response of a single pairing ofthe saccharin or saline with lithium chloride is stillstrong even after 20 days. We also showed that animalstrained in the Morris water maze are able to swimdirectly toward the place where the platform was on theday of the training 3 weeks earlier.

The role of the cholinergic system in learning pro-cesses is well-established [24,30,51,55,56] and the litera-ture showing behavioral impairments in different tasksafter lesions of the NBM has been attributed to deficitsin both processes. However, recent results from ourlaboratory demonstrate impairments in acquisition butnot retrieval of CTA and inhibitory avoidance afterbilateral cortical administration of antibodies for NGF[21]. This procedure led to a significant decrement ofcells in the NBM and a near total loss of cortical AChlevels. Furthermore, we have demonstrated that homo-topic fetal brain grafts are necessary for the acquisitionof CTA, but are not required for recalling this aversivestimulus [46]. This result confirms the previous findingswhere brain grafts (cholinergically rich) from the samecortical origin were able to induce recovery in learningCTA after IC lesions and showed a correlation between

ACh release and recovery induced by fetal neocortexgrafts [15,39]. However, in CTA intact animals therewas no significant correlation between ChAT levels inthe IC compared with CTA performance [32,33]. Nev-ertheless, Miranda and Bermudez-Rattoni [38] havedemonstrated an increase in ACh release in the IC ofawake free-moving rats during the consumption of anovel taste; the release of ACh seems to be related toearly stages of learning and the novelty of the stimulus,because temporary blockage of the NBM induced byTTX disrupts the acquisition but not the retrieval ofCTA.

These results strongly suggest that ACh plays animportant role only in the early stages of the CTA; forexample, it has been shown that shortly before theexposure of the rat to a novel taste, the muscarinicantagonist scopolamine blocks conditioned taste aver-sion, but this antagonist has no effect when microin-jected shortly after exposure or during recall [42].Additionally, it has been shown in the IC, but not inother brain areas, that after presentation of unfamiliartaste during conditioned taste aversion training, there isa rapid and marked enhancement of protein tyrosinephosphorylation of a set of neuronal and synapticproteins, including the NMDA receptor subunit 2B(NR2B) [50] a protein recently involved in learningenhancement [57].

The role of ACh in the early stages of learning hasbeen demonstrated in operant behaviors. Orsetti et al.[47] demonstrated that activation of the forebraincholinergic pathways occurs during the acquisition of arewarded operant response (lever pressing), while recallof the same behavior was not associated with theactivation of the cholinergic system. In this regard, ourresults showed that animals trained in the Morris watermaze are impaired only if they are trained after lesionsof the NBM, which represents further evidence of thedifferential role of the cholinergic system in spatialtasks. Recent works have shown that repeated dailytesting of spatial tasks generates differential activationof the cholinergic neurons, which suggests that thecortical projections arising from the NBM are playingan important role only in the early stages of learning[14]. In addition, the same authors have shown thatretrieval of different learning tasks is a cholinergic-inde-pendent process, either by posttraining vulnerability toscopolamine-induced amnesia, where they found thatadministration of the cholinergic antagonist with a 6-hdelay after the training was not effective in causingamnesia [13,58].

In conclusion, our results represent further evidencepointing to the hypothesis of two separate systemsinvolved in learning and memory that is: (1) choliner-gic-dependent in the early stages of memory formation;and (2) independent of the cholinergic system for re-trieval of aversive memories.

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Acknowledgements

Grants from CONACyT 31842-N and DGAPA IN-214399 supported this research. We acknowledge theassistance of Oreste Carbajal and Federico Jandete andgive thanks to Shaun Harris for his text review and toYolanda Dıaz de Castro for preparing the manuscript.

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