www.elsevier.com/locate/yexnrExperimental Neurology 189 (2004) 162–172

Cognitive and neurological deficits induced by early and prolonged

basal forebrain cholinergic hypofunction in rats

Laura Ricceri,a,* Luisa Minghetti,b Anna Moles,c Patrizia Popoli,d Annamaria Confaloni,b

Roberta De Simone,b Paola Piscopo,b Maria Luisa Scattoni,a

Monica di Luca,e and Gemma Calamandreia

aSection of Behavioural Neurosciences, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita, 00161 Rome, ItalybSection of Degenerative Inflammatory Neurological Diseases, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita, 00161 Rome, Italy

c Instituto di Neuroscienze, Laboratorio di Psicobiologia e Psicofarmacologia, CNR, 43 00137 Rome, ItalydSection of CNS Pharmacology, Department of Drug Research and Safety, Istituto Superiore di Sanita, 00161 Rome, Italy

eDepartment of Pharmacological Sciences, University of Milano, 9 20133 Milan, Italy

Received 1 April 2004; revised 17 May 2004; accepted 17 May 2004

Available online 2 July 2004

Abstract

In the present study we examined the long-term effects of neonatal lesion of basal forebrain cholinergic neurons induced by

intracerebroventricular injections of the immunotoxin 192 IgG saporin. Animals were then characterised behaviourally, electrophysiolog-

ically and molecularly. Cognitive effects were evaluated in the social transmission of food preferences, a non-spatial associative memory task.

Electrophysiological effects were assessed by recording of cortical electroencephalographic (EEG) patterns. In addition, we measured the

levels of proteins whose abnormal expression has been associated with neurodegeneration such as amyloid precursor protein (APP),

presenilin 1 and 2 (PS-1, PS-2), and cyclooxygenases (COX-1 and COX-2). In animals lesioned on postnatal day 7 and tested 6 months

thereafter, memory impairment in the social transmission of food preferences was evident, as well as a significant reduction of choline

acetyltransferase activity in hippocampus and neocortex. Furthermore, similar to what observed in Alzheimer-like dementia, EEG cortical

patterns in lesioned rats presented changes in a, h and y activities. Levels of APP protein and mRNA were not affected by the treatment.

Levels of hippocampal COX-2 protein and mRNA were significantly decreased whereas COX-1 remained unaltered. PS-1 and PS-2

transcripts were reduced in hippocampus and neocortex.

These findings indicate that neonatal and permanent basal forebrain cholinergic hypofunction is sufficient to induce behavioural and

neuropathological abnormalities. This animal model could represent a valid tool to evaluate the role played by abnormal cholinergic

maturation in later vulnerability to neuropathological processes associated with cognitive decline and, possibly, to Alzheimer-like dementia.

D 2004 Elsevier Inc. All rights reserved.

Keywords: 192 IgG saporin; Social transmission of food preferences; Memory; EEG; Cyclooxygenases; Presenilins

Introduction BFCN lesions in rodents, obtained by the selective immu-

Loss of basal forebrain cholinergic neurons (BFCN) is a

distinctive pathological feature of Alzheimer disease (AD),

and impaired cholinergic function appears to underlie age-

related memory loss (Auld et al., 2002; Davis et al., 1999;

Perry, 1988). In the last decade, paradigms employing

0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2004.05.025

* Corresponding author. Section of Behavioural Neurosciences,

Department of Cell Biology and Neurosciences, Istituto Superiore di

Sanita, Viale Regina Elena 299, 00161 Rome, Italy. Fax: +39-06-4957821.

E-mail address: laura.ricceri@iss.it (L. Ricceri).

notoxin 192 IgG saporin (192 IgG-Sap) have been used,

with the aim of clarifying the role of the cholinergic

dysfunction in the cognitive deficits associated to AD

(Rossner, 1997; Wiley et al., 1995). The 192 IgG-Sap

immunotoxin consists of a monoclonal antibody to the low

affinity/p75 nerve growth factor receptor (NGFr), 192 IgG,

that is coupled to the ribosome-inactivating protein saporin.

The immunotoxin exploits the fact that most BFCN express

high levels of the p75 NGFr relative to other cholinergic and

non-cholinergic neurons in nearby regions (Woolf et al.,

1989; Yan and Johnson, 1988). When injected intracerebro-

ventricularly (icv) or directly into basal forebrain cholinergic

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172 163

nuclei, 192 IgG-Sap selectively destroys neurons bearing p75

NGFr and induces dramatic cholinergic depletions (Book

et al., 1994; Wiley, 1992). Adult rats lesioned with 192 IgG-

Sap have been initially proposed as a valuable animal model

of AD because they mimic the cholinergic degeneration

found in endstage AD patients (Davis et al., 1999; Perry,

1988) and allow the evaluation of the role played by BFCN

hypofunction in the cascade of neural events leading to AD-

like neurodegeneration (Rossner, 1997; Wiley et al., 1995).

From a behavioural perspective, injection in basal fore-

brain nuclei of 192 IgG-Sap leads to limited alterations in

spatial learning and memory performances (Baxter and

Gallagher, 1996; Berger-Sweeney et al., 1994; Torres et al.,

1994), whereas clear deficits are evident in either more

complex spatial paradigms (Janis et al., 1998; Pizzo et al.,

2002) or olfactory-based non-spatial tasks, such as social

transmission of food preferences (Berger-Sweeney et al.,

2000; Vale-Martinez et al., 2002) and discriminative learn-

ing (Bailey et al., 2003). According to many authors,

behavioural effects of basal forebrain 192 IgG-Sap injec-

tions in adult rats suggest the involvement of the BFCN in

cognitive attentional processes, rather than in spatial learn-

ing and memory (Baxter and Chiba, 1999; Sarter et al.,

2003). In addition, neurochemical data concerning APP

metabolism or cholinergic markers failed to show either a

clear link between BFCN destruction and amyloid deposi-

tion or a robust correlation between magnitude of choliner-

gic depletion and memory loss. Altogether, these findings

have led to a re-examination of the role of BFCN, in that in

adult animals cholinergic degeneration per se is not able to

induce the full spectrum of cognitive and neuropathological

features of AD-like dementia.

Recently, early dysregulation of BFCN functions has

been proposed to represent an important risk factor for

age-related cognitive decline and possibly AD-like dementia

(Sarter and Bruno, in press). Several studies have shown

that neonatal lesion with 192 IgG-Sap successfully targets

BFCN, inducing a marked and long lasting selective loss of

cholinergic markers in both cortex and hippocampus

(Leanza et al., 1996; Pappas et al., 1996; Ricceri et al.,

1997; Robertson et al., 1998; Sherren et al., 1999). Devel-

opmental BFCN lesions also induces learning deficits and

changes in emotional responses as early as the second

postnatal week (Ricceri, 2003; Ricceri et al., 1997). At 2

months of age, spatial discrimination deficits were clearly

evident only in the spatial open field test (Ricceri et al.,

1999), whereas spatial memory in the water maze was

mildly affected (Leanza et al., 1996; Pappas et al., 1996;

Ricceri et al., 1999).

Our aim was to investigate at a longer time span from

the lesion than previously examined, the effects of neonatal

192 IgG-Sap on non-spatial cognitive responses as well as

on expression of proteins that have been reported to be

linked to neurodegenerative phenomena in AD. Our hy-

pothesis was that an animal model with reduced cholinergic

input cortical and hippocampal regions since the first

postnatal week could be a valid model to assess the role

played by abnormal cholinergic maturation in later vulnera-

bility to neuropathological processes associated with cogni-

tive decline.

To this aim, we used an integrated approach in which

behavioural, electrophysiological and neurochemical tech-

niques were combined. From a behavioural perspective, we

wanted to evaluate whether neonatal icv 192 IgG-Sap lesion

would induce learning and memory deficits in social trans-

mission of food preferences at 6 months of age. Along with

memory loss, we evaluated electrophysiological (EEG cor-

tical patterns) and neurochemical markers, which could be

relevant to characterise the escalation already reported in

humans (Petersen et al., 2001; Snowdon et al., 1996) from

early cognitive impairment to age-related decline and AD-

like dementia [amyloid precursor protein (APP), presenilin

(PS) 1 and 2, cyclooxygenase (COX) 1 and 2].

Our results show that the neonatal cholinergic lesion

induces memory impairment and EEG alterations, mild dec-

rease in PS-1 and PS-2 mRNA levels and also reduction in

COX-2 protein and mRNA levels.

Methods

Subjects

Wistar rats were purchased from Charles River Italia

(Calco, Italy). The animals were kept in an air-conditioned

room at 21 F 1jC and 60 F 10% relative humidity, with

a white/red light cycle (white light on from 8:30 a.m. to

8:30 p.m.). Males and multiparous females were housed

separately in couples in 42 � 27 � 14 cm Plexiglas boxes

with a metal top and sawdust as bedding. Pellet food

(enriched standard diet purchased from Mucedola, Settimo

Milanese, Italy) and tap water were continuously available.

Two weeks after their arrival, 12 breeding pairs were formed

and housed in 42 � 27 � 14 cm boxes. After 10 days, the

females were individually housed and subsequently inspected

daily at 9:30 a.m. for delivery (pnd 1).

Toxin administration

Eleven litters were culled at birth to four males and four

females to maintain an adequate gender composition (Alleva

et al., 1986). Four male pups in each litter were randomly

assigned to either the control (phosphate-buffered saline

0.1 M, PBS, 2 pups) or the 192 IgG-sap treatment condition

(2 pups). On pnd 7, between 9 and 12 a.m., pups were re-

moved from their mothers and anesthetised by hypothermia.

Pups to be lesioned were secured in a stereotaxic apparatus

(Stoelting, IL, USA) with a Plexiglas-mold holder for neo-

nate rats. Either PBS or 192 IgG saporin were injected over

1 min using a 30-gauge needle that was left in place for an

additional 1 min (0.5 Al of a solution 0.42 Ag/Al of 192 IgG

saporin, Chemicon International Inc., CA, USA, in PBS

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172164

0.1 M). A pair of injections were therefore made at the

coordinates AP = 0.0; ML = +2.0; DV = �3.5 relative to

bregma. Previous study performed on 7-day-old rats

showed that this procedure was an effective means of

administering 192 IgG-Sap into the third ventricle. After

surgery, animals were sutured with Histoacryl tissue glue

(Braun, Melsugen, Germany), transferred to a heating pad

for 20 min to regain normal body temperature, and

subsequently returned to their mothers.

Behavioural testing

Social transmission of food preferences

After surgery, rats were left with their mothers for 2 weeks

and subsequently housed in pairs. Each pair was constituted

by a control-treated demonstrator (DEM) and either a Sap

(n = 11) or a control (n = 6) observer (OBS). DEM and OBS

were always siblings.

At 6 months of age, both OBS and DEM were habituated

for a week to eat ground chow from two glass jars (5 cm

high � 5 cm diameter). The jars were secured to a squared

20 � 20 cm ceramic tray to collect spillage. These feeders

permitted an accurate and consistent measure of food intake

throughout the habituation phase. Experimental procedure

consisted of four steps:

Step 1—to ensure that demonstrators would eat during

step 3, the day before the test session, DEM subjects were

placed in a single cage and food deprived (18 h) until the

test session began at 13.00. Meanwhile, OBS animals

were left in their home cage with food and water ad li-

bitum. To ensure that all observers shared a comparable

foodmotivational state, theywere food deprived 6 h before

the food choice test. Two OBS animals, whose DEMs ate

less than 4 g of cued food were excluded from the analysis.

Step 2—DEM was moved in a separate room and was

offered for 30 min on either a powdered laboratory chow

adulterated with 2% by weight cocoa flavour (Cacao

Perugina, PG Italy), or 1% by weight ground Cinnamon

(PC Eda, VR, Italy). In each treatment group, half of the

DEMs was offered the cocoa- and half cinnamon adul-

terated diet.

Step 3—The DEM was returned to the OBS cage and

DEM and OBS were allowed to freely interact for 15 min.

Step 4—The DEM was removed from the experiment

and the OBS was placed for 24 h in a new cage and

offered two weighed food cups, one containing cocoa-

flavoured diet and one containing cinnamon-flavoured

diet. Food intake was measured after 30 min, 4 and 24 h.

This procedure followed the one established and exten-

sively characterised in the last two decades by Galef

(2002) and Galef and Wigmore (1983).

Neophobia

To evaluate possible differences in response to novel food

or differences in food motivation following neonatal cholin-

ergic lesion, thus biasing the expression of socially acquired

food preferences, OBS rats underwent a food neophobia test

3 weeks after the social transmission experiment (Rollins et

al., 2001). Rats were presented with Honey loops (Weetabix

Cereal, UK) in the previously described food cups. To assess

neophobia, latency to eat and amount of food consumed in a

25-min test were measured. Rats were timed with a stopwatch

to the nearest second. A condition of minimal food depriva-

tion (6 h) was instituted to ensure that the rats would eat

within a reasonable time period and that group differences

would not be obscured by extreme hunger.

EEG

One week after behavioural testing, 12 animals (6 192 IgG-

Sap and 6 control) were newly anaesthetised with Equithe-

sin, and screw cortical electrodes were implanted at the level

of the frontal cortex and fixed with dental acrylic to the skull

surface. Five to 6 days thereafter, the animals were individ-

ually placed in a cylindrical Plexiglas container in a sound-

proof experimental room. After a 30-min habituation period,

each animal was connected to an Ote polygraph (model

10b). The EEG was then continuously recorded over 45

min. The methods used for EEG recording and analysis

have been described previously (Reggio et al., 1999).

Briefly, sequential power spectra of 20 s EEG epochs (1

epoch every minute) were analysed by fast Fourier trans-

formation with a frequency resolution of 0.35 Hz (Staderini

software, IADA Sistemi, Rome, Italy). Power spectra rele-

vant to an EEG tracing were recorded on an optical disk and

then analysed to calculate the relative power in each freq-

uency band. Frequency bands were as follows: 1.2–4 Hz

(y), 4.35–7 Hz (u), 7.35–9.5 Hz (a1), 9.85–12.5 Hz (a2),

12.85–16 Hz (h1), and 16.35–30 Hz (h2).

Neurochemical analyses

Tissue dissection

One week after behavioural testing, 28 rats (14 control

and 14 192 IgG-Sap) were guillotined and the brains rapidly

removed onto an ice-cooled metal plate. Fronto-parietal

cortex was dissected bilaterally, followed by the hippocam-

pus and striatum. Dissected samples were immediately

frozen on dry ice and stored at �70jC until the time of

the neurochemical assays.

ChAT activity

Chat activity was assessed in six control and six 192 IgG-

Sap-lesioned rat brains. All assays were performed in trip-

licate. All reagents were obtained from Sigma-Aldrich

(Milano, Italy), except where indicated. ChAT activity mea-

surements were based on the method of Fonnum (1975).

Briefly, tissue samples were homogenised by sonication in

50 mM Tris buffer pH 7.4 containing 0.2% Triton X-100

(dilution 1:100 vol/w). Homogenates were spun at 10,000� g

for 10 min. Aliquots of the supernatants were incubated for

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172 165

20 min at 37jC in a mixture containing (final concen-

trations): 0.02 ACi 14C-acetyl coenzyme A (4 � 105 cpm,

55.9 ACi/mole, New England Nuclear), 225 AM acetyl

coenzyme A, 8 mM choline bromide, 100 AM physostig-

mine, 10 mM EDTA, 0.05% BSA, 0.3 M NaCl, and 50 mM

Na-phosphate (pH 7.4). The acetylcholine product was

separated from the Acetyl-CoA substrate via an organic:

inorganic separation using Kalignost solution (5 g Na-

tetraphenylboron in 850 ml toluene and 150 ml acetonitrile).

The supernatant was then transferred to scintillation cocktail

and radioactivity was measured. Protein content was deter-

mined according to the method of Bradford (1976), and

ChAT activity was calculated as nmol ACh/hr/mg protein.

Total RNA extraction from rat brain tissues

Fresh tissues from rat cortex and hippocampus were pre-

pared on ice from 16 rats, collected in RNA Later (QiagenR)and stored at �20jC until use. Briefly, tissue samples were

homogenised byMixer Mill (Retsch-Germany) in 1 ml Qiazol

Lysis Reagent (Qiagen). Total RNA was extracted from tis-

sues (10 mg) according to manufacturer’s protocol (RNeasy

Lipid Tissue (QiagenR) and quantified by GeneQuantkRNA/DNA Calculator (Amersham Pharmacia Biotech).

Semiquantitative RT-PCR analysis

To allow the assessment of APP, PS1, PS2, COX-1 and

COX-2 expression in neocortex and hippocampus by RT-

PCR, 2 Ag of denaturated total RNAwas converted into first-

strand cDNA using the SuperScript IIk (Rnase H� Reverse

Transcriptase-Life Technologiesk) and 2,5 AM of oli-

go(dT)16 in a total reaction volume of 20 Al under the

conditions provided by the manufacturer’s protocol.

For APP, PS1, PS2 and h-actin each single gene fragment

was amplified by a PCR reaction carried out in a total volume

of 50 Al that included 1 Al of each single template, 0,5 AM of

each primer, 10 AM of each dNTP, 1.5 mM MgCl2, DMSO

5% (SIGMA), 10� Buffer II and 2.0 U of Taq GoldR(Applied Biosystems). The primers were: PS-1 (GenBank

Accession Number, D82578), sense 5V-CAT TCA CAG

AAG ACA CCG AGA, antisense 5V-TCC AGA TCA

GGA GTG CAA CC, product length 261 bp; PS-2

(AB004454), sense 5V-CTT CAC CGA GGA CAC ACC

CT, antisense 5V-GAC AGC CAG GAA CAG TGT GG,

product length 256 bp. APP primers were published else-

where (Shi et al., 1997). h-actin was used as housekeeping

gene (NM031144), sense 5V-GTC GAC AAC GGC TCC

GGC ATG, antisense 5V-CTC TTG CTC TGG GCC TCG

TCG C, product length 158 bp. PCR included denaturation

at 94jC for 30 s, annealing for 30 s and extension at 72jCfor 60 s. The annealing temperature and number of cycles

are: PS-1, 54jC, 35 cycles; PS-2, 62jC, 35 cycles; h-actin62jC, 30 cycles; COX-1 and COX-2, 58jC, 30 cycles. PCR

products were electrophoresed in 2% agarose TBE gel

containing ethidium bromide. Pictures of gels were taken

and the signal intensities of bands were quantified using the

FX Imager (Bio-Rad).

For COX-1 and COX-2, oligonucleotide primers with

similar Tm were chosen to generate a PCR fragment of 887

bp for COX-1, and of 702 bp for COX-2 (Feng et al., 1993;

Tanaka et al., 2002). PCR conditions (number of cycles and

cDNA and primer concentration) that ensure the data to be

obtained within the exponential phase of amplification of

each template were carefully assessed. One and 3 Al of

cDNAs were amplified for h-actin, COX-1 and COX-2,

respectively. PCR-amplification was done in a final volume

of 50 Al containing 1� PCR buffer, the four dNTPs

(0,2 mM), MgSO4 (2 mM), 1 Units of Platinium Taq

DNA polymerase High Fidelity (Invitrogen), 1.25 pmol of

h-actin or 12.5 pmol of COX-1 and COX-2 primers. A

sample containing all reaction reagents except cDNA was

used as PCR negative control in each experiment. The PCR

conditions for h-actin were described above. The absence of

genomic DNA was verified using 3 Al of cDNA that was

reverse transcribed without the enzyme and used as a further

PCR negative control (-rt). PCR products were analysed by

electrophoresis in 1,2% (COX-1 and COX2) or 1.8% (PS-1,

PS-2, APP and h-actin)(w/v) agarose gel, stained with

ethidium bromide and photographed. Transcript levels were

analysed by Fluor-STM Multimager analyser (Biorad). For

each experiment, the ratio between optical density (arbitrary

units) of bands corresponding to tested genes and h-actin(used as internal standard) was calculated to quantify the

level of the transcripts for mRNAs.

The identity of amplified fragments was confirmed

by Southern blotting using a digoxigenin oligonucleotide

probe and the DIG Luminescent Detection Kit (data not

shown).

Protein extraction and Western blot analysis

APP and h secretase: for APP and BACE Western

blot analysis, tissue samples (cortex and hippocampus

from 6 control and 6 lesioned animals) were homoge-

nised with a potter homogeniser (Teflon/glass, 700 rpm)

in HEPES/Na+ 25 mM containing EDTA 2 mM, EGTA

1 mM, PMSF 0.1 mM, pH 7.4. Thereafter, homogenates

were centrifuged at 10,000 � g for 10 min to remove

crude nuclear material, and supernatants were analysed

by Western blot to measure total APP (monoclonal

antibody 22C11, Chemicon, CA, USA; dilution 1:3000)

and beta-secretase (polyclonal antibody anti-BACE CT,

Affinity Bioreagents Inc., Golden, CO, USA; dilution

1:500). An aliquot of supernatants was further centrifuged

(60 min at 100,000 g) to pellet holoAPP. Final supernatants

were used to measure soluble APPalpha by Western blot

(monoclonal antibody 6E10, Chemicon; dilution 1:3000).

After incubation with peroxidase-conjugated secondary anti-

bodies (Kirkegard, MD, USA; dilution 1:10,000), blots were

developed with enhanced chemiluminescence (ECL, Amer-

sham-Pharmacia Biotech, UK). The optical density of the

bands (integrated area, arbitrary units) was measured by

computer assisted imaging (Quantity-One System, Bio-Rad,

CA USA).

Fig. 1. Left panel: percentage of cued food eaten by OBSs 30 min, 4 and 24 h following the social interaction; *significant difference (P < 0.05) between 192

IgG-Sap and control animals. Right panel: total weight of food eaten at three different retention intervals.

Fig. 2. Effects of neonatal 192 IgG-Sap on relative EEG power distribution

in adult rat brains. *Significant difference (P < 0.05) between 192 IgG-Sap

and control animals, **significant difference (P < 0.01) between 192 IgG-

Sap and control animals.

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172166

Cyclooxygenases: tissue samples (cortex and hippocam-

pus from 8 control and 8 lesioned animals) were homoge-

nised in 50 mM Tris buffer, pH 7.5 supplemented with 1%

NP40, 0.1% SDS, 100 Ag/ml PMSF, 30 Ag/ml aprotinin,

100 AM leupeptin, 10 mM NaF, 1 mM EDTA, 1 mM EGTA,

100 AM Na3VO4) and unsoluble material removed by

centrifugation (10,000 � g at 4jC, 10 min) as previously

described (Calamandrei et al., 2003). Equals amounts of

proteins (50 Ag/lane) were separated by 10% SDS-PAGE

and transferred to nitrocellulose membranes, then blocked

with 10% non-fat milk and incubated with dilution of 1:500

of polyclonal anti-COX-1 or anti-COX-2 antibodies (Cay-

man Chemical, MI, USA) for 1 h at 25jC. Horseradishperoxidase conjugated anti-rabbit IgG (1:5000, 1 h at 25jC)and ECL reagents from Amersham (Buckingamshire, UK)

were used as detection system. Purified COX-1 and COX-2

were used as standard controls (0.5 Ag/lane). After severalwashes, membranes were stripped by a 10-min incubation in

0.2 M NaOH with vigour shaking at room temperature

(Suck and Krupinska, 1996), blocked with 5% non-fat milk,

incubated with the primary anti-GAPDH antibody (1:4000,

1 h, RT), then with horseradish peroxidase conjugated anti-

mouse IgG (1:5000, 1 h at 25jC) and processed for the

ECL detection as before. The optical density of the bands

(integrated area, arbitrary units) was measured by GS-700

Imaging Densitometer (Bio-Rad) and referred to the corres-

ponding control samples (taken as 100%), which were run

in the same gel.

Statistical analysis

A mixed-model ANOVA for repeated measures was used

to analyse social transmission data. One-way ANOVA was

used for total consumption data from neophobia experiment,

ChAT activity, EEG relative power and Western blot data.

Post hoc comparisons were performed using the Tukey’s

HSD test, which can be used in the absence of significant

ANOVA results (Wilcox, 1987). Before ANOVA, arcsin

transformation was applied to percentage data from the

social transmission experiment. Neophobia latency data

were analysed by Mann–Whitney non-parametric test. For

RT-PCR and Western blot experiments, comparison between

treatment groups was made by Student’s t test.

Results

Behavioural testing

Social transmission of food preferences

Repeated measure ANOVA was performed on the trans-

formed percentage of cued food eaten by OBSs (Fig. 1).

Treatment as main effect just missed statistical significance

[F(1, 13) = 3.65, P = 0.07]. Post hoc comparisons

performed on the interaction treatment � time interval

(30 min, 4 and 24 h) [F(2, 26) = 1.35, ns] showed that 30

min after the social interaction both control and 192 IgG-

Sap subjects showed a clear preference towards the cued

food and this preference was sustained throughout all the

experimental session (4 and 24 h) in control animals. By

contrast, this preference already disappeared at 4-h interval

Fig. 3. Effect of neonatal 192 IgG-Sap treatment on PS-1 mRNA levels in

the hippocampus. Representative semiquantitative RT-PCR analysis of PS-

1 and housekeeping gene h-actin mRNAs in the hippocampus of control

and IgG192-Sap treated animals is shown in the upper panel. The ratio of

PS-1 and h-actin mRNA was statistically analysed (lower panel). Data

shown are the means F SEM of five animals, analysed in duplicates.

*Significant difference (P < 0.05) between 192 IgG-Sap and control

animals.

Fig. 4. Effect of neonatal 192 IgG-Sap treatment on PS-2 mRNA levels in the cor

PS-2 and housekeeping gene h-actin mRNAs in the cortex and hippocampus of con

of PS-2 and h-actin mRNA was statistically analysed (lower panels). Data shown

differences (P < 0.05) between 192 IgG-Sap and control animals.

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172 167

in lesioned animals (Tukey HSD P < 0.01) reaching the

chance level in the 24-h interval as demonstrated by the

lower percentage of cued food eaten relative to control

(Tukey HSD, P < 0.01). No significant differences were

found in the total amount of food consumed by OBS of

both groups [F(1, 13) = 1.27, P = 0.28].

Neophobia

One-way ANOVAwas performed on the amount of novel

food eaten by OBSs. No significant effect of 192 IgG-Sap

was found [F(1, 14) = 0.34, P = 0.57]. Latency to eat the

novel food was not significantly affected by 192 IgG-Sap

[U = 28, P = 0.67].

EEG

As shown in Fig. 2, the EEG tracing of adult rats which

had been lesioned with IgG-Sap at neonatal age showed

significant EEG alterations with respect to control animals

(P < 0.05 according to one-way ANOVA and Tukey post

hoc test). Specifically, the relative EEG power lying in the

y band was significantly increased, while a decrease was

observed in the a and h bands.

ChAT activity

Cortical ChAT activity in the 192 IgG-Sap group (3.7 F0.05 nmol/h/mg) was decreased 78% relative to the control

group (17.3 F 0.6). Hippocampal ChAT activity in the 192

IgG-Sap group (4.9 F 0.7 nmol/h/mg) was decreased 89%

relative to the control group (47.3 F 1.8). ANOVA revealed

tex and hippocampus. Representative semiquantitative RT-PCR analysis of

trol and 192 IgG-Sap-treated animals is shown in the upper panel. The ratio

are the means F SEM of 5 animals, analysed in duplicates. *Significant

Fig. 5. Effect of neonatal 192 IgG-Sap treatment on COX-2 mRNA levels

in the hippocampus. Representative semiquantitative RT-PCR analysis of

COX-2 and of the housekeeping gene h-actin mRNAs in the hippocampus

of control and IgG192-Sap-treated animals is shown in the upper panel. The

ratio of COX-2 and h-actin mRNAwas statistically analysed (lower panel).

Data shown are the means F SEM of 5 animals, analysed in duplicates.

*Significant difference (P < 0.01) between 192 IgG-Sap and control

animals.

Fig. 6. Effect of neonatal 192 IgG-Sap treatment on COX-protein levels in

the hippocampus. Representative Western blot analysis of COX-2 and

GAPDH, used as internal control, in the hippocampus of control and IgG

192-Sap-treated animals is shown in the upper panel. The ratio of COX-2

and GAPDH expression in control and IgG 192-Sap-treated animals was

statistically analysed (lower panel). Data shown are the means F SEM of

four animals, analysed in duplicates. *Significant difference (P < 0.01)

between 192 IgG-Sap and control animals.

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172168

significant differences between the control and 192 IgG-Sap

group in both neocortex and hippocampus [F(1,10) = 404.8,

and F(1,10) = 450.8, P < 0.01, respectively].

Expression levels of APP, PS-1 and PS-2

A semiquantitative competitive RT-PCR assay was used

to investigate the effects of neonatal basal forebrain cholin-

ergic lesion on APP, PS-1 and PS-2 expression in both

cortex and hippocampus.

The analysis of APP mRNA levels by RT-PCR showed no

difference between control and 192 IgG-Sap groups (cortex:

APP/h-actin ratio: 3.4 F 1.4 and 3.1 F 1.5, n = 5, respec-

tively; hippocampus: APP/h-actin ratio: 1.7F 0.3 and 1.8F0.3, n = 5). The decrease in cortical PS-1 mRNA levels did

not reach statistical significance (PS-1/h-actin ratio: 5.5 F1.9 and 3.3F 0.6, n = 5, respectively). On the contrary, PS-1

expression levels in the hippocampus were significantly

decreased in the 192 IgG-Sap treatment compared to controls

[T(4) = 3.1, P = 0.03] (Fig. 3). Similarly, PS-2 expression

levels in both cortex and hippocampus were significantly

decreased in the 192 IgG-Sap group compared to controls

[T(4) = 3.6, P = 0.02; T(4) = 3.1, P = 0.03;] (Fig. 4).

APP and b secretase Western blot

The amount of total APP measurable in homogenates of

both cortices and hippocampi of treated rats (192 IgG-Sap)

did not show any significant difference as compared to

control animals (cortex: 192 IgG-Sap vs. control 8.73 F5.48 vs. 10.27 F 3.9, P = 0.478; hippocampi 192 IgG-Sap

vs. control 6.78 F 6.27 vs. 7.56 F 5.28, P = 0.738) ac-

cording to data reported for APP mRNA.

Similar results were obtained measuring amounts of

secretase: the immunoreactivity of BACE did not change

significantly following the treatment (cortex: 192 IgG-Sap

vs. control 7.23 F 1.17 vs. 6.29 F 4.52, P = 0.503; hip-

pocampi 192 IgG-Sap vs. control 9.61 F 2.47 vs. 10.2 F3.58, P = 0.669).

We then investigate whether metabolism of APP could be

affected by 192 IgG-Sap. Quantitative analysis of soluble

APP in both cortex and hippocampus did not reveal a

significant difference between control and treated animals

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172 169

(cortex: 192 IgG-Sap vs. control 9.58F 5.9 vs. 9.38F 1.58,

P = 0.961; hippocampi 192 IgG-Sap vs. control 7.6 F 2.44

vs. 6.87 F 0.84, P = 0.71) in agreement with lack of effects

of treatment on total APP and BACE.

COX-1 and 2 expression

The analysis of COX-1 mRNA levels by RT-PCR showed

no difference between control and 192 IgG-Sap groups

(COX-1/h-actin ratio: 1.0 F 0.19 and 0.85 F 0.20, n = 5

per group, respectively) in cortex. In the hippocampus, there

was a tendency of COX-1 mRNA reduction, which however,

was not significant (COX-1/h-actin ratio: 1.0 F 0.13 and

0.69 F 0.17, n = 4 per group, respectively). Similarly,

cortical COX-2 mRNA levels decreased but not to a signif-

icant extent (COX-2/h-actin ratio: 1.0 F 0.26 and 0.66 F0.27, n = 5 per group, respectively). By contrast, in the

hippocampus COX-2 mRNA levels were significantly de-

creased in 192 IgG-Sap group compared to controls [T(8) =

5.6, P = 0.0008] (Fig. 5).

The levels of COX-1 in homogenates from both cortical

and hippocampal regions were barely detectable by Western

blot analysis, preventing us from any further assessment.

Similarly, the quantification of cortical COX-2 levels was

not possible since the protein was expressed at very low

levels (not shown), as previously described (Calamandrei

et al., 2003). However, COX-2 expression in the hippocam-

pus was clearly detectable and COX-2 protein levels were

significantly decreased in the 192 IgG-Sap group compared

to controls [T(7) = 4.9, P = 0.0024] (Fig. 6).

Discussion

The results of the present study show that neonatal

damage to BFCN induces long-term behavioural and neu-

ropathological alterations. In particular, we observed (i) a

selected memory loss of a socially acquired olfactory

information; (ii) alterations in EEG patterns similar to those

found in Alzheimer patients (Auld et al., 2002); (iii) a mild

decrease in PS-1 and PS-2 gene expression; (iv) a signifi-

cant decrease in COX-2 expression in the hippocampal

region. However, no significant alterations were evidenced

in APP metabolism.

Memory impairment

In the present food preference task, after interacting with

a conspecific recently fed on a particular cued food (DEM),

an OBS rat exhibits an enhanced preference towards that

food lasting up to 1 month. This test is based on food related

olfactory messages passing from DEM to OBS through

DEM’s breath (Galef and Whiskin, 2003). This task was

chosen because it requires integrity of hippocampal function

(Bunsey and Eichenbaum, 1995; Winocur, 1990), it is

rapidly acquired (Galef, 2002) and appetitively, rather than

aversively, motivated (Galef and Whiskin, 2003; Moles and

D’Amato, 2000; Moles et al., 1999). One of the distinctive

features of this task is that it requires the animal to exploit

the cues associated with the conspecific during the social

interaction within a completely different context such as

binary food choice, whereas memory testing commonly take

place in the same context where training occurred. Just this

kind of flexible response has been proposed to make

performance in this task dependent on hippocampal and

subiculum regions (Bunsey and Eichenbaum, 1995).

Our results showed that while control rats clearly de-

veloped a socially acquired food preference lasting at least

24 h, in 192 IgG-sap rats this preference extinguished much

earlier, lasting less than 4 h. Data relative to the first 30 min

of food preference test are indicative that olfactory messages

had passed from DEMs to OBSs in both treatment groups

and we can therefore exclude 192 IgG-Sap-induced olfac-

tory deficits. Also, saporin-aspecific effects on either atti-

tude towards novel foods or food motivation can be ruled

out from the results obtained in the neophobia test in le-

sioned and control rats. Thus, the lack of preference towards

the cued-food observed in the 192 IgG-Sap animals was an

amnesic effect per se and not a lesion-induced reduction of

the neophobic response to novel food stimuli.

Altogether, our data strongly suggest that neonatal saporin

lesion induces a memory deficit of socially transmitted

information concerning food selection. These results are also

in partial agreement with previous investigations on the

effects of adult 192 IgG-Sap lesions (Berger-Sweeney et

al., 2000; Vale-Martinez et al., 2002). Rats lesioned as adults

with IgG 192 Sap, however, showed also diminished total

food consumption and a general increase of preference to-

wards cued food during the 24-h interval between the im-

mediate and the 24-h test. Thus, in line with previous evi-

dence with other learning tasks (Berger-Sweeney, 2003), it

appears that adult rats undergoing neonatal lesions showed

a more pronounced deficit in social memory than adult-

lesioned ones.

EEG alterations

The electrophysiological changes in response to selective

cholinergic deafferentation of cortex and hippocampus con-

sisted of an increase in cortical slow-wave power and a

decrease in high frequency power. Such a finding is con-

sistent with the well-accepted role of basal forebrain system

in the maintenance of activated EEG activity. Interestingly,

the EEG pattern of lesioned rats shows many similarities

with the typical EEG changes observed in AD [namely a

generalised slowing of neocortical EEG due to loss of h and

decrease in a activity, and increased power in y (<4 Hz)

(Dringenberg, 2000)]. Although AD patients also show

increased u activity, no such changes were found in lesioned

rats. It should be noted, however, that septo-hippocampal

cholinergic neurons are likely to be destroyed by icv IgG-

Sap and thus, given the essential role played by those

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172170

neurons in the generation of the u pattern, a decrease in

u activity should rather be anticipated in our model. On the

other hand, previous studies have shown that even when all

septo-hippocampal cholinergic neurons are destroyed by

IgG-Sap, the integrity of septo-hippocampal GABAergic

neurons is sufficient to maintain some u activity (Lee et al.,

1994). Whatever the mechanisms responsible for the pres-

ervation of theta activity, however, it is worth mentioning

that such a compensation does not correlate with a rescue in

the impairment of the associative task. Like the memory

deficit, also the EEG alterations found in our model are

more marked that those reported in rats lesioned at adult

age, in which less evident changes (Berntson et al., 2002;

Holschneider et al., 1999) or even no effect (Wenk et al.,

1994) have been reported. Our data, thus, indicate that a

very early removal of the ascending cholinergic projection

induces marked and long-lasting EEG alterations, while

lesions performed at later developmental stages may be

better compensated by other pathways involved in the

maintenance of cortical desynchronisation.

APP and presenilins

Neonatal 192 IgG-Sap lesions do not have a significant

impact on APP mRNA levels, protein levels of total and

soluble APP as well as h secretase. The lack of 192 IgG-Sap

effects is somehow in contrast with previous studies on adult

lesions reporting APP increase (Leanza, 1998; Lin et al.,

1998, 1999). However, other groups did not observe the same

effects on total APP levels, but rather a reduction of secreted

APP and an increase in membrane-bound APP (Apelt et al.,

1997; Rossner et al., 1997). In addition, a small, albeit

significant, reduction of APP695 mRNA in cortical and

hippocampal regions was found 30 days after the lesion

(Apelt et al., 1997). These apparent discrepancies could

probably reflect some developmental compensations occur-

ring in APP metabolism of rats receiving the basal forebrain

cholinergic lesion in the first postnatal week. Alternatively,

alterations in APP and BACE levels may occur at later time

points. In this respect, the reduction of presenilin mRNA

observed in our model could be indicative of an initial change

in APP metabolic cascade. Indeed, presenilins associated to

the multimeric g-secretase complex are intimately involved

in the cleavage of amyloid precursor protein to form beta-

amyloid peptides (De Strooper et al., 1997). They are highly

regulated (Theuns and Van Broeckhoven, 2000) and show

tissue-specific transcriptional differences. Some authors in-

vestigated PS1 role by genetically modified mice under-

expressing PS1 (PS1 +/� mice) and showed that chronic

reduction of PS1 activity leads to impaired synaptic plasticity

(Morton et al., 2002). Results from human AD brains also

revealed PS1 and PS2 decrease in hippocampus, frontal cor-

tex and basal forebrain followed by impaired synaptic plas-

ticity (McMillan et al., 2000; Takami et al., 1997). Our data

show that a selective cholinergic deafferentation induces PS

down-regulation in those brain regions where the highest

levels of PSs mRNA expression are observed (Benkovic

et al., 1997). It is noteworthy that in human studies of early-

stage AD cases, a prominent down-regulation of PS2 mRNA

levels in hippocampal regions has been observed, even in the

absence of marked neuropathology (McMillan et al., 2000).

COX-1 and COX-2 expression

COX enzymatic activity is responsible for the first

committed step in prostaglandin biosynthesis. COX-1 is

constitutively expressed in most of cell and tissue types,

whereas the inducible COX-2 is the major isoform ex-

pressed during inflammatory processes. The brain represents

one of the few exceptions to this general rule since COX-2

is constitutively expressed in discrete populations of excit-

atory neurones of cerebral cortex and hippocampus (Breder

et al., 1995; Kaufmann et al., 1997). Although the physio-

logical functions of neuronal COX-2 are poorly understood,

several lines of evidence suggest that COX-2 expression is

regulated by synaptic function and its activity involved in

synaptogenesis and consolidation of hippocampal-dependent

memory (Kaufmann et al., 1997; Teather et al., 2002).

Studies of COX-2 expression in AD brains have produced

varied and conflicting results, possibly because of con-

founding factors due to post-mortem delay and tissue hand-

ling. Interestingly, recent studies reported a down-regulation

of neuronal COX-2 that correlated with severity of clini-

cal dementia (Hoozemans et al., 2002; Yermakova and

O’Banion, 2001). Our results indicate that a long-lasting

selective cholinergic deafferentation induces a decrease in

the levels of COX-2 mRNA and protein in the hippocam-

pus, a brain region in which COX-2 is highly expressed in

neurons of dentate gyrus and fields CA1, CA2 and CA3 of

Ammon’s horn. COX-2 down-regulation could possibly

indicate a reduction of synaptic plasticity in the hippocam-

pal region due to early removal of the cholinergic input to

this region (Chen et al., 2002). Although the present find-

ings do not tell us whether COX-2 down-regulation is due to

a general decreased expression in the hippocampus or to a

selective loss of COX-2 positive neurons, preliminary

immuno-histochemical analyses (not shown) favour the lat-

ter hypothesis. This would be consistent with the human

studies previously reported (Hoozemans et al., 2002; Yer-

makova and O’Banion, 2001) showing a depletion of COX 2

positive neurons in the degenerated regions of AD brains.

Finally, in line with previous studies in AD patients, COX-1

expression was not affected by cholinergic depletion.

Concluding remarks

Altogether, the present study provides a snapshot of

the effects of early and long lasting cholinergic depletion

on cognitive responses and neuropathological changes in 6-

month-old (brains) rats. Our findings indicate that 6 months

after the neonatal lesion distinct pathological events take

place including memory loss, electrophysiological altera-

L. Ricceri et al. / Experimental Neurology 189 (2004) 162–172 171

tions, downregulation of COX-2, and reduction of PS

mRNAs. The neonatal lesion model here presented lends

itself to assess the contribution of early cholinergic system

dysfunction to the development of cognitive and neurological

deficits observed in age-related decline and human patholo-

gies such as AD. Finally, this lesion model appears suitable to

test the recent hypothesis of a developmental origin of age-

related cognitive decline (possibly escalating to AD-like

dementia), so far only supported by epidemiological evi-

dence, also considering different environmental risk factors

influencing cholinergic system maturation throughout life.

Acknowledgments

This research was supported by: a grant from the

Ministry of Health (Project ALZ 6, ‘‘Pathogenesis and repair

mechanisms in in vivo and in vitro models of Alzheimer

disease’’) to GC and LM, and PP; intramural ISS funding

Project 2149/RI ‘‘The proteolitic complex involved in

Alzheimer etiopathogenesis by in vivo and in vitro models’’

to AC. The excellent technical assistance of Antonella

Pezzola and Rosaria Reggio is gratefully acknowledged.

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