Study animals
Male Wistar rats (about 300 g and 2.5 months of age) kept in standard laboratory conditions (08.00:20.00 light, food and water ad libitum and controlled humidity and temperature) were used in the experiments. The animals were maintained according to the guidelines for ethical conduct developed by the European Union directive of 22 September 2010 (2010/63/EU). The study was approved by the local ethics committee of the IRCCS Fondazione Santa Lucia. Rats subjected to behavioral testing were randomly assigned to the following experimental groups: (1) donepezil-treated sham-lesioned (Don-Sham) rats (n = 7), which were treated with donepezil and then sham-lesioned; (2) donepezil-treated Sap-lesioned (Don-Sap) rats (n = 8), which were treated with donepezil and then Sap-lesioned; (3) saline-treated Sap-lesioned (Sal-Sap) rats (n = 8), which were treated with saline (NaCl 0.9%) and then Sap-lesioned; and (4) saline-treated sham-lesioned (Sal-Sham) rats (n = 12), which were treated with saline and then sham-lesioned. This group encompassed six intact rats treated with saline and six rats pre-treated with saline and then sham-lesioned. The behavioral performance of the two groups was not statistically different in all the following behavioral parameters (multivariate analysis of variance (group × parameter): group: F1,10 = 0.01, P = n.s.; parameter: F37,370 = 120.79, P < 0.000001; and group × parameter: F37,370 = 1.25, P = n.s.; Additional file 1). These animals were pooled in the Sal-Sham group. At the end of behavioral testing, all rats were killed to verify the lesion by choline acetyltransferase (ChAT) immunohistochemistry on the lesion sites (MS and NBM). Furthermore, an additional three rats per group were prepared to verify ChAT levels in target areas of cholinergic projections (hippocampus and neocortex) and synaptic impairment. In these rats, ChAT densitometry and caspase-3 activity were measured 2.5 weeks after Sap or sham surgery.
Drug
Donepezil hydrochloride, (RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one (Aricept; Pfizer Inc, New York, NY, USA), was intraperitoneally (i.p.) administered daily at a dosage of 0.75 mg/kg dissolved in 0.5 ml of 0.9% NaCl solution. The same volume of saline without the drug was administered daily to the animals used as controls (Sal-Sham and Sal-Sap). Pre-treatment started 15 days before Sap or sham lesions were created and was stopped after the surgery (Figure 1). The donepezil dosage was chosen on the basis of information from previous in vivo studies [12, 13].
Surgery
All rats were anesthetized with a mixture of tiletamine/zolazepam (50 mg/kg Zoletil 100 i.p.; Virbac s.r.l., Milan, Italy) and xylazine (10 mg/kg Rompun i.p.; Bayer s.p.a., Milan, Italy). In the animals to be lesioned, the immunotoxin 192 IgG-Sap (Chemicon International, Harrow, UK) was bilaterally injected through a 10-μl Hamilton syringe in the MS (dosage: 0.5 μg/side 192 IgG-Sap; coordinates: anteroposterior (AP) = +0.45 mm (from the bregma); mediolateral (ML) = ±0.6 mm (from the midline); and dorsoventral (DV) = −7.2 mm (from the dura) [36]) and in the NBM (dosage: 0.4 μg/side; coordinates: AP = +1 mm (from the bregma); ML = ±2.3 mm (from the midline); and DV = −8 mm (from the dura) [37] with doses and coordinates modified from those used by Frick et al. [38]). 192 IgG-Sap diluted in PBS (1 μg:1 μl) was injected at a rate of 0.1 μl/min. At the end of administration, the needle was left in situ for five minutes. In the remaining rats, only PBS solution was injected.
Behavioral testing
As shown in Figure 1, 2.5 weeks after the surgery (time required to reach a stable and permanent loss of cholinergic neurons using Sap [28]), all rats were subjected to the following tests: EPM, to assess anxiety levels; OF, to evaluate the ability to develop spatial and discriminative competencies; RAM, to analyze spatial working memory; sociability test, to analyze social motivation; PSNT, to evaluate social novelty discrimination; and contextual and tone FC with USV recordings, to analyze acquisition of aversive learning and emotional competencies. A pseudo-random order of test administration was used with FC always being the last test, given its high stress levels.
Elevated plus maze
The maze raised 90 cm above the ground was formed by a wooden structure in the shape of a cross with four 50 cm × 10 cm arms. The north and south arms were open, and the east and west arms were enclosed by walls 36 cm high. The parameters taken into account were frequency of entries into the open and closed arms, total time spent in the open and closed arms and number of defecations [20].
The parameters taken into account were: frequency of entries in the open and closed arms; total time spent in the open and closed arms; number of defecations [20].
Open field with objects
The apparatus consisted of a circular arena (diameter 140 cm) delimited by a 30-cm-high wall. During session 1 (S1), each rat was allowed to move freely in the empty open field and the baseline activity level was measured. During S2 to S4 (habituation phase), four objects were placed in a square arrangement in the middle annulus of the arena and a fifth one was placed in the central area. In S5 and S6 (spatial change), the spatial configuration was changed by moving objects 2 and 5 so that the initial square arrangement was changed to a polygon-shaped configuration without any central object. During S7 (novelty), the configuration was modified by substituting one object with another new one. Sessions lasted six minutes, and intersession intervals were three minutes long. All testing was recorded by a video camera whose signal was relayed to a monitor and to an image analyzer (EthoVision, Noldus Information Technology, Wageningen, The Netherlands). The parameters taken into account were total and peripheral distances traveled in S1, time spent contacting the objects, frequency of rearing, motionless time and number of defecations [20, 39].
Radial arm maze
The apparatus consisted of a central platform (30-cm diameter) from which eight arms (12.5 cm wide × 60 cm long) radiated like the spokes of a wheel. A food well (5-cm diameter, 2-cm depth) was located at the end of each arm [20]. A 40-W red light bulb provided the only source of illumination in the testing room. Testing was performed between 09:00 and 17:00 hours. After a habituation phase, all rats (whose food was restricted to decrease their weight by approximately 15%) were subjected to the RAM full-baited procedure in which all arms were baited with a piece of Purina chow (Purina Mills, Gray Summit, MO, USA) with the goal of having the rats collect the eight rewards in a maximum of 16 entries. The animals were subjected to two trials daily for five consecutive days. The intersession interval was four hours. The parameters taken into account were percentage of total errors (number of revisited arms divided by total number of visits × 100), mean spatial span (longest sequence of correctly visited arms in each session) and perseverations (sum of consecutive entries in the same arm or in a fixed sequence of arms).
Sociability and preference for social novelty test
The apparatus consisted of a white rectangular three-chamber wooden box (150 × 40 × 40 cm). The central chamber was 30 cm long, and the two lateral chambers were 60 cm long. The three chambers were divided by two transparent Plexiglas walls with an open middle door (height 10 cm, width 8 cm), which allowed free access to each lateral chamber. Each lateral chamber contained a small plastic cage (18-cm diameter) with meshlike holes in which stranger rats were confined for social interaction.
The test comprised 3 sessions: Habituation, Sociability and PSNT. During the habituation, each rat was allowed to freely move in the apparatus for 10 min. During Sociability, an unfamiliar juvenile (35 to 45 days old) male conspecific (Stranger 1) was placed inside the small cage in one of the lateral chambers (randomly selected and counterbalanced for each group). The experimental rat was placed in the apparatus and it was allowed to freely explore the three chambers and contact the small cages for ten minutes.
During PSNT, another unfamiliar rat (stranger 2) was placed inside the plastic cage of the opposite lateral chamber that had been empty during the Sociability session. The experimental rat was allowed to move freely and contact the plastic cages housing the strangers for ten minutes. Inter-session intervals lasted three minutes. Rats’ behavior was recorded by a video camera mounted on the ceiling. The resulting video signal was relayed to a monitor and to an EthoVision image analyzer. The parameters analyzed in each lateral chamber were frequency of entries, total duration (in seconds) and total distance traveled (in centimeters).
Context and tone fear conditioning
The apparatus consisted of a 21 × 21 × 49 cm conditioning chamber (model 7532; Ugo Basile, Comerio, Italy). Chamber walls were made of gray Plexiglas, and the ceiling was made of transparent Plexiglas to allow video recording. The grid floor (steel pieces spaced by 1.5 cm) was connected to a shock generator scrambler (conditioner 7531; Ugo Basile).
The FC test encompassed three sessions: training, context and tone. During training session, following a 120 seconds of acclimation to the conditioning apparatus (baseline), three trials consisting of the 30-second tone exposure (2 kHz, 90 dB) were carried out. The last 2 seconds of each tone were paired with a 1-mA foot shock. Tone- and shock-free 60-second inter-trial intervals were used.
After 24 hours, rats were placed for 240 seconds in the training chamber (context test). After 4 hours, the rats were submitted to a tone test in a white Plexiglas box (21 × 18 × 45 cm) with black stripes applied on the walls. After 120 seconds of acclimation, a 120-second tone identical to that used in the training session was sounded without any shock (tone test).
During training, context and tone tests, the rats’ behavior was recorded by a video camera mounted on the ceiling. The resulting video signal was relayed to a monitor and to an EthoVision image analyzer. In addition, 22-kHz USVs were recorded [38, 40].
Behavioral parameters taken into account were frequency and duration of freezing (behavioral immobility, except for respiration movements) and number of defecations.
USVs were collected via an ultrasound microphone (UltraSoundGate CM16; Avisoft Bioacoustics, Berlin, Germany) placed through a hole in the middle of the test chamber roof approximately 21 cm above the shock floor. The microphone was sensitive to 15 to 180 kHz frequencies with a flat frequency response (±6 dB) between 25 and 140 kHz. It was connected by an UltraSoundGate USB audio device to a personal computer, which recorded data at 250,000 Hz in 16-bit format and stored as .wav files for subsequent analysis. Sound files were transferred to SASLab Pro (version 5.2; Avisoft Bioacoustics) for sonographic analysis and a fast Fourier transform (FFT) was performed (512 FFT length, 100% frame, Hamming window and 75% time window overlap). USV parameters taken into account were number of calls emitted, peak amplitude and frequency, frequency modulation and call duration.
Histological analyses
At the end of behavioral testing, the animals were deeply anesthetized and transcardially perfused with saline, followed by 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (4°C, pH 7.5). Brains were removed and cryoprotected in 30% buffered sucrose and cut on a freezing microtome. The anterior part of the brain was cut into coronal sections of 40 μm and stored for ChAT immunohistochemistry.
Choline acetyltransferase immunohistochemical staining
Sections (40 μm) immunostained for ChAT were preincubated in PBS at 4°C, then in 0.4% Triton X-100 in PBS and finally in 0.1% Triton X-100 plus 1% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) plus normal goat serum (NGS; Vector Laboratories, Burlingame, CA, USA) in PBS. Sections were incubated for 16 hours at 4°C with 0.1% Triton X-100 and NGS in PBS with the primary antibody for ChAT diluted 1:1,000 (Chemicon International). Subsequently, sections were incubated with biotinylated secondary antibody (goat anti-rabbit IgG-biotin conjugate) and 3% NGS (Kit Elite PK-6101; Vector Laboratories) in PBS for 10 minutes at room temperature. Staining was visualized with 0.05% diaminobenzidine and ammonium nickel(II) sulfate (Sigma-Aldrich Chemie, Steinheim, Germany) after incubation with avidin and biotinylated peroxidase (Kit Elite PK-6101). The sections were then rinsed in PBS. Stained sections were mounted on slides, dehydrated and coverslipped. To exclude artefacts, in each case some random sections were processed as previously described. The only difference was the absence of the primary antibody.
Biochemical analyses
Total homogenate preparation from hippocampal and neocortical tissues
After the animals were decapitated, hippocampal and neocortical tissues were dissected and homogenized in lysis buffer (320 mM sucrose, 50 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 0.5 mM sodium orthovanadate, 5 mM β-glycerophosphate, proteases inhibitors), then incubated on ice for 30 minutes and centrifuged at 13,000 g for 10 minutes. The total protein content of the resulting supernatant was determined by the Bradford assay method.
Immunoblot analysis and antibodies
Proteins were subjected to SDS-PAGE and electroblotted onto a polyvinylidene fluoride membrane. Immunoblot analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using ImageQuant 5.0 software. Antibodies to anti-ChAT were purchased from Chemicon International (AB143), and anti-actin clone EP184E rabbit monoclonal antibody was obtained from EMD Millipore (04-1040; Billerica, MA, USA).
Fluorometric assay of caspase-3 activity
Total hippocampal and neocortical tissue was homogenized in lysis assay buffer (100 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (pH 7.4), 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (wt/vol), 1 mM ethylenediaminetetraacetic acid, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and lysed by freezing in liquid N2 and thawing at 37°C three times. After centrifugation at 11,500 g for 5 minutes, the protein concentration of resulting supernatant was determined and the same amount of protein was incubated at 37°C in lysis assay buffer containing 50 μM caspase-3 substrate II, fluorogenic (Ac-DEVD-AMC; Calbiochem, San Diego, CA, USA). The fluorescence was measured with 380-nm excitation wavelength and 460-nm emission wavelength.
Statistical analysis
All data were tested for normality (Shapiro-Wilk test) and homoscedasticity (Levene’s test). Behavioral data were analyzed using two-way analysis of variance (ANOVA; F-statistic) (with drug and lesion as between-animal factors) or a mixed model of three-way ANOVA (with drug and lesion as between-animal factors and arm/session/object/day/chamber as within-animal factors). Post hoc comparisons were performed by means of Tukey’s honestly significant difference test. When parametric assumptions were not fully met, data transformations (angular transformation for percentages) or nonparametric ANOVAs (Kruskal-Wallis test (H-statistic) and Mann-Whitney U test; Z-statistic) were used. Biochemical data of ChAT levels were analyzed by Student’s t-test, and data regarding caspase-3 activity were analyzed using the Bonferroni multiple comparisons test. Differences were considered significant at the P ≤ 0.05 level.