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  • Experiments were performed on adult male Swiss mice (3- to 5-month-old; 28–35 g) obtained from an authorized supplier (Animal House of the University of Granada, Granada, Spain). Mice were kept on a 12-h light/dark cycle with constant ambient temperature (21±1°C) and humidity (50±7%). Food and water were available ad libitum. Electrophysiological and behavioral studies were performed in accordance with the guidelines of the European Union Council (2003/65/CE) and Spanish regulations (BOE 252/34367-91, 2005) for the use of laboratory animals in chronic experiments. Experiments were also approved by the institutional committee for animal care and handling (Vicerrectorate of Research/Ethic Committee Code 13/01/11). The experimental animals were assigned to the following groups: (i) animals having stimulation in the reuniens nucleus and fEPSP recordings in the prefrontal cortex (n = 20); these animals were classically conditioned with a trace paradigm in the presence (n = 10) or absence (n = 10) of two HFS sessions; (ii) animals stimulated in the reuniens nucleus and with fEPSP recordings carried in the hippocampal CA1 layer (n = 20); half of these animals were presented with two HFS sessions before the first two conditioning sessions; (iii) mice (n = 20) presented with an object discrimination test in the presence (n = 10) or absence (n = 10) of an HFS stimulation session; (iv) animals (n = 10) presented with two HFS sessions, and the fEPSPs evoked by pairs of pulses (40 ms of interval) in the PFC and in the CA1 were followed for up to 3 d; and (v) ten additional animals used for the histological study. Once anesthetized (ketamine, 35 mg/kg and xylazine, 2 mg/kg, i.p. ), mice were placed in a multi-arm stereotaxic frame. Body temperature was maintained at 37°C with a water blanket. A pair of stimulating electrodes was implanted stereotactically in the right reuniens nucleus (−0.82 mm posterior, +0.2 mm lateral, and −4.5 mm below bregma [54]). These bipolar stimulating electrodes were made from 50 µm, tungsten Teflon-coated wire (A-M Systems, Carlsborg, WA). Separation between tips was <1.0 mm, with the cathodic tip ∼0.5 mm ventral to the anodic one. For experiments included in groups (i) and (ii), animals were also implanted with a recording electrode aimed at the right mPFC (+1.94 mm anterior, +0.25 mm lateral, and −3.12 mm below bregma) for half of the animals or at the right CA1 layer of the hippocampus (−3.16 mm posterior, +3.2 mm lateral, and −2 mm below bregma) for the other half. The mPFC and CA1 recording electrodes were made from a single Teflon-coated tungsten wire (A–M Systems). The final depths of stimulating and recording electrodes were adjusted to achieve a maximal fEPSP response [2], [5]. In the same surgical step, animals included in groups (i) and (ii) were implanted with four electrodes in the upper eyelid of the left eye. Electrodes were also made from 50 µm, Teflon-coated, annealed stainless steel wire (A–M Systems), with their tips bared of the isolating cover for 0.5 mm and bent as a hook. Two of the electrodes were aimed at the supraorbital nerve, and served for the application of electrical stimuli. The second electrode pair was implanted in the ipsilateral orbicularis oculi muscle and served for recording its EMG activity. Another electrode was soldered to a screw and fixed in the skull, serving as ground. The eight electrodes were soldered to two 4-pin sockets (RS-Amidata, Madrid, Spain) and the whole assembly was fixed with dental cement to the cranial bone. After surgery, animals were kept in independent cages, with free access to food and water, for the rest of the experiment. Experiments were started one week after surgery. The EMG activity of the orbicularis oculi muscle was recorded with Grass P511 differential amplifiers (Grass-Telefactor, West Warwick, RI, USA) at a bandwidth of 0.1 Hz–10 kHz. Field EPSP recordings were also made with Grass P511 differential amplifiers through a high-impedance probe (2×1012 Ω, 10 pF). For input/output curves, animals were stimulated in the reuniens nucleus with paired pulses (40 ms of interstimulus interval) at increasing intensities (0.1–1.8 mA). The effects of paired pulses at different (20, 40, 100, 200, and 500 ms) interstimulus intervals were also checked. In this case, we used intensities corresponding to 35-50% of the amount necessary to evoke a saturating response [25]. To avoid any cumulative effect, intensities and intervals were presented at random. For the range of intensities used here, population spikes were observed rarely in the collected recordings. For LTP induction, each animal was presented with one or two HFS sessions. An HFS session consisted of five 200 Hz, 100 ms trains of pulses at a rate of 1/s. This protocol was presented six times, at intervals of 1 min. Thus, a total of 600 pulses were presented during the HFS session. Unless otherwise indicated, and in order to avoid evoking large population spikes and/or the appearance of EEG seizures, the stimulus intensity during HFS was set at the amount necessary to evoke about 1/3 of the maximum fEPSP response (0.4–0.9 mA) – that is, well below the threshold for evoking a population spike [2], [55]. An additional criterion for selecting stimulus intensity was that a second stimulus, presented 40 ms after a conditioning pulse, evoked a larger (>20%) synaptic field potential than the first [2], [56]. In addition, fEPSP evolution after HFS sessions was checked with pairs of pulses (40 ms of interval) presented to the reuniens nucleus for up to 72 h following the first HFS session. Conditioning sessions were carried out with two animals at a time. Animals were placed individually in a small (5×5×10 cm) plastic chamber located inside a larger (30×30×20 cm) Faraday box to eliminate electrical interferences. A trace conditioning paradigm was used. For this, animals were presented with a tone (2400 Hz, 70 dB, 20 ms) as CS, followed 500 ms later by an electrical stimulation (250 µs, 3 x Threshold, cathodic pulse) as US. Pairs of CS-US presentations were separated at random by 30±5 s. In total, two habituation, 10 conditioning, and three extinction sessions were carried out per animal. A conditioning session consisted of 60 CS–US presentations, and lasted ∼30 min. In order to determine the complete EMG profile of CRs, in 10% of the cases the CS was presented alone. For habituation and extinction sessions, only the CS was presented, also for 60 times per session at intervals of 30±5 s. As a criterion, we considered a “CR” the presence, during the CS-US interval, of EMG activity lasting ∼10 ms and initiated ∼50 ms after CS onset. In addition, the integrated EMG activity recorded during the CS-US interval had to be at least 2.5 times greater than the averaged activity recorded immediately before CS presentation [2], [21]. Synaptic field potentials in the mPFC or in the hippocampal CA1 layer were evoked during habituation, conditioning, and extinction sessions by a single 100 ìs, square, biphasic (negative–positive) pulse applied to the reuniens nucleus 250 ms after CS presentation. Stimulus intensities ranged from 0.4 mA to 0.9 mA. In the object recognition task, mice were individually habituated to an open field (40×25×15 cm), under low-illumination conditions and with no objects, for 5 min. Mice behavior was videotaped using a digital camera (Airis N729, Madrid, Spain) mounted 1 m above the open field. During the training session, two unknown but identical objects (O1 and O2) were put into the open field, and the mouse was allowed to explore them freely for 10 min. The time spent exploring each object and the total approach time (i.e., the time spent exploring both objects) were quantified. As criterion, object exploration was determined as the time spent by the animal close to the object and touching it with the nose or the vibrissae. After each trial, the apparatus and the objects were thoroughly cleaned with 70% ethanol. One hour after the first training, mice were allowed to explore the open field for another 10 min, when one of the two familiar objects (O1 or O2) was replaced with an identical object (O3), and the other (O1 or O2) with a novel object (B1). The time spent exploring each object and the total approach time were quantified again. Within each experimental group, the object positions were interchanged between mice to avoid location bias. After 24 h, mice were tested again, with a new object (C1) and an object identical to the previous one (B2). At the end of the behavioral studies, mice were divided in three groups: naïve control animal (without electrode), electrode controls (animals without stimulation) and stimulated animals (animals with stimulation). Mice were deeply re-anesthetized (sodium pentobarbital, 50 mg/kg), and perfused transcardially with saline and 4% phosphate-buffered paraformaldehyde. Selected sections (50 µm) including the thalamus, ventral hippocampus, and prefrontal cortex were mounted on gelatinized glass slides and stained using the Nissl technique with 0.1% cresyl violet. That allowed us to determine first, the exact location of the stimulating and recording electrodes in the brain (Fig. 1) and second, to verify whether neuronal loss or atrophy occurs in the reuniens nucleus. Quantification of neuronal density and reuniens nucleus area was carried out using the free Image J Software. For the sake of homogeneity, only data collected from animals that completed all the required tests were stored and analyzed (n≥7 per group and task). Data were stored directly on a computer through an analog/digital converter (CED 1401 Plus, Cambridge, England), at a sampling frequency of 11–22 kHz and an amplitude resolution of 12 bits. Data were analyzed off-line for quantification of CRs, fEPSP amplitude, and object recognition times with the help of homemade representation programs [2], [21], [23]. Collected eyelid data were quantified, through a purpose-designed Excel worksheet, as the percentage of CRs per session — namely, the proportion of stimulations within a session of 60 presentations that generated an EMG activity satisfying the above-mentioned criteria. For fEPSP analysis, five successive evoked field synaptic potentials were averaged, and the mean value of fEPSP amplitude was determined between the lower and upper inflection points of the evoked field potential [2]. Time expended on each object during the object recognition test was quantified from the collected videos. Statistical differences between groups were compared across conditioning and extinction sessions using the two-way repeated measures analysis of variance (ANOVA) test, performed with the SPSS 13.0 for Windows package (SPSS Inc, Chicago, IL). Unless otherwise indicated, data are represented by the mean ± s.e.m. Collected data were analyzed using a two-way ANOVA test, with time or session as repeated measure, coupled with contrast analysis when appropriate. One-way ANOVA allowed checking the statistical differences between different groups. In all of the cases, the corresponding statistical significance test (i.e., F[(m-1), (m-1) x (n-1)] statistic) were reported where m and n indicate number of groups and number of animals, respectively.
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