Young (3 months old) and aged (18–24 months old) male Sprague Dawley rats were obtained from the Universidad de Valparaíso animal facility. Food and water were provided ad libitum. Lights were maintained on a 12–12 light/dark cycle and all experiments were performed in the light phase. Animals were handled in the experimental room for 10 min per day for 3 days prior to the first exposure to the behavioral apparatus. All experimental protocols used in this work complied with the “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society and were approved by the Bioethics Committee for Investigation in Animals of the Universidad de Valparaíso, Valparaiso, Chile.

Both the object-location and the novel-object recognition memory tasks were performed as described, with minor modifications (Uekita and Okanoya, 2011; Wimmer et al., 2012). The behavioral procedure included three consecutive phases: open field exploration, sample phases and recognition memory tasks. During the open field exploration phase, rats were habituated to the arena for 5 min during three consecutive days, using as behavioral apparatus a polyethylene black box (50 cm × 40 cm × 63 cm). During the sample phases, each rat was placed in the apparatus and was allowed to explore four different objects during 5 min for three consecutive sessions, separated by 5 min intervals. Four groups of young and four groups of aged rats were evaluated independently in the object-location or the novel-object recognition memory tasks at short-term (5 min) or long-term (24 h). To test object-location memory, two of the four objects previously presented in the sample phases were repositioned and the times exploring the repositioned and the non-relocated objects, and the quiescent times were determined as detailed below. To test object recognition memory, one familiar object was replaced with a novel-object and the times exploring the novel and the familiar objects, and the quiescent times were determined as described below. To eliminate odor cues between trials, the experimental apparatus and all objects were cleaned with 75% ethanol after each trial. Rat behavior was recorded with a video camera positioned over the behavioral apparatus and the collected videos were analyzed with the ANY-MAZE software (Stoelting Co., Wood Dale, IL, USA).

Six hours after concluding the behavioral procedures, animals under halothane anesthesia were sacrificed by decapitation and their brains were quickly removed. The hippocampal tissue was removed, dissected, immersed in cold dissection buffer (in mM: 212.7 sucrose, 5 KCl, 1.25 NaH₂PO₄, 2 MgCl₂, 1 CaCl₂, 26 NaHCO₃ and 10 glucose, pH 7.4) and cut into 400 μm transversal slices with a vibratome (Vibratome 1000 plus, Ted Pella Inc., Redding, CA, USA). Likewise, the perirhinal and surrounding cortex were immersed in cold dissection buffer and cut into 400 μm slices as described (Ziakopoulos et al., 1999). The hippocampal slices used for electrophysiological experiments were transferred to an immersion storage chamber and were kept at room temperature for 1 h in artificial cerebrospinal fluid (ACSF) solution (in mM: 124 NaCl, 5 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 26 NaHCO₃, 10 glucose, pH 7.4), bubbled with 95% O₂/ 5% CO₂. For mRNA or protein determinations (see below), the CA1 region or the PrhC from slices collected from controls or trained rats were micro-dissected and stored at -80°C for subsequent analysis.

Electrophysiological experiments were performed in an immersion-recording chamber. To evaluate fEPSP hippocampal slices were superfused at 30 ± 2°C with artificial cerebro-spinal fluid (ACSF) bubbled with 95% O₂ / 5% CO₂, at a rate of 2 ml/min. To evoke fEPSP, square current pulses (0.2 ms) were delivered with a concentric bipolar stimulating electrode (FHC Inc., Bowdoinham, ME, USA) located in the Schaeffer collateral–commissural fibers; fEPSP were recorded with ACSF-filled glass microelectrodes (2–3 MΩ) placed into the stratum radiatum layer of the CA1 region. To evaluate basal excitatory synaptic transmission, pulses of 25, 50, 75, 100, 150, or 200 microamperes were applied to generate an input/output curve. Results are presented as stimulus intensity versus FV amplitude or fEPSP slope. To evaluate pre-synaptic response components, two pulses were applied every 15 s, with inter-stimulus intervals starting at 20 ms and ending at 640 ms, doubling the interval after each trial. Results from paired-pulse stimulation experiments are presented as the ratio between the values of the initial fEPSP slope evoked by the second over the first stimulus. After monitoring both basal synaptic transmission and pre-synaptic responses, we evaluated LTP adjusting fEPSP to half of the maximal evoked response. Pulses were applied every 15 s until a stable baseline was recorded for at least 15 min. To induce LTP, we used the TBS protocol, comprised of four trains of 10 bursts at 5 Hz each, where each burst comprised four pulses at 100 Hz. To induce LTD, we used LFS protocols (1 Hz / 900 pulses). In all experiments, fEPSP were recorded for 60 min after applying the TBS or LFS protocols. Recordings were filtered at 10 kHz and digitized at 5 kHz using Igor Pro (WaveMetrics Inc., Lake Oswego, OR, USA). Synaptic responses were quantified as the initial slope of the evoked fEPSPs and were plotted as percentage of basal change, defining as 100% the slope measured during baseline recording.

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA); DNA digestion with DNA-free^TM Kit (Ambion, Austin, TX, USA) was included to remove any contaminating genomic DNA. RNA purity was assessed by the 260/280-absorbance ratio and RNA integrity by gel electrophoresis (Adasme et al., 2011). cDNA was synthesized using 2 μg total RNA with the Improm TM II reverse transcriptase (Promega, Madison, WI, USA). Two hundred nanogram of cDNA in a final volume of 20 μl was used for qPCR amplification, which was performed in the Stratagene MX3000P QPCR System (La Jolla, CA, USA) using the DNA binding dye SYBR green (Brilliant III Ultra-fast SYBR^® Green QPCR Master Mix; Carlsbad, CA, USA). For each analyzed gene, 10 picomoles of forward and reverse primers were used. The primer sequences used are listed in Table 1. To determine the level of RyR relative to that of β-actin mRNA we used the 2^-ΔΔCT method (Pfaffl, 2001).

Micro dissections of the hippocampal CA1 region and the PrhC from young and aged rats were homogenized in lysis buffer A (in mM: 300 sucrose, 2 EDTA, 2 EGTA, 1 BAPTA, 20 MOPS, pH 7.0, 1% Nonidet P-40, 0.1 % SDS) containing protease inhibitors (Calbiochem, La Jolla, CA, USA). Lysates were sonicated five times for 20 s with 20 s intervals, and were centrifuged at 4.000 rpm for 20 min to remove debris. After determining protein concentration with the sulfosalicylic acid Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), the resulting supernatants were diluted with Laemmli buffer and loaded on SDS-containing discontinuous polyacrylamide gradient gels (4.5 and 15%). After electrophoresis, gels were transferred to PVDF membranes (Millipore Corp., Bedford, MA, USA) using Transfer-Blot^® Turbo System (BIO-RAD, Hercules, CA, USA); membranes were subsequently blocked for 1 h at room temperature with 5% non-fat milk for β-actin, RyR2 and IP₃R1 or with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS), pH 7.4, for RyR3 and washed with TBS-Tween 20% (TBS-T). Membranes were incubated under constant shaking with primary antibodies: mouse anti-RyR2 (1:4000, Thermo, Waltham, CA, USA), rabbit anti-IP₃R1 (1:4000, Thermo, Waltham, CA, USA), or mouse β-actin (1:20000, Sigma, San Luis, MI, USA). Incubations were performed at 4°C overnight in 5% non-fat milk containing 0.2% Tween-20. For RyR3 immunodetection, membranes were incubated as above with rabbit anti-RyR3 (1:4000, Millipore, Bedford, MA, USA) dissolved in TBS-T containing 5% BSA. After washing three times with TBS-T for 10 min, membranes were incubated for 1.5 h at room temperature with appropriate secondary antibodies. Finally, membranes were washed three times with TBS-T for 10 min and immunoreactive proteins were detected with enhanced chemiluminescence (ECL) reagents according to the manufacturer instructions (Amersham Biosciences, Piscataway, NJ, USA). Signals were captured with the ChemiDoc system (Bio-Rad, Hercules, CA, USA). The IMAGE J image program (National Institutes of Health, USA) was used to quantify optical band intensity.

The whole hippocampus was homogenized in lysis buffer B (0.3 M sucrose, 20 mM Tris-MOPS, pH 7.0, plus protease inhibitors: 20 μg/ml benzamidine, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 10 μg/ml trypsin inhibitor and 20 μg/ml phenylmethylsulfonyl fluoride). The homogenate was sedimented at 4,000 g for 10 min at 4°C. The resulting supernatant was sedimented at 100,000 × g for 1 h, the pellet was homogenized in lysis buffer B. After determining protein concentration with the sulfosalicylic acid Protein Assay Kit, the resulting suspension was bubbled with argon gas and stored at -80°C. To label free SH residues, frozen aliquots were thawed and diluted to 2 mg/ml with Tris-HCl buffer, pH 7.4, and were incubated for 1 h on ice with 0.4 nmol/mg protein of EZ-Link Maleimide-PEG2-Biotin (Thermo Scientific, Rockford, IL, USA), followed by further incubation for 30 min with glutathione (2 nmol/mg of protein). After protein separation by SDS-PAGE electrophoresis and transfer to PVDF membranes as above, blots were blocked with 5% non-fat milk in TBS-T and incubated for 1 h at room temperature with streptavidin reagent (1:50,000, Thermo Scientific, Rockford, IL, USA). Membranes were washed with TBS-T, incubated with ECL reagents and signals were captured in the ChemiDoc system (Bio-Rad, Hercules, CA, USA). Membranes were subsequently stripped and incubated with anti-RyR2 antibodies as described above.

Values represent Mean ± SEM. Comparison between two groups was performed with Student’s t-test, and for multiple groups with one-way ANOVA followed by Tukey’s post hoc test or with two-way ANOVA followed by Bonferroni’s post hoc test; p < 0.05 was considered statistically significant.

To assess spatial memory retention in an object-location memory task, rats were tested 5 min or 24 h after concluding the sample phases, during which rats explored four different objects during 5 min for three consecutive sessions separated by 5 min intervals (Figure 2A). When tested after 5 min both young and aged rats recognized the two spatially modified objects and did not present significant differences in performance (Figure 2B). In contrast, only young rats recognized the spatial rearrangement of the objects when tested after 24 h (Figure 2C). We also evaluated possible changes over time in the ability to recognize novel objects, a PrhC-dependent task, and carried out short (5 min) and long-term (24 h) assays (Figure 2D). When tested at short-term, both young and aged rats recognized the novel object (Figure 2E). In contrast, 24 h after exposure to the sample phases, young rats discriminated the novel object but aged rats failed this task and presented significant differences in novel-object recognition compared to young rats (Figure 2F). Of the total 180 s allowed for the OL and the OR tasks, young and aged rats did not present significant differences between stationary times (Table 2). Altogether, these findings reveal that aged rats lose after 24 h the ability to perform both memory recognition tasks.

The hippocampal CA1 region has a key role in learning and memory processes (Moser et al., 1995). After performing the long-term object-location task, young rats exhibited no changes in RyR1 mRNA levels in CA1 hippocampal micro dissections (Figure 3A), but presented significant increments in RyR2 mRNA levels (Figure 3B) and a more modest but significant increase in RyR3 mRNA levels (Figure 3C). Of note, the CA1 region from naive aged rats displayed significantly higher RyR2 (1.7-fold) mRNA levels relative to naïve young rats, which did not change after testing aged rats in the object-location task at long-term (Figure 3D). No changes in RyR1 and a modest increase (1.4-fold) in RyR3 mRNA levels in the CA1 region were also detected (data not shown); these levels did not change after long-term exposure to the object-location task.

After performing at long-term the novel-object recognition task, which engages the PrhC, young rats did not exhibit significant differences in RyR1 (Figure 3E), RyR2 (Figure 3F), or RyR3 (Figure 3G) mRNA levels in the PrhC compared to naïve rats. In contrast to the increase in RyR2/RyR3 mRNA levels exhibited by the CA1 hippocampal region from naive aged rats, the PrhC from naïve aged rats had similar RyR1 (Figure 3H), RyR2 (Figure 3I), and RyR3 (Figure 3J) mRNA levels as naïve young rats. Moreover, these levels remained unaltered in the PrhC from aged rats tested after 24 h in the novel-object recognition task (Figures 3H–J).

After performing the long-term object-location task, the CA1 region from young rats exhibited significantly higher RyR2 (Figures 4A,C) and RyR3 (Figures 4B,D) protein contents. In contrast, neither the RyR2 (Figures 4A,C) nor the RyR3 (Figures 4B,D) protein levels changed after performing the novel-object recognition task. Naive aged rats exhibited significantly higher RyR2 (Figures 4A,C) and RyR3 (Figures 4B,D) protein contents in the CA1 region relative to young rats (Table 3); these levels did not change following exposure after 24 h to the object-location or the novel-object recognition tasks (Figure 4). We did not detect measurable RyR1 protein levels in the hippocampus from young or aged rats when using a highly specific antibody for the RyR1 isoform.

The PrhC isolated from young or aged rats had similar RyR2 protein contents. Likewise, both young and aged rats had similar RyR3 protein contents; these levels did not change after exposure to the object-location or the novel-object recognition tasks at long-term (Figure 5).

After performing the object-location, but not the novel-object recognition task, young rats displayed a small but significant increment in IP₃R1 protein levels in the hippocampal CA1 region, whereas aged rats did not present this task-related increase (Figures 6A,C). Naïve aged displayed similar IP₃R1 protein contents as naïve young rats (Table 3 and Figures 6A,C). The IP₃R1 protein levels in young rats did not change in the PrhC after performing the object-location or the novel-object recognition tasks (Figures 6B,D). Interestingly, naïve aged rats displayed lower IP₃R1 protein levels in the PrhC compared to young rats and these low levels did not change after exposure to the object-location or the novel-object recognition tasks at long-term (Figures 6B,D).

Aging entails impairments in synaptic plasticity processes (Barnes, 2003; Rosenzweig and Barnes, 2003; Hidalgo and Arias-Cavieres, 2016). To inquire into the synaptic basis of these defects, we examined the strength and plasticity of CA3-CA1 synapses. To this purpose, we isolated rat hippocampal slices from naïve rats or 6 h after the conclusion of behavioral procedures and registered field fEPSP in the CA1 region (Figure 7A). Slices from aged and young rats showed similar increments in FV amplitude with increasing stimulus intensity (Figure 7B), but slices from aged rats displayed lower fEPSP slopes versus stimulus intensity when compared to slices from young rats (Figure 7C). Differences between fEPSP slopes measured in slices from young and aged rats at the stimulus intensity of 150 μA, a value that produced maximal responses, were statistically significant (Figure 7D), suggesting that aged rats present defects in basal synaptic transmission.

To test whether a decreased probability of neurotransmitter release contributes to the reduced basal transmission recorded in slices from aged rats, we evaluated the paired-pulse ratio, which reflects the quantal release of neurotransmitter from the presynaptic neurons (Schulz et al., 1994). Hippocampal slices from either young or aged rats did not exhibit differences in facilitation ratio (Figures 7E,F), suggesting that defective post-synaptic events underlie the reduction in basal synaptic transmission displayed by aged rats. In addition, hippocampal slices isolated from young or aged rats exposed at long-term to the object-location task did not exhibit differences in FV amplitude (Figure 7B), fEPSP responses (Figure 7C) or facilitation ratio (Figures 7E,F), when compared to their respective naïve counterparts.

Next, we evaluated TBS-induced LTP at the CA3-CA1 synapses in naïve rats and in rats exposed to the object-location task after 24 h. Compared to their naïve counterparts, trained young rats displayed significantly increased LTP (Figures 8A,B) and higher fEPSP responses measured 1 h after TBS (Figure 8C), suggesting that this particular behavioral protocol enhances synaptic plasticity as reported for other types of training protocols (Whitlock et al., 2006). In contrast, hippocampal slices from aged rat displayed impaired LTP (Figures 8A,B) and significantly lower fractional increase in fEPSP slopes regardless of previous exposure to the object-location task (Figure 8C).

In addition, we assessed LTD induction by LFS of slices from naïve young or aged rats. Slices from aged rats exhibited significantly increased LTD (Figures 8D,E), with significantly higher fractional decrease in fEPSP slopes (evaluated at the end of the record) in comparison to slices from young rats (Figure 8F).

We evaluated RyR2, RyR3, and IP₃R1 protein contents in CA1 hippocampal micro dissections from slices isolated from young or aged rats that were exposed to LTP- or LTD-inducing protocols. After 1 h of exposure to the TBS protocol, slices from young rats displayed significant increases in RyR2 (Figures 9A,D), RyR3 (Figures 9B,E), and IP₃R1 (Figures 9C,F) protein contents in the CA1 region. In contrast, basal stimulation or exposure to the LTD-inducing protocol did not modify the content of these three proteins in CA1 from young rat slices, measured after 1 h (Figure 9). As exhibited by the CA1 region isolated from the whole hippocampus of naive aged rats (Table 3), CA1 micro dissections from slices obtained from naïve aged rats also displayed increased RyR2 (Figures 9A,D) and RyR3 (Figures 9B,E) protein contents (Table 3). These levels did not change after basal stimulation or after exposure to either the LTP- or the LTD-inducing protocols.

In agreement with the results presented in Table 3, the IP₃R1 protein content of the CA1 region from slices obtained from naive aged rats was not significantly different from that exhibited by naïve young rats (Table 3 and Figures 9C,F). These levels did not change in response to basal stimulation or after exposure to the LTD-inducing protocol (Figure 9). In contrast, after LTP-inducing stimulation the IP₃R1 protein content showed a modest but significant increase in CA1 dissections from hippocampal slices isolated from young but not from aged rats (Figures 9C,F).

We determined if the RyR2 channels present in aged rat hippocampus displayed increased oxidation levels, an unexplored subject to our knowledge. We found that the RyR2 channels expressed in the hippocampus isolated from aged rats exhibited significantly higher oxidation levels relative to the levels displayed by RyR2 channels present in young rat hippocampus (Figure 10).

Previous reports have shown that hippocampal-dependent spatial memory deteriorates during aging (Rosenzweig and Barnes, 2003; Veng et al., 2003; Wimmer et al., 2012; Kumar and Foster, 2013; Hopp et al., 2014). In agreement with these reports, we found that aged rats presented impaired hippocampal-dependent spatial long-term memory. In contrast to the general agreement on the deleterious effects of aging on hippocampal-dependent spatial memory, studies on the impact of aging on PrhC-dependent object recognition memory have yielded contradictory information. Previous reports using short delay intervals between the sample phase session and the object recognition task indicate that aged rats effectively discriminate novel objects (Cavoy and Delacour, 1993; Lukaszewska and Radulska, 1994). In contrast, studies using longer delay intervals report that aged rats fail this task (Burke et al., 2010; Gamiz and Gallo, 2012). Our results contribute to solve this apparent contradiction, since we found that the interval duration between the sample phase session and the object recognition task determines the response of aged rats. Thus, when examined after 5 min aged rats discriminated the novel object but failed to do so when evaluated after 24 h.

Synaptic plasticity is likely to represent the neuronal substrate for learning and memory (Gruart et al., 2006; Whitlock et al., 2006). Aging causes a progressive decline in synaptic function and causes significant LTP impairments, which correlate with the impaired ability to process and store information (Robillard et al., 2011; Haxaire et al., 2012). Here, we report reductions in synaptic strength and faulty LTP in aged animals; these responses strongly correlate with their spatial cognitive dysfunction. These deleterious changes probably arise from defects in post-synaptic mechanism, since presynaptic-dependent properties, such as the amplitude of FVs and the paired pulse responses were similar in young and aged rats. We also confirmed that aged rodents display increased susceptibility to LTD induction, as previously reported (Norris et al., 1996; Kumar and Foster, 2005).

A significant increase in hippocampal RyR2 (Zhao et al., 2000; Adasme et al., 2011) and RyR3 (Adasme et al., 2011) mRNA and protein levels occurs in young rats trained in the Morris water maze, a widely accepted protocol to evaluate hippocampal-dependent spatial memory (Morris et al., 1982). In addition, increased levels of RyR1, RyR2, and RyR3 mRNA correlate negatively with the performance of aged rats in this maze (Hopp et al., 2014). Our results expand these previous findings by showing that successful performance of a different hippocampal-dependent spatial memory task promoted significant increases in the mRNA and protein levels of RyR2/RyR3 in young rat hippocampus. The IP₃R1protein content also increased in the hippocampal CA1 region from young rats after successful performance at long-term of the spatial memory task. Up-regulation of these Ca²⁺ release channels caused by the spatial memory task was restricted to the hippocampus, since their levels did not change in the PrhC isolated from the same groups of trained young rats. Moreover, successful performance at long-term of the PrhC-dependent novel-object recognition task did not modify the expression of these channels in the PrhC from young rats. Consequently, we propose that successful performance of the spatial memory task selectively engages hippocampal cellular pathways that promote RyR2/RyR3 and IP₃R1 up-regulation in young rats. In addition, sustained LTP (1 h) but not LTD resulted in increased RyR2, RyR3, and IP₃R1 protein contents in hippocampal slices from young rats. To our knowledge, this is the first description of hippocampal up-regulation of these two types of calcium release channels induced both by LTP and by a spatial memory task.

The expression of several genes changes during the aging process (Kadish et al., 2009). In particular, aging entails decreased levels of FK506-Binding Protein 12.6 (FKB12.6), a RyR-associated protein that inhibits RyR channel activity; this decrease correlates with cognitive decline while FKBP12.6 overexpression reverses it (Gant et al., 2015). Synaptic dysfunction correlates with altered Ca²⁺ signaling in aged rodents, affecting in turn neuronal excitability and learning (Disterhoft et al., 1996; Thibault and Landfield, 1996; Foster and Norris, 1997; Gant et al., 2015). Enhanced RyR-mediated CICR (Kumar and Foster, 2005; Gant et al., 2006) and increased L-type Ca²⁺ channel expression (Veng et al., 2003) and activity (Thibault and Landfield, 1996) contribute to age-related Ca²⁺ signaling dysregulation. Our results reveal increased RyR2/RyR3 protein contents in the hippocampus but not in the PrhC from naive aged rats. In contrast, aged rats had similar hippocampal IP₃R1 protein content but decreased levels of this protein in the PrhC relative to young rats.

In addition to defective Ca²⁺ signaling, enhanced ROS generation is a characteristic trait of the aging process, which if uncontrolled leads to neuronal oxidative stress. Age-related changes in synaptic plasticity correlate with oxidative stress and with post-synaptic shifts toward oxidation in the intracellular oxidation-reduction environment (Bodhinathan et al., 2010a,b; Robillard et al., 2011; Haxaire et al., 2012; Kumar and Foster, 2013). In particular, the increased oxidative tone present in aged neurons decreases the synaptic responses induced by activation of N-methyl-D-aspartate receptors and affects as well other mechanisms underlying synaptic plasticity (Bodhinathan et al., 2010a; Kumar and Foster, 2013). We show here that RyR2/RyR3 protein content and RyR2 oxidation levels are higher in the hippocampus from aged naïve rats compared to naive young rats. Of note, RyR oxidative modifications strongly stimulate RyR-mediated Ca²⁺ release whereas reducing agents have the opposite effects (Hidalgo and Donoso, 2008; Hidalgo and Arias-Cavieres, 2016). Accordingly, we propose that the increase in RyR2/RyR3 hippocampal protein content and in RyR2 oxidation displayed by aged neurons, plus their decreased FKB12.6 levels (Gant et al., 2015), jointly contribute to produce the enhanced RyR-mediated Ca²⁺ signals that cause the prolonged sAHP phase exhibited by aged CA1 pyramidal neurons (Bodhinathan et al., 2010b). Since reducing agents significantly ameliorate the RyR activity-dependent prolonged sAHP phase exhibited by aged neurons (Bodhinathan et al., 2010b), we suggest that restoring RyR-mediated Ca²⁺ release to normal levels would reverse, at least partially, the deleterious effects of aging on hippocampal neuronal function.