Aging-related impairments of hippocampal mossy fibers synapses on ...

Neurobiology of Aging 49 (2017) 119e137

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Aging-related impairments of hippocampal mossy fibers synapses on CA3 pyramidal cells Cindy Villanueva-Castillo, Carolina Tecuatl, Gabriel Herrera-López, Emilio J. Galván* Departamento de Farmacobiología, Cinvestav Sede Sur, México City, México

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 March 2016 Received in revised form 15 September 2016 Accepted 17 September 2016 Available online 28 September 2016

The network interaction between the dentate gyrus and area CA3 of the hippocampus is responsible for pattern separation, a process that underlies the formation of new memories, and which is naturally diminished in the aged brain. At the cellular level, aging is accompanied by a progression of biochemical modifications that ultimately affects its ability to generate and consolidate long-term potentiation. Although the synapse between dentate gyrus via the mossy fibers (MFs) onto CA3 neurons has been subject of extensive studies, the question of how aging affects the MF-CA3 synapse is still unsolved. Extracellular and whole-cell recordings from acute hippocampal slices of aged Wistar rats (34 2 months old) show that aging is accompanied by a reduction in the interneuron-mediated inhibitory mechanisms of area CA3. Several MF-mediated forms of short-term plasticity, MF long-term potentiation and at least one of the critical signaling cascades necessary for potentiation are also compromised in the aged brain. An analysis of the spontaneous glutamatergic and gamma-aminobutyric acid-mediated currents on CA3 cells reveal a dramatic alteration in amplitude and frequency of the nonevoked events. CA3 cells also exhibited increased intrinsic excitability. Together, these results demonstrate that aging is accompanied by a decrease in the GABAergic inhibition, reduced expression of short- and long-term forms of synaptic plasticity, and increased intrinsic excitability. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Aging MF-CA3 synapse Feed-forward inhibition Frequency-dependent facilitation MF LTP PKA signaling cascade

1. Introduction One of the earliest cognitive dysfunctions associated with the natural process of aging is the hindering of learning and memory performance. The decreased ability to form new episodic memories and contextual knowledge (Craik and Simon, 1980; Hedden and Gabrieli, 2004, 2005; Small et al., 1999) as well as the decline in the spatial memory abilities (Newman and Kaszniak, 2000) have been associated with modifications in the strengthening capabilities of the central synapses involved in mnemonic tasks. In the hippocampus, the diminished capacity for pattern separation, the neural activity associated with the storage of new episodic memories, the increased propensity for pattern completion, and the ability to recall memories from partial or degraded cues emerge from the computational imbalance that occurs in the dentate gyrus and CA3 network (Holden and Gilbert, 2012; Wilson et al., 2006; Yassa et al., 2011). In area CA3, pattern separation requires coordinated activation of 2 glutamatergic inputs to pyramidal cells: the perforant path (PP) * Corresponding author at: Departamento de Farmacobiología, Cinvestav Sede Sur, Calz. Tenorios No. 235, Col. Granjas Coapa, México City 14330, México. Tel.: þ52 55 5483 2852; fax: þ52 55 5483 2863. E-mail address: [email protected] (E.J. Galván). 0197-4580/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2016.09.010

that conveys compressed representations of the neocortical activity (Barnes and McNaughton, 1980; Do et al., 2002; Hunsaker et al., 2007; Treves and Rolls, 1992, 1994); and the mossy fibers (MFs), involved in the activation of subpopulations of CA3 pyramidal cells to form nonoverlapping representations of new memories via the strengthening of their commissural/associational (C/A) synapses (Lassalle et al., 2000; Lee and Kesner, 2004; Marr, 1971; McNaughton and Morris, 1987; Treves and Rolls, 1992, 1994). The aging-related alterations of the PP are well documented. Thus, a series of subtle modifications of multiple PP components (Adams et al., 2001; Barnes, 1979; Barnes and McNaughton, 1980; Foster et al., 1991; Geinisman et al., 1992; Huang et al., 2008; Magnusson, 1998; Smith et al., 2000) are believed to underlie the deficits in the maintenance of long-term potentiation (LTP) observed in the aged PP synapses onto dentate and CA3 pyramidal cells (Barnes et al., 2000; Burke and Barnes, 2006; Diana et al., 1994; Dieguez and Barea-Rodriguez, 2004; Gallagher et al., 2006; reviewed in Rosenzweig and Barnes, 2003). On the other hand, less is known about the modifications of the MF-CA3 synapse in the aged brain. For example, the MF-axonal sprouting in response to kainic acid administration exhibits a dramatic reduction in the aged brain, suggesting diminished axonal plasticity capability associated to age (Shetty and Turner, 1999). Furthermore, the CA3 region exhibits decreased levels of trophic

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and transcriptional factors (Hattiangady et al., 2005; Shetty et al., 2004) required for induction and expression of MF LTP (GómezPalacio-Schjetnan and Escobar, 2013; Gooney et al., 2004; Huang et al., 2008; Schildt et al., 2013). The aged MF buttons show a slight reduction in the number of mitochondria and vesicles, in addition to decreased number of excitatory synapses on CA3 thorny excrescences in the stratum lucidum of area CA3 (Ojo et al., 2013). Also, the MF bouton has a decreased capacity to release glutamate (Stephens et al., 2011). Finally, it is well known that aged hippocampal neurons suffer a loss of homeostatic calcium control (reviewed in Toescu et al., 2004) and decreased responsiveness to second messenger activation (Araki et al., 1995; Asanuma et al., 1996; Bach et al., 1999; Karege et al., 2001a,b; Nicolle et al., 2001). Together, the aforementioned cellular modifications can be an indicator of aging-related impairments in the synaptic transmission of area CA3. By combining electrophysiological and immunohistochemical techniques, we addressed the issue of how aging affects the strengthening capabilities of the MF-CA3 synapse. Our results show a dramatic alteration in the MF-CA3 feed-forward mechanism and the MF-mediated facilitation. Tetanic stimulation of the aged MFs yielded transitory potentiation and the pharmacological activation of the 30 ,50 -cyclic adenosine monophosphate signaling transduction pathway, consistently failed to trigger enhancement of the fEPSPs. Paradoxically, the decreased strengthening capability of the MF-CA3 synapse is accompanied by an increase in the intrinsic excitability of CA3 pyramidal cells, as previously reported (Simkin et al., 2015). Our results support the hypothesis that strengthening of the CA3 commissural/associational (C/A) synapses during aging (Wilson et al., 2006) occurs at the cost of reducing the capability for encoding new information via the mossy fibers to CA3 cells. 2. Materials and methods 2.1. Animals The animal procedures were deeply reviewed before approbation in accordance with the Mexican Official Norm for the use and care of laboratory animals “NOM-062-ZOO-1999” by the local Ethics Committee of our Institution (Cinvestav-IPN). Wistar rats were group housed (3 per cage), maintained in a climate-controlled facility (22 2 C) under a 12-hour light-dark cycle, with ad libitum access to food and water and periodic veterinary care. Twenty-one young (4e6 weeks old) and 22 aged Wistar rats (33 5 months old) were used to assess the effect of the nonpathological senescence on the synaptic strength and plasticity of the MF synapse on pyramidal cells in the hippocampus of the Wistar rat. 2.2. Hippocampal slice preparation The animals were anesthetized (pentobarbital intraperitoneal, 60 mg/1 kg body weight) and decapitated. The skull was exposed, and a caudal-to-rostral cut along the occipital suture was performed. The 2 plates of the skull were carefully removed and the brain was exposed in less than 30 seconds; placed in an ice-cold sucrose slicing solution containing (in mM): 210 sucrose, 2.8 KCl, 2 MgSO4, 1.25 Na2HPO4, 26 NaHCO3, 6 MgCl2, 1 CaCl2, and 10 D(þ)-glucose with pH 7.2e7.35 and saturated with O2 (95%)/CO2 (5%) carbogen mixture. After 30e45 seconds in the ice-cold sucrose solution, the hemispheres were separated with a midsagittal cut. The resulting blocks of tissue were glued to the plate of a VT1000S microtome (Leica, Nussloch, Germany) and transverse hippocampal slices (400-mm thick) were obtained using a cut frequency and advance speed of 90 Hz/0.75 mm/s (for young animals) or 95e100 Hz/1e1.25 mm/s (for aged animals) in the next 20 minutes.

The slicing method followed for the hippocampal slices has been previously published (Galván et al., 2010, 2015). The slices were transferred and maintained for 30 minutes at 33 2 C in an incubation solution with the following composition (in mM): 125 NaCl, 2.5 KCl, 1.2 Na2HPO4, 25 NaHCO3, 2 MgCl2, 1 CaCl2, 0.4 ascorbic acid, and 10 D-(þ)-glucose; pH 7.3 and continuously bubbled with O2 (95%)/CO2 (5%). Following the incubation period, the tissue was allowed to stabilize at room temperature for 1 hour before its use. Individual slices were transferred to a submersion recording chamber (total vol. 600 mL) for at least 15 minutes before the beginning of the experiments. The slices were maintained at a constant flow (3.5e4 mL/min) with a standard artificial cerebrospinal fluid (ACSF) solution containing (in mM): 125 NaCl, 3 KCl, 1.25 Na2HPO4, 25 NaHCO3, 2.5 CaCl2, 2.5 MgCl2, 0.5 ascorbic acid, and 10 glucose, heated to 33 2 C with the help of an inline solution heater coupled to a temperature controller (TC-324C, Warner Instruments). 2.3. Extracellular recordings Extracellular responses were recorded from the stratum lucidum of area CA3b with pipettes pulled from borosilicate glass with resistances of 1e2 MU when filled with a NaCl (3M) solution. Extracellular stimulation evoked MF EPSPs and commissural/ associational fEPSPs via bipolar electrodes made of nichrome wire (38 mM bare diameter). The electrodes were positioned on the suprapyramidal blade of the dentate gyrus and in the stratum radiatum, near the border between CA3b and CA3a. In some experiments, the MF electrode was placed in the hilus and the C/A electrode in the stratum pyramidale of CA3b/a (see Fig. 1A). Systematically, the stimulation electrodes were positioned on either side of the recording pipette. Test stimuli (0.06 Hz, unless otherwise indicated) consisted of paired monopolar pulses (15e45 mA intensity; 100e125 ms duration) that evoked 30%e50% of the maximal response. These values were previously determined with I-O curves (gray box in Fig. 1B). The test stimuli were delivered with a high voltage isolator unit (A365D; World Precision Instruments, Sarasota, FL, USA), controlled with a Master-8 pulse generator (AMPI, Jerusalem, Israel). The responses were amplified with a Dagan BVC-700A amplifier (Minneapolis, MN, USA) coupled with an extracellular headstage (Dagan 8024) and high-pass filtered at 0.3 Hz. Additional electrical noise suppression was achieved with a Humbug noise eliminator (Quest Scientific Instruments; North Vancouver, British Columbia). The resulting responses were displayed on a personal computer-based oscilloscope and digitized for storage and off-line analysis with custom-written software (Lab View system, National Instruments, Austin, TX, USA). Because of the complicated circuitry underlying the mossy fiber responses, we used the next criteria to identify and accept MF fEPSPs (Calixto et al., 2003; Claiborne et al., 1993; Urban and Barrionuevo, 1996). (1) The negative-evoked response (sink) was restricted to the stratum lucidum, and the duration was 4 ms; (2) the fEPSP onset latency was 0.06; at 90 minutes after HFS, 106 16%; p > 0.1;

n ¼ 12; Fig. 6A and B). Importantly, we also noticed in 3 AA slices that HFS yielded a persistent increase in the MF fEPSP slope that was sensitive to DCG-IV (PTP ¼ 231.8 53; MF fEPSP at 30 minutes after HFS, 161 8% of baseline; p < 0.001; at 60 minutes, 150 9%; p < 0.001; at 90 minutes after HFS, 154 14%; p < 0.05; MF fEPSP in the presence of DCG-IV ¼ 88.3 21 of depression; n ¼ 3). This observation suggests that aging did not affect in a similar way to the population of aged animals included in this study. In 5 additional AA slices, HFS did not cause PTP or long-lasting changes in the synaptic strength; those experiments were discarded from the study.


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Fig. 4. Decreased expression of the GAD-67 enzyme in the hippocampal formation. (A) Photomicrographs of GAD-67 immunoreactivity of horizontal slices from the hippocampus of young (left) and aged animals (right). (Top panels) Nuclei were counterstained with DAPI. (Middle panels) GAD-67 immunoreactivity for young and aged animals. (Bottom panels) Merged images. (B) Bar graphs summarizing the changes in the relative fluorescence intensity (FI; left bars) and normalized GAD-67 FI per area (mM; right bars) of the entire hippocampus of young and aged animals (n ¼ 4 for each condition). (C) Bar graphs of FI and normalized FI per area occurring solely in the DG-CA3 subregion. Error bars indicate SEM. **p < 0.01; ***p < 0.001 or higher statistical significance. Scale bar, 500 mM. Abbreviations: DAPI, 40 ,6-diamidine-20 -phenylindole dihydrochloride; GAD, glutamic acid decarboxylase; SEM, standard error of the mean.

3.7. FSK-induced MF potentiation is reduced in the aged animals Among the multiple downstream components of the signal transduction pathway required for induction of MF LTP, presynaptic activation of protein kinase A (PKA), plays a critical role (Calixto et al., 2003; Huang et al., 1994; Villacres et al., 1998; Weisskopf et al., 1994). Therefore, the failure in the induction of MF LTP in

the AA slices could be a consequence of alterations in the adenylyl cyclase-cAMP/PKA signaling cascade located at the presynaptic mossy boutons, as PKA signaling decreases its activity during aging (Araki et al., 1995; Bach et al., 1999; Karege et al., 2001a,b; Reis et al., 2005). Therefore, we explored the chemical induction of MF potentiation by bath application of the adenylyl cyclase activator, FSK (50 mM) in combination with the cyclic nucleotide

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Fig. 5. Aging-related changes in the paired-pulse facilitation. (A) Scatter plot summarizing the PPF ratio (explored between 50e1000 ms ISI) of young (empty circles; n ¼ 8) and aged (filled circles; n ¼ 7) animals. In the young animal group, MF PPF was consistently observed up to 1000-ms ISI. The facilitation of aged animal slices was restricted up to 200 ms ISI. (B) Scatter plot of S2 versus S1 amplitude (ISI ¼ 60 ms). Each dot in the graph represents the average of 5 continuous sweeps of 1 independent experiment (YA ¼ 55 slices; AA ¼ 61 slices). The values distributed above the dotted line indicate facilitated responses, whereas young animal responses were mainly distributed above the dotted line, the responses of aged animals were located below the line, exemplifying the loss of paired-pulse facilitation. (C) Representative paired pulse facilitation traces (S2) of young (top) and aged (bottom) animals at the indicated ISIs. (D) DCG-IV application at the end of the experiments depressed the synaptic responses. Each S2 trace is the average of 5 continuous sweeps. Error bars indicate SEM. **p < 0.01; ***p < 0.001 or higher statistical significance. Abbreviations: MF, mossy fiber; PPF, paired pulse facilitation; SEM, standard error of the mean.

phosphodiesterase inhibitor, and IBMX (25 mM) (Duffy and Nguyen, 2003; Huang et al., 1994). For those experiments, a second stimulation electrode was placed in the stratum radiatum (Fig. 7A, right panel) and C/A fEPSPs were also acquired as a negative control. A stable 25e30 minutes baseline was obtained in YA and AA slices; then, FSK þ IBMX was bath perfused for 15 minutes. As previously reported (Calixto et al., 2003; Huang et al., 1994; Villacres et al., 1998; Weisskopf et al., 1994), the coapplication of these drugs yield a sustained potentiation of MF fEPSP slope in YA (325 60% of baseline; p < 0.001). After the washout of the drugs, the MF fEPSP gradually decayed to a plateau level and remained potentiated up to 2 hours (FSK-induced potentiation at 130 minutes ¼ 180 23% of baseline; p ¼ 0.001; n ¼ 7; Fig. 7A and B). The MF-origin of the responses was confirmed by bath application of a higher dose of DCGIV, 10 mM (99 1% of synaptic depression). This higher concentration was used because previous studies demonstrated that adenylyl cyclase stimulation with FSK cause phosphorylation of the mGluR2, counteracting the inhibitory effect of mGluR2 activation on MF boutons (Kamiya and Yamamoto, 1997; Schaffhauser et al., 2000). In AA slices, the coapplication of FSK þ IBMX also yield a strengthening of the MF fEPSP slope (AA maximal response to FSK þ IBMX ¼ 178 27% of baseline; p < 0.001; Fig. 7A and B). However, after the removal of FSK þ IBMX, the MF fEPSP slope slowly returned to baseline values (95.2 11%; n ¼ 6). DCG-IV 10 mM applied at the end of the experiments confirmed the MF nature of the evoked responses (76.3 21% of depression; p ¼ 0.01; Fig. 7D, filled bars). As expected, the C/A fEPSPs were insensitive to the application of FSK þ IBMX (Calixto et al., 2003; Weisskopf et al., 1994) or DCG-IV in all the tested slices (upper time-course graph; Fig. 7B). We also monitored the paired-pulse facilitation ratio through the length of the experiments. In YA, during the perfusion

of FSK þ IBMX, a persistent decrease in the MF PPF was observed after the washout of the drugs (YA MF PPF in control ¼ 2.5 0.5; during FSK þ IBMX: 1.07 0.04; at 150 minutes washout: 1.07 0.04; Fig. 7D, empty bars). In contrast, AA did not exhibit changes in the paired-pulse facilitation during the perfusion nor during the drugs washout (AA MF PPF in control: 1.19 0.02; during FSK þ IBMX: 0.97 0.13; at 150-minute washout: 1.08 0.24; Fig. 7D, filled bars). Together, these results show that the adenylyl cyclasecAMP/PKA signaling required for the induction of hippocampal MF LTP is compromised in the aged animals. Moreover, the lack of changes in the MF-facilitation ratio point toward alterations in the presynaptic mechanisms controlling the MF-mediated transmitter release (Weisskopf et al., 1994; Zalutsky and Nicoll, 1990). 3.8. The aged CA3 pyramidal neurons show increased intrinsic excitability Simultaneous to the extracellular recordings, visually guided, patch clamp whole-cell recordings were also performed in aged CA3b pyramidal neurons (n ¼ 38). The cells included in the study exhibited a stable RMP after the initial break-in and action potentials with an amplitude z70 mV. The RMP of YA CA3 cells was within a range of 65 to 80 mV (average ¼ 71.24 1.1; n ¼ 15) and the RMP was in the range of 62 to 86 mV (average ¼ 70.5 1.9 mV; n ¼ 19). Both experimental groups exhibited similar membrane properties, as previously reported (Simkin et al., 2015; Fig. 8A; Table 2). The measurements of membrane time constant (sm) and input resistance (RN) indicated lower values (Fig. 8A and Table 2) compared to previous studies (Hemond et al., 2009; Spruston and Johnston, 1992). Nevertheless, sm and RN are membrane measurements that exhibit significant


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Fig. 6. Aging-related impairment in the induction of MF LTP. (A) Representative traces (averaged from 10 continuous sweeps) of MF fEPSP obtained at the times indicated by the numbers in the time-course graph on panel B, of aged (top traces) and young animals (bottom traces). LTP was induced with HFS (100 Hz, repeated 3 times at 10-second interval). In both groups, the slopes of the fEPSPs were depressed by bath application of DCG-IV. (B) Averaged time course and magnitude of LTP of young (empty circles; n ¼ 8) contrasted with the responses of aged (filled circles; n ¼ 12) animals. Arrowhead at time ¼ 0 indicates delivery of tetanic stimulation. Tetanic stimulation resulted in a robust post-tetanic potentiation (PTP) of the slope of the fEPSP in YA and, in a lesser extent, in the aged animals group. (C) Time course decay of MF fEPSPs slopes after tetanic stimulation. The nonlinear regressions are the mathematical best fits of data from young and aged (gray traces) animals adjusted from the time course graph (upper panel B). (D) Summary of the changes in the MF fEPSP slopes of young (empty bars) and aged (filled bars) animals during PTP, at 90 minutes after HFS and after the application of DCG-IV. (E) Cumulative probability distribution plot of normalized fEPSPs slopes in young (open circles) and aged (filled circles) animals. The probability plot was constructed excluding data from PTP on both conditions. Each point represents the magnitude of change relative to the normalized baseline slope values (dashed line). The arrowhead corresponds to the minimum criteria for LTP (stable synaptic enhancement >25% above baseline for at least 30 continuous minutes. Error bars indicate SEM. **p < 0.01; *** p < 0.001 or higher statistical significance. Abbreviations: DCG, (2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl)glycine; HFS, high-frequency stimulation; LTP, long-term potentiation; MF, mossy fiber; PTP, post-tetanic potentiation; SEM, standard error of the mean.

variations among multiple studies (YA sm coefficient of variation ¼ 0.55; for AA ¼ 0.46; YA RN coefficient of variation ¼ 0.31; for AA ¼ 0.36) (see, e.g., Hemond et al., 2009). Also, RN and sm can be modulated by the main Kþ carrier of the intracellular solution (Kaczorowski et al., 2007). In this study, the recorded CA3 pyramidal neurons displayed a bursting fire pattern (YA, n ¼ 1; AA, n ¼ 2), a single action potential during the sustained depolarization (YA, n ¼ 12; AA, n ¼ 15) or a regular or weak adaptive fire pattern (YA, n ¼ 3; AA, n ¼ 2) in response to the respective rheobase current injection (see Table 2). However, in response to a 400-pA current step, CA3b cells from both groups displayed regular or adaptive fire pattern (Fig. 8B) (Hemond et al., 2009; Masukawa et al., 1982).

Our experiments did not reveal a significant modification in the total number of action potentials of AA compared to YA (YA mean AP number ¼ 4.18 0.69; n ¼ 13; in AA CA3b cells ¼ 5.11 1.56; n ¼ 11; p ¼ 0.065; Fig. 8B and C); however, the injection of the square pulse unmasked a unique characteristic of AA cells. The Fig. 8D shows the individual fitting to an exponential decay function of the frequency adaptation (sadapt) of 15-YA and 18-AA cells to determine the time constant of adaptation. sadapt was significantly faster in AA (YA sadapt ¼ 91.1 21.5 ms; AA sadapt ¼ 21.4 10.5 ms; p < 0.05) and the attenuation factor (Fadapt) was significantly higher in AA neurons (YA Fadapt ¼ 0.62 0.07; n ¼ 15; AA Fadapt ¼ 0.81 0.05; n ¼ 18; p < 0.05) indicating that AA CA3b cells exhibit a stronger adaptive fire pattern. The time constant of adaptation and

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Fig. 7. Aging-related impairment of the forskolin-induced potentiation of MF fEPSPs. (A) Representative traces (averaged from 5 consecutive sweeps) of MF fEPSPs obtained at the times indicated by the numbers of aged (top traces) and young animals (bottom traces). The inset shows the stimulation and recording configuration in which a second stimulation electrode was positioned in the C/A fibers as a negative control. (B) Time-course graph showing the simultaneous recordings of C/A fEPSPs and MF fEPSPs. C/A fEPSPs were stable responses through the length of the experiments, whereas the MF fEPSPs exhibited a robust potentiation of young (empty circles; n ¼ 7 slices) and mild potentiation in the aged (filled circles; n ¼ 6 slices) animals. In the young animal group, FSK þ IBMX caused a persistent enhancement of the MF fEPSP; in the aged animals, the chemical stimulation of the AC with FSK failed to enhance persistently the MF fEPSPs and the evoked responses decayed to baseline levels within 25 minutes after the washout of the drugs. DCG-IV applied at the end of the experiments confirmed the MF nature of the evoked responses. The time course of the upper panel (C/A fEPSPs) was constructed from the aged animals group (n ¼ 6 slices). (C) Summary of the changes in MF fEPSP slopes of young and aged animals (empty and filled bars, respectively), during FSK þ IBMX stimulation, at 90 minutes after the washout and in the presence of DCG-IV. (D) Summary of the changes in the PPF ratio in the young and aged animal groups. PKA stimulation caused a persistent decrease in the MF PPF in the young but not in the aged animal group. (E) Cumulative probability distribution of the MF fEPSPs potentiation for YA and AA. The arrowhead indicates the minimum criteria for LTP (stable synaptic enhancement > 25% above baseline for 30 minutes). Error bars indicate SEM. **p < 0.01; ***p < 0.001 or higher statistical significance. Abbreviations: DCG, (2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl)glycine; FSK, forskolin; MF, mossy fiber; PPF, paired pulse facilitation; SEM, standard error of the mean.

the average adaptation frequency is shown in Fig. 8D and inset graph. We determined that the faster sadapt and the increased Fadapt of AA CA3b cells were a consequence of the early onset of action potentials (indicated with arrowheads in Fig. 8B) that also alters the IFF within the first interspike interval (YA maximal IFF at the

first interspike interval ¼ 47.8 12.04 Hz; n ¼ 15; AA ¼ 105.5 18.8 Hz; n ¼ 18; p < 0.001; Fig. 8E and F). The increase in the fire pattern of aged CA3b cells was consistently observed from trial to trial in all the cells tested and supports the notion that aged CA3b cells exhibit higher firing rates (Simkin et al., 2015; Wilson et al., 2005, 2006).


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Fig. 8. Increased excitability of aged CA3 pyramidal cells. (A) Scatter plot. Aging does not alter the relationship among input resistance (RN) and membrane time constant (sm) in CA3b pyramidal cells. Each dot represents the input resistance and membrane time constant of individual young and aged CA3b pyramidal cell recorded using the whole-cell technique (YA, n ¼ 15; AA, n ¼ 19 neurons). (B) Representative traces showing the fire pattern of CA3b pyramidal cells (young and aged, respectively). The dotted line represents the action potential overshooting. The arrowheads indicate the increase in the instantaneous fire frequency of aged cells. (C) Bar graph summarizing the mean number of spikes in response to 1-second duration, 400-pA depolarizing square pulse. No difference was observed in the number of action potentials in both groups. (D) Individual decay adjustment of the instantaneous fire frequency in response to 1-second current step (700 pA; YA, n ¼ 15; AA, n ¼ 18, red lines) showing the increase in the instantaneous firing frequency of aged CA3 cells. Inset, average response constructed from the same cells. The IFF is significantly higher and decays faster in aged cells. (E) Scatter plot of instantaneous fire frequency of the first interspike interval versus increasing current intensity (YA, n ¼ 15; AA, n ¼ 18). (F) Bar graph summarizing the changes in the mean instantaneous frequency. Compared to young, aged animals exhibited a robust increase in the instantaneous fire frequency (YA, n ¼ 15; AA, n ¼ 18). (G) Representative example of the fire pattern elicited with a 5-Hz sine-wave current injection (150 pA; 50-pA increase step) in young and aged CA3b pyramidal neurons. (H) Mean number of action potentials evoked in function of the current intensity and the number of the sine-wave crest of the stimulus for both groups. Note the increased excitability of aged CA3 pyramidal cells. (I) Box plots summarizing the action potential number evoked on the top of each sine-wave crest at different current intensities. The dashed line indicates the mean for each group. *p < 0.5; **p < 0.01; ***p < 0.001 or higher statistical significance. Abbreviation: IFF, instantaneous fire frequency. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Because the injection of square pulses can inactivate multiple ionic conductances (Chitwood and Jaffe, 1998), we also injected a sine-wave current pulse (0e450 pA at 5 Hz; 2-second duration), a manipulation that increases the probability of spike appearance avoiding the inactivating effect of the square pulse (Sheffield et al., 2010). As expected, the sine-wave current injection revealed a significant increase in the rate of spikes per sine-wave crest in AA (YA ¼ 0.76 0.22 spikes; n ¼ 10 cells; AA ¼ 1.49 0.39 spikes; n ¼ 9 cells; p < 0.001). In contrast to the square-pulse injection, the sine-wave pulse revealed an increase in the number of AP on aged CA3b cells (Fig. 8GeI). To finish with this study, we also performed an analysis of sEPSCs and sIPSCs, respectively, of CA3b cells. The spontaneous

activity was recorded in voltage clamp mode while cells were held at 80 mV in the presence of D-AP5 (50 mM) and bicuculline (10 mM). In another group of cells, the holding potential was 0 mV and the recording was performed in the presence of kynurenic acid (4 mM). Under these conditions, we recorded sEPSCs or sIPSCs for 5 minutes, and the amplitude and frequency (expressed as the inverse of interevent interval) of the events was compared. A previous report indicated that tonic levels of glutamate release are barely affected with aging in the DG and CA3 (Stephens et al., 2011). In agreement with this observation, we found that the frequency of the sEPSCs of the AA group was similar to the frequency of YA cells. In contrast, the AA CA3b cells displayed a slight but significant reduction in sEPSCs amplitude. (YA sEPSCs

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Table 2 Intrinsic membrane properties of young and aged CA3 pyramidal cells

Resting membrane potential (mV) Input resistance (RN) (MU) Sag ratio (mV) Membrane time constant (sm) (ms) Rise tau (ms) Rheobase current (pA) Max AP freq (Hz) AP threshold first spike (mV) AP threshold second spike (mV) Max first spike rising dV/dt (mV/ms) Max first spike falling dV/dt (mV/ms) First spike height (mV) Second spike height (mV) AHP amplitude first to second AP (mV)

Young CA3 cells (n ¼ 15)

Aged CA3 cells (n ¼ 19)

71.2 81.5 3.2 41.8 42 251.5 10.2 42 41.3 201 106.7 82.4 81.8 9.3

70.6 80.4 3.2 40.9 40.9 255 13.7 41 40 180 106.6 79.3 78.5 12.8

1.1 8.1 0.4 5.3 5.3 22 1.2 0.6 0.9 8 3.2 1.2 1.1 1.1

1.2 5.4 0.5 7 7 25 2.9 1 1 6 4 1.4 1.5 1.1*

Key: AHP, afterhyperpolarization; AP, action potential. * p < 0.05.

amplitude ¼ 36.49 0.34 pA; sEPSCs frequency ¼ 3.5 0.96 Hz; n ¼ 10 cells; AA sEPSCs amplitude ¼ 32.05 0.4 pA; p < 0.001; AA sEPSCs frequency ¼ 2.9 1 Hz; p > 0.05; n ¼ 11 cells; Fig. 9A and B). Perfusion of kynurenic acid (4 mM) at the end of the recordings confirmed the excitatory nature of the evoked responses (Fig. 9A, bottom panel). On other hand, the sIPSCs of AA CA3b cells, recorded at 0 mV in the presence of kynurenic acid (4 mM),

exhibited a reduction in amplitude and frequency (YA sIPSCs amplitude ¼ 51.17 0.61 pA; AA sIPSCs amplitude ¼ 39.18 0.73 pA; p < 0.001; n ¼ 9. YA sIPSCs frequency ¼ 1.94 0.48 Hz; AA sIPSCs frequency ¼ 0.69 0.14 Hz; p < 0.05; n ¼ 9; Fig. 9C and D). Together, the whole cell recordings indicate that the increased intrinsic excitability of aged CA3b cells is accompanied by a reduction in the amplitude and frequency of inhibitory events impinging on CA3 pyramidal cells. 4. Discussion 4.1. Summary The aging-related alterations of the perforant path making synaptic contact with dentate granule cells and CA3 neurons are documented (Barnes and McNaughton, 1980; Dieguez and BareaRodriguez, 2004; Foster et al., 1991). Nevertheless, the information regarding the aging-related electrophysiological alterations of the mossy fibers onto CA3 neurons is limited. Here, we provided a detailed description of the changes observed in the aged Wistar rat. A reduction in the strength of the interneuron-mediated inhibition was observed. The presynaptic short-term plasticity including frequency-facilitation and MF LTP are also compromised in the aged animals. Moreover, at least one of the critical signaling cascades required for MF LTP, the adenylyl cyclaseecAMP/PKA, fails to potentiate the MF fEPSP. In agreement with previous reports

Fig. 9. Aging-related alterations of the spontaneous synaptic activity of CA3 pyramidal neurons. (A) Representative traces from a continuous acquisition (5 minutes) in voltage clamp of spontaneous excitatory postsynaptic currents recorded at 80 mV in the presence of D-AP5 and bicuculline. Kynurenic acid application at the end of the acquisition suppressed the spontaneous glutamatergic events. The solid lines in the upper trace indicate the expansion in the bottom panels. (B) Cumulative distribution of amplitude (left) and interevent intervals (frequency) of sEPSCs (right) in young and aged cells. The amplitude exhibited a significant decrease, whereas the frequency of the spontaneous glutamatergic events remains constant in the aged CA3b cells. The inset box plot summarizes the changes in the amplitude (dash line inside the boxes represents the mean and the continuous line, the median). The distribution was constructed from the individual analysis of 10 and 11 cells (YA and AA, respectively). (C) Representative traces and expansions of spontaneous inhibitory postsynaptic currents recorded at 0 mV in the presence of kynurenic acid. (D) Cumulative distribution of amplitude (left) and interevent intervals (frequency) for young and aged CA3b cells. In contrast to the observed with the spontaneous glutamatergic transmission, both amplitude and frequency of the spontaneous GABAergic inputs are significantly decreased in the aged CA3b cells. The inset box plot summarizes the changes of the inhibitory activity and the bar graph, amplitude (YA, n ¼ 9; AA, n ¼ 9 cells). *p < 0.05; ***p < 0.001 or higher statistical significance. Abbreviation: sEPSCs, spontaneous excitatory postsynaptic currents.


(Simkin et al., 2015; Wilson et al., 2005, 2006), our whole-cell experiments indicated that aging is associated with an increase in the intrinsic excitability without changes in the passive membrane properties of CA3 pyramidal neurons. We also found a reduction in the amplitude but not in the frequency of spontaneous glutamatergic postsynaptic currents and a systematic decrease in the amplitude and frequency of spontaneous GABAergic currents. 4.2. Electrophysiological alterations of the MF-CA3 synapse during aging The analysis of the composite MF responses in which presynaptic fiber volleys preceded the fEPSPs revealed an amplitude reduction for both components. However, we found no significant change in the latency of the evoked MF responses. Therefore, the combination of decreased volleys and MF fEPSP amplitudes without changes in latency suggests that aged MF-CA3 synapses have a decreased number of active MF axons. In agreement with this, Ojo et al. (2013) reported a loss in the number of asymmetrical excitatory synapses, a reduced number of presynaptic vesicles and mitochondria loss in the aged mossy boutons (however, see Poe et al., 2001; Smith et al., 2000). In comparison to young, aged dentate granular cells are more efficient in eliciting action potential discharges, a phenomenon proposed as a compensatory mechanism to preserve the synaptic strength of the PP-granule cell synapse (Barnes and McNaughton, 1980; Burke and Barnes, 2006; Foster et al., 1991). Thus, it is possible that granular cells require discharging faster (Barnes and McNaughton, 1980) to compensate the reduction in the synaptic density within the stratum lucidum of area CA3 (Ojo et al., 2013) or the decreased capability of aged mossy fibers to release glutamate (Stephens et al., 2011). 4.3. Decreased response to the activation of the mGluR2 in the aged hippocampus Hippocampal excitability is highly regulated by the activation of the Gi/o-protein-coupled receptors, group II mGluRs. Thus, presynaptic activation of mGluR2/mGluR3 suppresses the glutamatergic transmission by inhibiting the adenylyl cyclase activity, increasing activation of Kþ channels and inhibiting the N and P/Q calcium channels involved in glutamate release (Brunner et al., 2013; Kamiya and Ozawa, 1999; Kamiya et al., 1996; Swartz, 1993). On the contrary, our data revealed that the pharmacological activation of the presynaptically located mGluR2 with DCG-IV, did not suppress the MF-fEPSP in the aged as it did in the young animal slices, an observation that can be explained by different mechanisms. The mGluR2 is an autoreceptor located extrasynaptically, and, elevated concentrations of glutamate are required to activate it (Scanziani et al., 1997). As glutamate release from the aged mossy fibers decrease with age (Stephens et al., 2011), it can be speculated that, in response to the lower levels of glutamate, the mGluR2 effector system decreases its activity as a compensatory mechanism during aging. Also, as mGluRs of group I and II shows alterations in expression and binding during aging (Magnusson, 1998; however, see Nicolle et al., 1999; Simonyi et al., 2000), we cannot discard that those alterations also occur at the aged mossy bouton. If this is the case, it would explain the elevated concentrations of DCG-IV required for the partial depression observed in our study. Finally, the mGluR2-mediated suppression of glutamate release from the mossy fibers is due to a reduction of presynaptic Ca2þ influx and/or downregulation of the subsequent exocytotic machinery (Kamiya and Ozawa, 1999). Thus, it is also likely that the disregulation of the intracellular calcium during aging negatively affects the inhibition of calcium entry or the activation of the release machinery.

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Albeit all these possibilities, we did not perform experiments to examine directly the expression levels or transductional pathways of the mGluRs in the mossy fibers, an issue that requires further investigation. 4.4. Aging-related loss of inhibition The axons of dentate cells or mossy fibers are comprised of a series of small presynaptic terminals contacting interneurons that outnumber the large mossy buttons contacting CA3 pyramidal cells (Acsády et al., 1998; Frotscher, 1989; Henze et al., 2000; Mcbain, 2008). This cytoarchitectonic arrangement confers a unique characteristic to the DG-CA3 network, a strong disynaptic feedback and feed-forward inhibitory mechanism (Buzsáki, 1984; Lawrence et al., 2004). Our experiments revealed alterations of the inhibitory network. The reduction in paired-pulse inhibition indicates a diminished synchronous activity of interneurons during the granule cell discharge; suggesting a loss of the interneuron-mediated recurrent or feedback inhibition (Andersen et al., 1966; Sloviter, 1991). The reduction in the MF-driven, frequency-dependent facilitation indicates decreased control of CA3 pyramidal cells excitability driven by granular cell firing (Torborg et al., 2010). Previous works demonstrated that the aging-related loss of inhibition could be ascribed to a dysfunction of subpopulations of GABAergic interneurons and a reduction of GAD-67 immunoreactivity in CA1 (Potier et al., 2006; Stanley et al., 2012). In agreement with this, we did observe that decreased inhibition of area CA3 is accompanied by a reduction in the expression levels of the GAD-67 enzyme in addition to the reduced spontaneous GABAergic currents on CA3 cells. It is physiologically relevant that the unmasked alterations observed with the stimulation train delivered to the mossy fibers correspond to the granule cell’s place field discharge rate observed in vivo (10e40 Hz; Henze et al., 2002; Jung and McNaughton, 1993; Mori et al., 2004; Pelkey and McBain, 2008). Notably, the 40-Hz train unmasked several alterations at the MF synaptic transmission, including (1) a dramatic reduction in the frequencydependent facilitation; (2) decreased GABA release in response to rising the external calcium and additionally; and (3) decreased responsiveness to the pharmacological blockade of GABAA receptors. The latter phenomenon was consistently observed with the paired-pulse inhibition and the 40 Hz trains and can be ascribed to alterations in the GABAA receptor composition (Post-Munson et al., 1994; Rizzo et al., 2015). The compromised frequency-dependent facilitation has been observed at the aged CA1 synapses (Landfield and Lynch, 1977; Landfield et al., 1986) and is associated with an increase in the density of L-type calcium channels and elevated postsynaptic Ca2þ concentration (Ouanounou et al., 1999; Thibault et al., 2001, 2007). Conversely, the facilitation processes at the MF-CA3 synapse has a predominant presynaptic component (Regehr et al., 1994; Salin et al., 1996). Thus, the diminished facilitation observed in this study suggests alterations at the presynaptic level. The compromised responsiveness of the aged mossy fibers during the repetitive stimulation may include deficiencies in the cellular mechanisms controlling the presynaptic calcium homeostasis; alterations in the presynaptic release machinery or decreased efficiency of the remaining synaptic contacts (Geinisman et al., 1986; Ojo et al., 2013; Poe et al., 2001; but see Smith et al., 2000). However, the potential alterations of the aged mossy boutons still require experimental demonstration. The decreased GABAergic activity during the repetitive stimulation also suggests a loss of functionality of specific subpopulations of GABAergic cells during aging. Despite the fact that hippocampal interneurons represent a heterogeneous population of cells (Galván et al., 2011), they can be grouped into functional subpopulations that restraint in an input-specific fashion,

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the excitability of the different subcellular compartments of CA3 principal cells (Klausberger and Somogyi, 2008). Thus, the perisomatic inhibitory interneurons, mainly located in the stratum lucidum, restrain the somatic firing of action potentials of principal cells; whereas the dendritic-targeting inhibitory interneurons play a central role in the control of the dendritic Ca2þ signaling (Galván et al., 2008). The recordings of this study were performed in the stratum lucidum, a region in which the basket and axo-axonic cells are somatically located (Spruston et al., 1997; Vida and Frotscher, 2000). Therefore, the decreased inhibitory response of the aged animals also indicates that perisomatic interneuron functionality is compromised in the aged CA3 region of the hippocampus, a subject that we are currently investigating. To finish with this, previous works also demonstrated that gamma oscillations, a form of rhythmic electrical activity involved in the encoding and retrieval of episodic memories, are impaired in the aged hippocampus (Kanak et al., 2011; Lu et al., 2011; Vreugdenhil and Toescu, 2005). Gamma oscillations (20e80 Hz) occur in the hippocampus during the exploratory behavior (Bragin et al., 1995; Csicsvari et al., 2003) and require an intact GABAergic network (Hájos and Paulsen, 2009). Remarkably, the coherence, dominant frequency, and waveform of gamma oscillations are preserved through the lifespan and only exhibit a significant reduction in the power of the oscillation during aging (Driver et al., 2007; Vreugdenhil and Toescu, 2005). The decreased capability of the aged interneurons to induce electrical oscillations in area CA3 has been ascribed to a dysregulation of intracellular calcium control, altered expression of L-type calcium, and increased Kþ-dependent slow AHP (Lu et al., 2011; Vreugdenhil and Toescu, 2005). Interestingly, induction of LTP on CA3 interneurons is highly dependent on postsynaptic calcium and L-type calcium channels activation during the repetitive stimulation of the mossy fibers (Galván et al., 2008), suggesting that plastic processes of aged interneurons including LTP and LTD might exhibit alterations during aging (Galván et al., 2011).

Thus, presynaptic L-type calcium channels and activation of presynaptic autoreceptors modulate the strength of the synaptic transmission (Bortolotto et al., 2005; Lerma, 2003). Among the latter, the kainate receptors play a central role in the MF-mediated facilitation and synaptic plasticity (Kamiya et al., 2002; Mori-Kawakami et al., 2003; Schmitz et al., 2001), as induction of MF LTP require a kainate receptoredependent component that triggers calcium-induced calcium release (Bortolotto et al., 1999; Contractor et al., 2001; Lauri et al., 2001a,b, 2003). Unsurprisingly, L-type calcium channels, kainate receptor expression, and the calcium-induced calcium release machinery are compromised in the aged hippocampus (Clark et al., 1992; Gallagher and Nicolle, 1993; Lynch and Voss, 1994; Nagahara et al., 1993; Nicolle et al., 1996; Núñez-Santana et al., 2014). Another plausible source of calcium disturbance that cannot be discarded is the reduction in the expression of Ca2þ binding proteins on the aged granule cells. Changes in the expression levels of those proteins alter the buffering capabilities of the mossy buttons and the strength of the synaptic responses including PPF and LTP (Blatow et al., 2003; Dumas et al., 2004; Müller et al., 2005). Notably, Calbindin D28k is one of the calcium-binding proteins that reduce its expression during aging in the dentate gyrus (Kishimoto et al., 1998; Lally et al., 1997). Lastly, the transient rise in Ca2þ triggers the calcium-activated adenylyl cyclase-cAMP/PKA pathway, a signaling cascade required for the maintenance of the synthesis-dependent late phase of the MF LTP (Huang et al., 1994). Our experiments revealed that continuous stimulation of the adenylyl cyclase with FSK yields to a significant but transitory potentiation that failed to trigger a long-lasting enhancement of the MF fEPSP, suggesting that protein and RNA synthesis necessary for the late maintenance of MF LTP is also impaired in the aged brain (reviewed in Schimanski and Barnes, 2010). Consistently, the decreased functionality of PKA during aging has been observed in area CA1 (Bach et al., 1999; Reis et al., 2005) as well as in the entorhinal cortexeDG synapses (Kelly et al., 2000).

4.5. Impairments in the plasticity of the MF-CA3 synapse

4.6. The impact of aging on the physiology of the mossy fibers synapses

The reduction of the fEPSP frequency facilitation, paired pulse facilitation (PPF) ratio, PTP, and decreased levels of LTP shed lights on the mechanistic disturbance caused by aging on the MF-CA3 synapses. PPF, frequency facilitation, and PTP are presynaptic forms of short-term enhancement that depend on a rise in the intracellular Ca2þ concentration (Regehr et al., 1994; Salin et al., 1996; reviewed in Zucker and Regehr, 2002). The PTP that follows tetanic stimulation causes a transitory increase in presynaptic Ca2þ that activates PKA and induce presynaptic MF LTP (Langdon et al., 1995; Regehr et al., 1994; Urban and Barrionuevo, 1996; Weisskopf et al., 1994; Zalutsky and Nicoll, 1990). Given the relevance of presynaptic calcium in the induction and expression of MF LTP, our results favor the hypothesis of the disturbance in the mechanisms involved in the homeostatic control of calcium during aging (Thibault et al., 1998; Toescu et al., 2004; Verkhratsky and Toescu, 1998); and more specifically, our results indicate a disturbance in the control of calcium levels following the repetitive firing of the granule cells. Additionally, the reduced temporal integration of synaptic activity observed during the short-term forms of synaptic enhancement points out to a diminished capability of the DG-CA3 network to be instructed by specific temporal pattern of neural activity to encode new information, a phenomenon observed in aged DG-CA3 synapses of rodents and humans (Gallagher et al., 2006; Yassa et al., 2010). The pronounced synaptic facilitation of the MFs during repetitive stimulation involves multiple cellular mechanisms.

MFs are unique for its anatomical localization and synaptic capabilities: their pronounced short-term synaptic enhancement and frequency-dependent facilitation allow the MFs to work as temporal integrators of synaptic activity (Evstratova and Tóth, 2014; Henze et al., 2000; Nicoll and Schmitz, 2005; Salin et al., 1996). Moreover, MFs have been referred as a detonator synapse (Henze et al., 2000, 2002) that might work as a discriminator capable of categorizing and distinguish fire frequencies from granule cells (Urban et al., 2001). Therefore, the reduced inhibition and responsiveness of the MF synapses observed in this study provide details of the synaptic alterations that contribute to the reduced reaction of the DG-CA3 network to encode new memories in the aged brain. Disclosure statement The authors have no competing financial interest. The experiments carried out in this study complied with National Institutes of Health guidelines and with the Mexican Official Norm for the use and care of laboratory animals “NOM-062-ZOO-1999.” Acknowledgements The authors thank Dr Isabel Sollozo-Dupont for participating in data evaluation, fitting selection, and statistical analysis. This work was supported by CONACYTeMéxico grants to Emilio J. Galván: CB-2011-01-166241 and INFR-2012-01-187757.


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