Neuroscience 277 (2014) 474–485

INTRACELLULAR ACTIVITIES RELATED TO IN VITRO HIPPOCAMPAL SHARP WAVES ARE ALTERED IN CA3 PYRAMIDAL NEURONS OF AGED MICE H. MORADI-CHAMEH, a,c J. PENG, a C. WU a AND L. ZHANG a,b*

our present observations are supportive of the notion that excitability of hippocampal CA3 circuitry is increased in aged mice. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada b Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario M5T 2S8, Canada c Department of Physiology, Faculty of Medical Science, University of Tarbiat Modares, Tehran, Iran

Key words: aging, network, rhythm, subiculum.

Abstract—Pyramidal neurons in the hippocampal CA3 area interconnect intensively via recurrent axonal collaterals, and such CA3-to-CA3 recurrent circuitry plays important roles in the generation of hippocampal network activities. In particular, the CA3 circuitry is able to generate spontaneous sharp waves (SPWs) when examined in vitro. These in vitro SPWs are thought to result from the network activity of GABAergic inhibitory interneurons as SPW-correlating intracellular activities are featured with strong IPSPs in pyramidal neurons and EPSPs or spikes in GABAergic interneurons. In view of accumulating evidence indicating a decrease in subgroups of hippocampal GABAergic interneurons in aged animals, we test the hypothesis that the intracellular activities related to in vitro SPWs are altered in CA3 pyramidal neurons of aged mice. Hippocampal slices were prepared from adult and aged C57 black mice (ages 3–6 and 24–28 months respectively). Population and single-cell activities were examined via extracellular and whole-cell patch-clamp recordings. CA3 SPW frequencies were not significantly different between the slices of adult and aged mice but SPW-correlating intracellular activities featured weaker IPSC components in aged CA3 pyramidal neurons compared to adult neurons. It was unlikely that this latter phenomenon was due to general impairments of GABAergic synapses in the aged CA3 circuitry as evoked IPSC responses and pharmacologically isolated IPSCs were observed in aged CA3 pyramidal neurons. In addition, aged CA3 pyramidal neurons displayed more positive resting potentials and had a higher propensity of burst firing than adult neurons. We postulate that alterations of GABAergic network activity may explain the reduced IPCS contributions to in vitro SPWs in aged CA3 pyramidal neurons. Overall,

INTRODUCTION Rodent hippocampal neurons are capable of generating spontaneous rhythmic field potentials of 1–4 Hz when examined in vitro. These spontaneous potentials are now commonly referred to as in vitro sharp waves (SPWs) as they share some common features with electroencephalographic (EEG) hippocampal SPWs seen in vivo (Chrobak et al., 2000). In vitro SPWs arise from the CA3 area and their intracellular activities are characterized by strong IPSPs in pyramidal neurons and EPSPs/spikes in GABAergic inhibitory interneurons (Papatheodoropoulos and Kostopoulos, 2002; Wu et al., 2002, 2006; Kubota et al., 2003; Maier et al., 2003; Behrens et al., 2005; Ellender et al., 2010; Ha´jos et al., 2013; Pangalos et al., 2013). As such, in vitro SPWs are thought to result from the network activity of GABAergic inhibitory interneurons and present inhibition-based population potentials (Ho et al., 2012). In vitro SPWs may serve as a valuable model for examining hippocampal network activities. Like EEG SPWs seen in vivo (Chrobak et al., 2000), in vitro SPWs can spread from the CA3 area toward downstream structures (Both et al., 2008; Kanak et al., 2013); specially the subicular circuitry is thought to play a critical role in the hippocampal-entorhinal spreading of in vitro SPWs (Wu et al., 2006; Norimoto et al., 2013). In addition, the generation of in vitro SPWs from the CA3 circuitry is facilitated by a process similar to that underlying the long-term potentiation (LTP; Behrens et al., 2005). The presence of in vitro SPWs negatively regulates the magnitudes of CA1 LTP (Colgin et al., 2004). Furthermore, in vitro SPWs and associated ripples (oscillations of 200 Hz) are sensitive to genetic or neurochemical manipulations related to GABAergic synapses and GABAergic inhibitory interneurons (Maier et al., 2002; Behrens et al., 2011; Papatheodoropoulos and Koniaris, 2011; Giannopoulos and Papatheodoropoulos, 2013; Viereckel et al., 2013).

*Correspondence to: L. Zhang, 7KD-407, Toronto Western Research Institute, Toronto Western Hospital, 60 Leonard Avenue, Toronto, Ontario M5T 2S8, Canada. Tel: +1-416-603-5800x2209 (lab), +1-416-603-5800x2702 (office); fax: +1-416-603-5745. E-mail address: [email protected] (L. Zhang). Abbreviations: ACSF, artificial cerebrospinal fluid; EEG, electroencephalographic; LTP, long-term potentiation; MK-801, MK-801 hydrogen maleate; NBQX, 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide; SE, standard error; SPWs, sharp waves. http://dx.doi.org/10.1016/j.neuroscience.2014.07.048 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 474

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Aging is known to be associated with a decline in hippocampal memory functions and alterations of hippocampal neuronal activities (Burke and Barnes, 2006; Wilson et al., 2006). While numerous studies have examined hippocampal CA1 neurons and dentate granule neurons of aging/aged animals (Disterhoft and Oh, 2006; Burke and Barnes, 2010), relatively less information is available regarding the cellular and synaptic activities of aged CA3 neurons and their network activities. In view of the accumulating evidence indicating an association between GABAergic interneuronal activity and in vitro SPWs (see references above) as well as a decrease of subgroups of hippocampal GABAergic interneurons in aged animals (Shetty and Turner, 1998; Cadacio et al., 2003; Vela et al., 2003; Shi et al., 2004; Stanley and Shetty, 2004; Potier et al., 2006; Kuruba et al., 2011; Stanley et al., 2012; Spiegel et al., 2013), it would be of great interest to know whether in vitro SPWs are altered by reduced GABAergic interneuronal activity in aging/ aged animals. Two previous studies have tackled the above issue prior to our present experiments. Hermann et al. (2009) report that the frequency of CA1 SPWs and the power of CA1 ripples are decreased in aging mice (17-monthold) compared to adult mice (10-month-old). Kanak et al. (2013) report that while the frequency of CA3 SPWs is increased, the timing of CA1 SPWs is delayed in aged mice (21-month-old) when compared to adult mice (4-month-old). Their data also show that multi-unit activities associated with CA1 SPW-ripples are reduced in aged mice. Overall, these studies suggest that the network activities underlying in vitro SPWs are altered in aging/aged mice. However, intracellular activities underlying in vitro SPWs in aging/aged hippocampal neurons remain to be examined. The aim of our present study is to further examine in vitro SPWs in adult and aged mice (ages 3–6 months and 24–28 months respectively), with a particular focus on SPW-related intracellular activities in CA3 pyramidal neurons. Specifically, we test the hypothesis that IPSC contributions to SPW-correlating intracellular activities are weakened in aged CA3 pyramidal neurons. In the following text, we simply refer to in vitro SPWs as SPWs unless otherwise specified.

EXPERIMENTAL PROCEDURES Animals C57 black mice (C57BL/6N, Charles River Laboratory, Saint-Constant, Quebec, Canada), ages 3–6 months and 24–28 months, were used in the present experiments. We refer to these animals as ‘‘adult’’ and ‘‘aged’’ as they correspond roughly to humans aged 20–30 and P70-year-old respectively (Flurkey et al., 2007). These animals were housed in a vivarium that was maintained at 22 °C and with a 12-h light on/off cycle. Food and water were available ad libitum. All experimental procedures described below were reviewed and approved by the animal care committees of the University Health Network in accordance with the guidelines of Canadian Council on Animal Care.

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Hippocampal slices Slices were similarly prepared as we recently described (El-Hayek et al., 2013). The animal was anesthetized via an intra-peritoneal injection of sodium pentobarbital (70-mg/kg, Somnotol, WTC Pharmaceuticals, Cambridge, Ontario, Canada) and trans-cardiacally infused with cold (4 °C), low-Na+/Ca2+ artificial cerebrospinal fluid (ACSF) before decapitation. The brain was quickly dissected and hemi-sectioned, and brainstem tissues were removed to extend the curved hippocampus. Cortical–hippocampal tissues were glued onto an agar block, with the hippocampal longitudinal axis perpendicular to the horizontal cut plane. Transverse hippocampal slices of 0.5-mm thickness were obtained via a vibratome in the presence of ice-cold, low-Na+/Ca2+ ACSF. After sectioning, the slices were stabilized in standard ACSF at 35 °C for 30 min. During the stabilizing period, 2.5 mM kynurenic acid (a general ionotropic glutamate receptor antagonist) was included in ACSF as an attempt to reduce potential dissection-related excitotoxicity. After the 30-min stabilization, slices were rinsed twice and then maintained in standard ACSF at 22 °C for at 1–6 h before recording. The low-Na+/Ca2+ ACSF contained (in mM): sucrose 300, KCl 3.5, NaH2PO4 2, CaCl2 0.5, MgCl2 7, glucose 20 and HEPES 5 (pH adjusted to 7.4). The standard ACSF contained (in mM): NaCl 125, KCl 3.5, NaH2PO4 1.25, NaHCO3 25, CaCl2 2, MgSO4 1.3 and glucose 10 (pH of 7.4 when aerated with 95%O2–5%CO2). In vitro recordings and afferent stimulation The slice was placed in a submerged chamber and perfused with standard, oxygenated (95%O2–5%CO2) ACSF at a high rate (15 ml/min). Both the top and bottom surfaces of the slice were exposed to ACSF. Previous studies including works from our lab have shown that a fast, top and bottom perfusion of the slice is important for maintaining spontaneous population activities under submerged conditions (Wu et al., 2005; Zhang et al., 2008; Ha´jos et al., 2009; Ha´jos and Mody, 2009). All recordings were done at 36 °C. Recording and stimulating electrodes were positioned under a dissecting microscope. A ‘‘blind’’ method was used to approach individual cells. Recording electrodes were made with thin-wall glass tubes (World Precision Instruments, Sarasota, Florida, USA). Extracellular electrodes were filled with a solution that contained 150 mM NaCl and 2 mM HEPES (pH 7.4; resistance of 1–2 MO). Electrodes for whole-cell patchclamp recordings were filled with a solution that contained 140 mM potassium gluconate, 10 mM KCl, 2 mM HEPES and 0.1 mM EGTA (pH 7.25 and resistance of 5 MO). To raise ‘‘intracellular’’ Cl in recorded neurons, we modified the pipette solution by decreasing potassium gluconate to 125 mM and increasing KCl to 25 mM while keeping the other components unchanged (Zhang et al., 1991; DeFazio et al., 2000). Extracellular and intracellular signals were recorded via a dual channel amplifier (700A or 700B, Molecular Devices/Axon Instruments, Sunnyvale, California, USA).

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Data acquisition, storage and analysis were done using PClamp software (version 9 or 10, Molecular Devices). These signals were recorded in frequencies of 0–5 kHz and digitized at 50 kHz (Digidata 1320, Molecular Devices). For assessing regional SPWs and evoked field potentials, dual extracellular recordings were made from distal CA3a (near CA3b) and middle CA1 area or from distal CA3a and proximal subicular (near distal CA1) areas. For assessing SPW-correlating intracellular activities, CA3 pyramidal neurons were recorded via whole-cell voltage-clamp recordings together with extracellular monitoring of local SPWs. A bipolar electrode, made of polyimide-insulated stainless steel wires (outer diameter 0.1 mm; Plastics One, Ranoake, Virginia, USA), was placed in the distal CA3a oriens area for afferent stimulation. Constant current pulses (0.1-ms duration, 20–150 lA) were generated by a Grass stimulator (S88, Natus Neurology Incorporated – Grass Products, Warwick, Rhode Island, USA) and delivered through an isolation unit every 30 s. Data analysis Spontaneous SPWs were examined in slices that were obtained from the ventral pole of the hippocampus (2–3 slices per hippocampus). Basic extracellular and intracellular parameters were measured as previously described (Zhang et al., 1991; Wu et al., 2005, 2006; Zhang et al., 2008). Only slices with stable SPWs and/ or evoked CA3 synaptic potentials of P0.5 mV were included for further analyses. Basic intracellular parameters were measured from neurons that had resting membrane potentials more negative than 55 mV and action potential amplitudes greater than 80 mV. Synaptic currents recorded from individual neurons within 15 min after forming whole-cell configuration and with series resistance of 620 MO were analyzed. Evoked field potentials were measured from averages of 4–5 consecutive responses in individual slices. Frequencies of spontaneous SPWs were determined from 60 consecutive events collected from individual slices. SPWs were first recognized via the event detection function (threshold search method) of PClamp program. Original data were treated with a band-passing filter (0.5–100 Hz, Bessel) to minimize the influences of baseline shifts and high-frequency signals prior to SPW detection. Detected events were visually inspected, false events rejected and undetected events were manually added. To estimate reversal potentials of SPWcorrelating synaptic currents, areas (pA  ms) under these currents were measured from periods of 100 ms, which were 20–30 ms longer than the waveforms of corresponding local SPWs. Averages from 25 to 30 consecutive SPW-correlating currents were obtained at each voltage, and mean areas were plotted vs. voltages ( 70 to 40 mV. 10 mV steps) and fitted via the linear regression function (r2 > 0.9). The voltage at which the linear regression line intersected zero charge level was taken as the reversal potential of SPW-correlating currents (Wu et al., 2006; Zhang et al., 2008). Reversal potentials of pharmacologically isolated IPSCs were estimated using the same method. Evoked IPSCs were

recorded at different voltages (from 80 mV to 30 mV, 10 mV steps, 4 consecutive responses at each voltage), and averaged IPSC amplitudes were plotted vs. holding voltages. ‘‘Spontaneous’’ inward synaptic currents or EPSC-like events were analyzed via Mini Analysis Program (version 6.0, Synaptosoft, Decatur, GA, USA). EPSC events with amplitudes of P10 pA, onset times of 610 ms, decay times of 625 ms and mono-phasic/complex waveforms were detected in 60-s data segments from individual neurons. Detected events were visually inspected, and false events or events correlating with local extracellular SPWs were excluded from further analysis. Statistical tests were performed via Sigmaplot software (Systat Software Inc., San Jose, California, USA). Mean and standard error (SE) of the mean are presented throughout the text and figures except where indicated. To minimize erroneously skewing contributions by specific animals, data were collected from P3 animals in each age group except where indicated. If sample sizes were sufficient, multiple measurements from a single animal were first averaged and these averages were then grouped for analysis between adult and aged mice (Barnes, 1994). Pharmacological agents 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline7-sulfonamide (NBQX), MK-801 hydrogen maleate (MK-801) and kynurenic acid were obtained from Sigma/ Research Biochemicals Inc. (Mississauga, Ontario, Canada). MK-801 was initially dissolved in DMSO; the final DMSO dose was 650 ll/l ACSF. Stock solutions for NBQX and kynurenic acid were made of deionized distilled water.

RESULTS CA3 SPWs and their spread to CA1 and subicular areas We examined SPWs in ventral hippocampal slices of adult and aged mice. Extracellular recordings revealed spontaneous SPWs in both adult and aged slices. These SPWs displayed amplitudes up to 0.2 mV and frequencies of 0.5–5 Hz, and their waveforms were positive (upward) or negative (downward) when recorded from the cell body or apical dendritic layer of CA3/CA1 pyramidal neurons respectively (Fig. 1A). The frequencies of CA3 SPWs were measured in 52 slices obtained from 26 adult mice and in 35 slices from 19 aged mice. Overall, CA3 SPW frequencies were not significantly different between adult and aged slices (1.9 ± 0.1 Hz vs. 1.6 ± 0.1 Hz; p = 0.165, Mann– Whitney rank sum test; Fig. 1C). Averaging multiple SPW measurements from a single animal and then grouping the averages for analysis between adult and aged mice revealed a similar trend (1.9 ± 0.1 Hz vs. 1.6 ± 0.2 Hz, p = 0.096, independent t test; Fig. 1C). To assess the temporal relation of regional SPWs, we recorded SPWs simultaneously from CA3 and CA1 areas or from CA3 and subicular areas and measured the onset times of corresponding regional SPWs (see Methods). To ensure the preservation of the Schaffer collateral and

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Fig. 1. SPWs observed from ventral hippocampal slices of adult and aged mice. (A) Extracellular traces collected simultaneously from CA3 and CA1 areas. Left, field potentials evoked by stimulating CA3 oriens area and recorded from CA3 somatic and CA1 apical dendritic areas. Each illustrated trace was averaged from 4 consecutive responses. Middle, representative SPWs collected from same CA3 and CA1 sites. Original data were band-pass filtered (0.5–200 Hz) for illustrative purposes. SPW events in gray boxes expanded at right. (B) Evoked field potentials and SPWs recorded simultaneously from CA3 and subicular areas and similarly illustrated as in A. (C) CA3 SPW frequencies measured from two groups of slices and animals. In the latter, measurements from 3 to 4 slices per animal were averaged and these averages were pooled together for adult and aged mice. Numbers of slices or animals examined are indicated in parentheses. (D, E) Onset time lags of regional SPWs and evoked field potentials measured from adult and aged slices. (F) Onset time lags between CA3 and subicular SPWs were presented in histograms. Measurements from 12 adult slices and 7 aged slices were pooled together. Onset time lags were binned every 5 ms. Time-advanced CA3 or subicular events were presented by positive or negative time values. *, adult vs. aged, Chi-square test, p < 0.001.

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CA1-to-subicular projections in individual slices, we also measured onset time lags of evoked field potentials as a reference. These field potentials were evoked by a single stimulation of the CA3 oriens area and were recorded from the same sites where spontaneous SPWs were monitored (Fig. 1A, B). Coherent CA3 and CA1 SPWs were analyzed in 14 slices obtained from 8 adult mice and in 9 slices from 6 aged mice. There was no significant group difference in the onset time lags of CA3 and CA1 SPWs; CA3 SPWs led CA1 SPWs by 6.8 ± 1.0 ms and 10.5 ± 2.4 ms in the slices of adult and aged mice respectively (p = 0.226, independent t test; Fig. 1D). The onset time lags of evoked CA1 and CA3 field potentials were comparable between the two groups of slices (2.4 ± 0.5 ms vs. and 2.3 ± 0.4 ms, p = 0.352, independent t test; Fig. 1D). CA3 and subicular SPWs were analyzed in 12 slices obtained from 5 adult mice and in 7 slices from 4 aged mice. The onset time lags of CA3 and subicular SPWs were not significantly different between the two groups of slices; CA3 SPWs led subicular SPWs by 15.3 ± 0.9 ms and 16.5 ± 2.4 ms in the slices of adult and aged mice respectively (p = 0.11, independent t test; Fig. 1E). Evoked CA3 and subicular field potentials showed a similar trend; CA3 potentials were time advanced by 9.7 ± 0.9 ms and 9.4 ± 0.8 ms in the slices of adult and aged mice respectively (p = 0.585, independent t test; Fig. 1E). However, temporal relations between CA3 and subicular SPWs were noticeably variable in the aged slices; some subicular SPWs seemed to coincide with or preceded CA3 SPWs (Fig. 1B, bottom). When the onset time lags between CA3 and subicular SPW were pooled together for each group of slices, their distributions were significantly different between the adult and aged slices (Fig. 1F; Chi-square test, p < 0.001). Taking the above observations together, we suggest that while the ability to generate SPWs is largely preserved in the aged CA3 circuitry, intrinsic subicular rhythmicity may be altered in some aged mice. SPW-correlating intracellular activities in CA3 pyramidal neurons We conducted simultaneous extracellular and whole-cell voltage-clamp recordings to examine SPW-correlating synaptic currents. A solution with 10 mM Cl was used for these whole-cell recordings as ‘‘resting’’ levels of intracellular Cl are approximately 10 mM in hippocampal and cortical neurons of young adult mice (Glykys et al., 2014). Individual neurons were monitored at different potentials ( 70 mV to 40 mV, 10 mV steps) to assess relative contributions of glutamatergic and GABAergic activities to SPW-correlating currents. When monitored at near 70 mV (which was to the calculated Cl reversal potential, see below), both adult and aged CA3 pyramidal neurons displayed inward currents or EPSC components in phase with local extracellular SPWs; strong EPSC components were noticeable in some aged neurons (Fig. 2A, B, bottom panels). When monitored at near 60 mV, these neurons exhibited inward–outward currents or EPSC–IPSC-like complexes in correlation with local SPWs (Fig. 2A, B, middle panels).

SPW-correlating currents were largely outward or dominated by IPSC-like events at 50 mV and 40 mV, but overall IPSC-like events were less evident in aged neurons than adult neurons (Fig. 2A, B, top panels). To estimate reversal potentials of SPW-correlating currents, the areas of these currents were measured from 25 to 30 consecutive events at each potential and averaged areas were then plotted vs. holding potentials (Fig. 2C; see Methods). The estimated reversal potentials of SPW-correlating currents were significantly more positive in aged neurons ( 51.6 ± 1.8 mV, n = 11 from 6 mice) than in adult neurons ( 59.0 ± 1.3 mV, n = 12 from 4 mice; p = 0.03, independent t test; Fig. 2D). This difference might be largely due to a reduction in SPW-correlating IPSC components in aged neurons, as the areas of SPW-correlating currents at 40 mV and 50 mV were significantly smaller in aged neurons (2226.0 ± 928.5 pA  ms and 416.7 ± 618.9 pA  ms) than in adult neurons (6634.6 ± 1330.1 pA  ms and 2825.1 ± 700.9 pA  ms; p 6 0.0183, independent t-test) whereas there were no significant group differences in the areas of SPWcorrelating currents at 60 mV and 70 mV ( 962.6 ± 497.6 pA  ms and 2386.3 ± 639.9 pA  ms for aged neurons and 1811.2 ± 506.4 pA  ms and 4359.8 ± 994.8 pA  ms for adult neurons; p P 0.246, independent t-test; Fig. 2E). To assess glutamatergic activities related to SPW generation in individual CA3 pyramidal neurons, we analyzed ‘‘spontaneous’’ inward currents or EPSC-like events that occurred between adjacent SPWs events. Data segments collected at 70 mV (which was close the calculated Cl reversal potential, see below) were analyzed in an attempt to minimize the influences of GABAergic activities, and amplitudes, inter-event intervals and areas of EPSC-like events were detected in 60-s data segments, excluding the events in phase with local extracellular SPWs. Repetitive EPSC events with variable amplitudes and complex waveforms were recognizable in both adult and aged neurons. There were no significant group differences in EPSC amplitudes (31.6 ± 4.1 pA vs. 32.5 ± 4.3 pA), interevent intervals (44.4 ± 13.2 ms vs. 35.1 ± 6.9 ms) and areas (370.4 ± 70.3 pA  ms vs. 326.2 ± 39.7 pA  ms; n = 5 in each group, p P 0.599, independent t-test or Mann–Whitney rank sum test). When all EPSC events were pooled together from each group of neurons, distributions of EPSC amplitude (bin size 10 pA), intervals (bin size 20 ms) and areas (bin size 20 pA  ms) were also not significantly different between the adult and aged neurons (p P 0.257, Chi-square test). Based on the above observations, we suggest that SPW-correlating IPSC components are reduced in aged CA3 pyramidal neurons compared to adult neurons. We then explored whether such a reduction is associated with general alterations of evoked GABAergic responses. Evoked GABAergic responses of CA3 pyramidal neurons Synaptic currents were evoked by single stimulation of the CA3 oriens area and monitored together with

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Fig. 2. SPW-correlating currents in CA3 pyramidal neurons. (A, B) Whole-cell voltage-clamp recordings were made from two neurons together with extracellular monitoring of local SPWs. Illustrated current traces were collected at indicated holding potentials. (C) Areas of SPW-correlating currents were measured for the two neurons in A (adult) and B (aged). Averages from 25–30 consecutive SPW-correlating currents were obtained at each holding potential and then plotted vs. holding potentials. Lines through data points were computed by linear regression function. The voltage at which the linear regression line intersected with zero charge level (doted horizontal line) was considered to be the reversal potential of SPWcorrelating currents. (D) Reversal potentials of SPW-correlating currents estimated for individual neurons. The mean reversal potentials were 59.0 ± 1.3 mV and 51.6 ± 1.8 mV for adult and aged neurons respectively (n = 12 and 11). ⁄p = 0.030, independent t test. (E) Areas of SPWcorrelating currents measured at different potentials for adult and aged neurons. ⁄p 6 0.018, independent t-test.

SPW-correlating currents at same potentials. When recorded with an internal solution with 10 mM Cl , both adult and aged CA3 pyramidal neurons displayed inward-outward currents or EPC-IPSC like complexes following CA3 stimulation. The outward or IPSC responses were evident at 60 mV and 50 mV (Fig. 3A), and their amplitudes were not significantly different between adult and aged neurons (98.3 ± 26.3 pA vs. 79.8 ± 43.9 pA at 60 mV and 245.0 ± 60.9 pA vs. 233.8 ± 72.2 pA at 50 mV; n = 5 and 6; p > 0.601, independent t-test; Fig. 3B). SPWcorrelating currents were observed shortly before or after evoked IPSC responses and were dominated by IPSC components in adult neurons (Fig. 3A, top) but by EPSC components in aged neurons (Fig. 3A, bottom). These

observations suggest that a lack or reduction of SPWcorrelating IPSC components in aged neurons is not associated with general alterations of GABAergic synapses. To pharmacologically isolate GABAergic responses, we evoked synaptic currents in the presence of ionotropic glutamate receptor antagonists (20 lM NBQX plus 20 lM MK-801 or 3 mM kynurenic acid). Under these conditions, spontaneous SPWs and correlating synaptic currents were blocked, and evoked IPSCs with monophasic waveforms were observed in adult and aged CA3 pyramidal neurons (Fig. 3C). When recorded with the internal solution with 10 mM Cl , estimated reversal potentials of these evoked IPSCs were comparable between adult and aged neurons

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Fig. 3. Evoked IPSCs observed from CA3 pyramidal neurons. (A) Whole-cell voltage-clamp recordings were made from two neurons together with extracellular monitoring of local field potentials. Holding potentials for illustrated current traces were indicated. Synaptic responses were evoked by stimulating the CA3 oriens area (indicated by arrows at bottom) and in the absence of additional pharmacological manipulation. An internal solution with 10 mM Cl ([Cl ]in) was used for whole-cell recordings. (B) Amplitudes of evoked outward IPSC responses were measured from two groups of neurons under the conditions described in A. (C) Evoked IPSCs recorded from another 2 neurons and in the presence of 20 lM NBQX and 20 lM MK801. Recordings were made with the internal solution with 10 mM Cl . Corresponding current–voltage plots for the 2 neurons are shown below the traces. Lines through data points were computed by linear regression function. IPSC reversal potentials estimated from two groups of neurons under similar conditions are presented below the plots. (D) Evoked IPSCs were recorded from another 2 neurons in the presence of 3 mM kynurenic acid. Recordings were made an internal solution with 25 mM Cl . Data were similarly illustrated as in (C).

( 71.1 ± 3.0 mV vs. 72.5 ± 0.5 mV, n = 4 from 2 animals in each group; p = 0.885, Mann–Whitney rank sum test; Fig. 3C). Previous studies have shown that hippocampal and cortical neurons of young adult animals are effective at extruding excessive ‘‘intracellular’’ Cl when recorded with internal solutions containing high Cl (P20 mM;

Zhang et al., 1991; DeFazio et al., 2000). As SPW-correlating intracellular activities involve strong IPSC components (Fig. 2A, B), frequent IPSCs may lead to intracellular Cl accumulation in aged neurons if their ability to extrude excessive intracellular Cl is compromised. To explore this, we used an internal solution with 25 mM Cl in an attempt to raise ‘‘intracellular’’ Cl in recorded

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neurons and to test their abilities in extruding excessive Cl . When recorded under this high ‘‘intracellular’’ Cl condition, evoked IPSCs appeared to be more variable in aged CA3 pyramidal neurons than in adult neurons, and estimated IPSC reversal potentials were approximately 6 mV more positive in aged neurons than in adult neurons ( 38.1 ± 2.6 mV vs. 44.4 ± 1.5 mV, n = 4 from 2 mice in each group; Fig. 3D). However, these reversal estimations were not significantly different, most likely due to the small sample size (p = 0.1068, independent t-test). Taking into account the above observations, we argue that the reduction of SPW-correlating IPSC components in aged CA3 pyramidal neurons is not a result of general alterations of GABAergic synapses. However, further experiments are needed to characterize age-dependent changes in CA3 GABAergic synapses and intracellular Cl regulation for our model (see Discussion). Basic intracellular parameters of CA3 pyramidal neurons We also measured basic intracellular parameters of CA3 pyramidal neurons via current-clamp recordings. The internal solution with 10 mM Cl was used in these experiments. To signify age-dependent alterations in individual animals, data collected from 3 to 4 neurons per animal were averaged and these averages were then pooled together for aged and young mice (Barnes, 1994). The resting membrane potentials of CA3 pyramidal neurons were significantly more positive in aged than adult mice ( 58.3 ± 1.0 mV vs. 62.5 ± 0.8 mV, n = 13 and 15 animals; p = 0.009, independent t-test), but there were no significant group difference in measurements of input resistance (97.4 ± 8.2 and 87.5 ± 8.8 MO), action potential amplitudes (98.8 ± 2.6 mV and 99.6 ± 2.5 mV) and action potential durations (0.86 ± 0.03 ms and 0.91 ± 0.05 ms; p P 0.356, independent t-test or Mann–Whitney rank sum test; Fig. 4A). In response to intracellular injections of positive current pulses (100–600 pA, durations of 0.5 s), CA3 pyramidal neurons exhibited two types of discharge patterns: ‘‘regular spiking’’ and ‘‘burst firing’’. The regular spiking neurons displayed repetitive spikes with gradually increasing inter-spike intervals (Fig. 4B, C, left panels), and the burst firing neurons manifested in clusters of 8–15 spikes with inter-spike intervals of 615 ms (Fig. 4B, C, right panels). Of 13 CA3 pyramidal neurons recorded from 7 aged mice, burst firing was recognizable in 7 neurons from 5 animals, with interspike intervals of the first 5 spikes ranging from 5.66 ± 0.54 ms to 7.94 ± 1.61 ms. In contrast, of 15 CA3 pyramidal neurons sampled from 6 adult mice, only one neuron exhibited burst firing. The proportion of burst firing neurons was significantly greater in the aged CA3 pyramidal neurons examined (p = 0.011, Fisher exact test; Fig. 4D). Burst firing rendered a high firing rate in aged CA3 neurons. When the total number of induced spikes were plotted vs. the intensities of positive current pulses (in a range of 100–500 pA), overall firing rates were

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significantly higher in aged CA3 pyramidal neurons (n = 7, 3 burst firings and 4 regular spiking neurons) than in adult neurons (n = 8, 1 burst firing neuron and 7 regular spiking neurons; Fig. 4E; p = 0.042, ANOVA on ranks). Together the above observations suggest that intrinsic ionic activities of CA pyramidal neurons are altered in aged mice.

DISCUSSION In our experiments, CA3 SPWs were reliably recorded in ventral hippocampal slices of aged mice. The frequencies of CA3 SPWs and the time lags of CA3 and CA1 SPWs were not significantly different between adult and aged mice. We thus suggest that the ability to generate SPWs is largely preserved in aged CA3 circuitry. However, there are some discrepancies between our present observations and a previous study by Kanak et al. (2013). Kanak et al. report that the frequency of CA3 SPWs is increased and that the timing of CA1 SPWs is delayed in aged mice compared with adult mice. Differences in experimental conditions may partly explain these discrepancies. Hippocampal slices were recorded in an interfaced chamber in the Kanak study but were recorded in a submerged chamber in our experiments. As SPW expression is generally more robust in slices recorded in an interfaced chamber, we might have underestimated CA3 SPWs, particularly in the hippocampal slices of aged mice. In addition, Kanak et al. used C57BL/129 mice and we used C57BL mice for SPW assessments. It is plausible that the aging process has varying effects on hippocampal network activities in different mouse strains (Flurkey et al., 2007). Furthermore, although we recorded regional SPWs in similar hippocampal subfields in adult and aged slices, it was difficult to determine the exact distance of dual recording sites under our experimental conditions. Thus, experimental errors might have masked potential age-dependent differences in SPW spread from the CA3 to CA1 area. While the temporal relation between CA3 and CA1 SPWs remains to be further investigated in our model, there were noticeable group differences in subicular SPWs. In ventral hippocampal slices of adult mice, subicular SPWs lagged CA3 SPWs in a relatively consistent fashion, which is in keeping with the synaptic signal flow through the Schaffer collateral and CA1subicular projections. The time lags of CA3 and subicular SPWs varied a wide range in ventral hippocampal slices of aged mice, and some subicular SPWs did not follow CA3 SPWs. In addition, spontaneous subicular SPWs or SPW-like events, but not CA3 SPWs, were observed in dorsal hippocampal slices of some aged mice, whereas such subicular events were not found in dorsal hippocampal slices of adult mice (data not shown). Considering that the subicular circuitry is capable of generating intrinsic oscillatory activities when preserved in relatively large forms (such as in thick slices or intact hippocampal preparations) (Wu et al., 2006; Jackson et al., 2011), we speculate that the intrinsic network activity of subicular neurons may be altered or strengthened in aged mice

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Fig. 4. Basic intracellular parameters and discharge patterns of CA3 pyramidal neurons. (A) Resting membrane potentials, input membrane resistance, action potential amplitudes and durations measured from two groups of neurons. Data collected from 3 to 4 neurons per animal were averaged, and these averages were pooled together for adult and aged mice. Numbers of animals examined are indicated in parenthesis. ⁄ p = 0.009, independent t-test. (B, C) Voltage traces collected from 4 neurons at indicated resting potentials. Recordings were made with the internal solution with 10 mM Cl and in the absence of additional pharmacological manipulation. Voltage responses to intracellular current pulses (durations of 500 ms and 100 pA per step) were superimposed for individual neurons. (D) Discharge patterns were examined in 15 neurons from 6 adult mice and in 13 neurons from 7 aged mice. Proportions of regular spiking and burst firing CA3 pyramidal neurons were compared between the two groups of neurons. ⁄p = 0.011, Fisher exact test. (E) Numbers of induced spikes were plotted vs. positive current pulses for adult neurons (n = 8, 1 burst firing neuron and 7 regular spiking neurons and aged neurons (n = 7, 3 burst firings and 4 regular spiking neurons). ⁄p = 0.042, ANOVA on ranks.

and may facilitate the generation of SPW-like events independent of CA3 SPWs. In light of the importance of the subicular network in memory and cognitive functions (O’Mara et al., 2009), further works are needed to examine the cellular and synaptic activities of subicular neurons in aged mice.

We found that the mean reversal potential of SPWcorrelating currents was about 7 mV more positive in aged than adult CA3 pyramidal neurons. In addition, SPW-correlating IPSC components were smaller in aged CA3 pyramidal neurons compared to adult neurons when measured at 50 mV and 40 mV,

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whereas SPW-correlating EPSC components were not significantly different between the two groups of neurons when measured at more negative potentials. Collectively, these observations suggest that there is a reduction of SPW-associated GABAergic activities in the CA3 circuitry of aged mice. It is unlikely for such a reduction to be explained by general alterations of GABAergic synapses, as evoked IPSC responses and pharmacologically isolated IPSCs were observed in aged CA3 pyramidal neurons and appeared to be similar to those observed from adult CA3 pyramidal neurons. Previous studies have reported that the number of hippocampal GABAergic interneuron subgroups is decreased in aged animals (Shetty and Turner, 1998; Cadacio et al., 2003; Vela et al., 2003; Shi et al., 2004; Stanley and Shetty, 2004; Potier et al., 2006; Kuruba et al., 2011; Stanley et al., 2012; Spiegel et al., 2013). However, the paired pulse inhibition of evoked CA3 field potentials is not substantially impaired in aged mice (Vreugdenhil and Toescu, 2005; Lu et al., 2011). Taking the above information together, we hypothesize that alteration in GABAergic network activity may largely account for reduced IPSC contributions to SPWcorrelating intracellular activities in aged CA3 pyramidal neurons. In our experiments, we evoked IPSCs or IPSC responses by stimulating the CA3 oriens area as an attempt to activate GABAergic synapses in the somatic and basal dendritic areas of CA3 pyramidal neurons. However, this stimulation protocol might not have activated GABAergic synapses in apical CA3 dendritic areas. The location of the stimulating electrode in relation to the recorded CA3 pyramidal neuron also varied in different experiments. In addition, evoked IPSCs or IPSC responses were recorded from a small cohort of aged CA3 pyramidal neurons due to a limited availability of aged mice, and evoked IPSCs were highly variable in aged CA3 pyramidal neurons, particularly when recorded with the internal solution with 25 mM Cl . Overall, our present observations were quite limited at assessing the properties of CA3 GABAergic synapses. In light of the decrease of subgroups of hippocampal GABAergic interneurons in aged animals (see references above), further studies that examine ‘‘spontaneous’’ and focally evoked IPSCs in CA3 pyramidal neurons as well as synaptic/ionic activities of GABAergic interneurons in CA3 subfields are needed to reveal age-dependent changes in CA3 GABAergic synapses. Moreover, perforated patch-clamp recordings or intracellular Cl imaging (Glykys et al., 2014) together with local application of GABA agonists (DeFazio et al., 2000) are needed to evaluate the ability of intracellular Cl regulation in aged CA3 pyramidal neurons. Colgin et al. (2004) have reported that CA1 LTP is relatively weak in SPW-exhibiting ventral hippocampal slices and that surgical separation of CA3 and CA1 areas abolishes CA1 SPWs and greatly increases the magnitude and time course of CA1 LTP. We speculate that SPWs may also regulate the activity of the CA3 circuitry. Specifically, SPW-correlating IPSP/IPSC components may entrain CA3 pyramidal neurons and reduce overall

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excitability of the CA3 circuitry. In this context, the reduction of SPW-correlating IPSC components that we observed from aged CA3 pyramidal neurons may weaken the entrainment and lead to a heightened hyperexcitable state in the aged CA3 circuitry. This potential mechanism may partly explain the high tendency of aged ventral CA3 neurons to generate epileptiform field potentials following repeated afferent stimulation (Patrylo et al., 2007; El-Hayek et al., 2013). We found that aged CA3 pyramidal neurons displayed more positive resting potentials and had a higher propensity of burst firing relative to adult neurons. Both phenomena may potentially increase the excitability of aged CA3 circuitry by promoting spontaneous discharges in CA3 pyramidal neurons (Cohen and Miles, 2000) and hence CA3 recurrent glutamate synapses. Previous studies have shown that positive resting potentials of cortical pyramidal neurons are closely related to cortical EEG discharges in a mouse model of Alzheimer’s disease (Minkeviciene et al., 2009) and that hippocampal CA1 pyramidal neurons of adult rats have a high rate of burst firing following pilocarpineinduced status epilepticus (Chen et al., 2011). We have recently shown that aging/aged mice, but not adult mice, frequently exhibit aberrant hippocampal EEG spikes (El-Hayek et al., 2013). Based on the above information, we speculate that the positive resting potentials and burst firing may serve as cellular readouts of increased hippocampal excitability in aged mice. It remains to be examined whether alterations of voltage-gated Na+ currents and/or calcium-dependent K+ currents previously recognized in hippocampal CA1 pyramidal neurons (Matthews et al., 2009; Chen et al., 2011; Randall et al., 2012) facilitate the burst firing of aged CA3 pyramidal neurons. In summary, we report here that relative IPSC contributions to SPWs are reduced in CA3 pyramidal neurons of aged mice. In addition, intrinsic ionic activities of aged CA3 pyramidal neurons are featured with positive resting potentials and a high propensity of burst firing. Taking these observations together with our recent EEG and extracellular assessments (El-Hayek et al., 2013), we postulate that the excitability of hippocampal CA3 circuitry is increased in aged mice. Acknowledgment—This study was supported by a grant from the Natural Science and Engineering Research Council of Canada to L.Z. (RGPIN 217479-10).

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(Accepted 9 July 2014) (Available online 1 August 2014 )