1. HIPPOCAMPAL SHARP WAVES AND RIPPLES: EFFECTS OF AGING
AND MODULATION BY NMDA RECEPTORS AND L-TYPE CA2+
CHANNELS
S. KOUVAROS, D. KOTZADIMITRIOU
AND
C. PAPATHEODOROPOULOS *
Laboratory of Physiology, Department of Medicine, University
of Patras, 26504 Rion, Greece
Abstract—Aging is accompanied by a complicated pattern
of changes in the brain organization and often by alterations
in specific memory functions. One of the brain activities with
important role in the process of memory consolidation is
thought to be the hippocampus activity of sharp waves
and ripple oscillation (SWRs). Using field recordings from
the CA1 area of hippocampal slices we compared SWRs as
well as single pyramidal cell activity between adult (3–6-
month old) and old (24–34-month old) Wistar rats. The slices
from old rats displayed ripple oscillation with a significantly
less number of ripples and lower frequency compared with
those from adult animals. However, the hippocampus from
old rats had significantly higher propensity to organized
SWRs in long sequences. Furthermore, the bursts recorded
from complex spike cells in slices from old compared with
adult rats displayed higher number of spikes and longer
mean inter-spike interval. Blockade of N-methyl-D-aspartic
acid (NMDA) receptors by 3-((R)-2-carboxypiperazin-4-yl)-
propyl-1-phosphonic acid (CPP) increased the amplitude
of both sharp waves and ripples and increased the interval
between events of SWRs in both age groups. On the con-
trary, CPP reduced the probability of occurrence of
sequences of SWRs more strongly in slices from adult than
old rats. Blockade of L-type voltage-dependent calcium
channels by nifedipine only enhanced the amplitude of
sharp waves in slices from adult rats. CPP increased the
postsynaptic excitability and the paired-pulse inhibition in
slices from both adult and old rats similarly while nifedipine
increased the postsynaptic excitability only in slices from
adult rats. We propose that the tendency of the aged hip-
pocampus to generate long sequences of SWR events might
represent the consequence of homeostatic mechanisms
that adaptively try to compensate the impairment in the
ripple oscillation in order to maintain the behavioral out-
come efficient in the old individuals. The age-dependent
alterations in the firing mode of pyramidal cells might under-
lie to some extent the changes in ripples that occur in old
animals. Ó 2015 IBRO. Published by Elsevier Ltd. All rights
reserved.
Key words: hippocampus, aging, sharp waves, ripple oscilla-
tion, complex spike, NMDA receptor.
INTRODUCTION
Brain aging is a complex, heterogeneous and poorly
understood phenomenon (Kirkwood et al., 2003). Brain
changes during aging appear to be selective and region-
specific (Burke and Barnes, 2006; Kelly et al., 2006;
Kumar et al., 2009; Burger, 2010) and some of them
may represent the result of compensatory mechanisms
(Boric et al., 2008; Kumar et al., 2009; Burke and
Barnes, 2010). One behavioral attribute of brain aging is
impairment in forming new memories (Crook et al.,
1986; Burke and Mackay, 1997; Balota et al., 2000;
Beason-Held and Horwitz, 2002), especially those that
depend on the hippocampus (Rosenzweig and Barnes,
2003). Thus, aged rats may have impaired hippocam-
pus-dependent memory (Markowska et al., 1989;
Gallagher and Rapp, 1997) and specifically episodic-spa-
tial memory (Monacelli et al., 2003). In addition, the hip-
pocampus appears to undergo structural and functional
changes during aging (Rosenzweig and Barnes, 2003;
Wilson et al., 2006; Oh et al., 2010). However, not all
old individuals exhibit cognitive deficits and there is ample
inter-individual difference to age-associated memory
impairment both in humans and rats (Crook et al., 1986;
Markowska et al., 1989).
Hippocampus plays a crucial role on the long-term
establishment of episodic memories participating in the
process of memory consolidation (Wang and Morris,
2010), coordinated by communication between the hip-
pocampal and neocortical circuits (Buzsaki, 1996;
Siapas and Wilson, 1998; Sirota et al., 2003; Battaglia
et al., 2004; Wierzynski et al., 2009). During this process
the information stored in the hippocampal circuit is trans-
ferred to the neocortical circuit where it contributes to the
induction of plastic synaptic changes that result in alter-
ations of the circuit in which the memory content is
embedded (Buzsaki, 1989; Wang and Morris, 2010;
Inostroza and Born, 2013). Accumulating evidence sug-
gests that the contribution of the hippocampus to the pro-
cess of memory consolidation is realized through its
http://dx.doi.org/10.1016/j.neuroscience.2015.04.012
0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Laboratory of Physiology, Medical
School, University of Patras, 26 500 Rio, Patras, Greece. Tel: +30-
2610-969117; fax: +30-2610-997215.
E-mail address: cepapath@upatras.gr (C. Papatheodoropoulos).
Present address: Medical Research Council Anatomical Neu-
ropharmacology Unit, Department of Pharmacology, Oxford University,
Oxford, UK.
Abbreviations: ACSF, artificial cerebrospinal fluid; CPP, 3-((R)-2-
carboxypiperazin-4-yl)-propyl-1-phosphonic acid; CS, complex spikes;
DMSO, dimethyl-sulfoxide; fEPSP, field excitatory postsynaptic
potentials; ICI, intra-cluster interval; IEI, inter-event interval; ISI, inter-
spike interval; L-vdcc, L-type voltage-dependent calcium channel;
MUA, multiunit activity; NMDAR, N-methyl-D-aspartic acid receptor;
PS, population spikes; SWRs, sharp waves–ripples.
Neuroscience 298 (2015) 26–41
26

2. population activity called sharp waves–ripples (SWRs)
(O’Keefe and Nadel, 1978; Buzsaki, 1986). Recently, a
direct correlation between SWRs and learning and
memory has been demonstrated (Eschenko et al., 2008;
Ramadan et al., 2009; Singer and Frank, 2009).
Accordingly, memory performance declines when SWRs
are disrupted (Girardeau et al., 2009; Ego-Stengel and
Wilson, 2010).
The generation of SWRs involves activation of GABAA
receptor-mediated transmission (Papatheodoropoulos
and Kostopoulos, 2002; Wu et al., 2002b; Maier et al.,
2003; Papatheodoropoulos, 2010). In addition, specific
classes of interneurons are preferentially activated during
SWRs (Csicsvari et al., 1999a; Klausberger and Somogyi,
2008). Results from previous studies have suggested that
some aspects of the GABAergic transmission in the
hippocampus declines with old age (de Jong et al.,
1996; Stanley and Shetty, 2004; Potier et al., 2006;
Moradi-Chameh et al., 2014) Furthermore, among several
age-related changes with expected impact on hippocam-
pus function occur in N-methyl-D-aspartic acid (NMDA)
receptors (NMDARs) (Magnusson et al., 2010). Some
important aspects of SWRs including amplitude and pat-
terned occurrence in sequences involve activation of
NMDARs (Colgin et al., 2005; Papatheodoropoulos,
2010). In addition, aging is accompanied by alterations
in another cellular component involved in learning and
memory, the L-type voltage-dependent calcium channels
(L-vdcc), (Moyer and Disterhoft, 1994; Kumar et al., 2009;
Nunez-Santana et al., 2013).
In the present study we aimed to characterize the
activity of SWRs between adult and old rat slices and to
examine the effects of NMDARs and L-vdc on this
activity. We found that aging was accompanied by
decline in the ripple oscillation and an increased
tendency for generation of SWRs in the form of
sequences. Furthermore, the age-dependent changes in
the ripple oscillation were accompanied by
corresponding alterations in complex spike cell activity.
EXPERIMENTAL PROCEDURES
Slice preparation
Transverse slices were prepared from adult (3–6 months,
398.9 ± 5.6 gr) and aged (24–34 months,
632.8 ± 17.6 gr) Wistar male rats housed under
conditions of controlled temperature (21–23 °C), a 12-h
light/dark cycle with access to food and water ad libitum.
All animal treatment and experimental procedures were
conducted in accordance with the Directive Guidelines
for the care and use of Laboratory animals of the
European Communities Council Directive Guidelines
(86/609/EEC) for the care and use of Laboratory
animals and approved by the Prefectural (Achaia)
Animal Care and Use Committee (No: EL 13BIO04).
Accordingly, all measures were taken to minimize
animal suffering and to reduce the number of animals
used. The animals were deeply anesthetized with
diethyl-ether and decapitated by a guillotine. The brain
was removed and placed in cooled (4 °C) artificial
cerebrospinal fluid (ACSF) containing (in mM): NaCl
124, KCl 4, MgSO4 2, CaCl2 2, NaH2PO4 1.25, NaHCO3
26 and glucose 10 at pH 7.4, equilibrated with 95% O2
and 5% CO2 gas mixture. The two hippocampi were
excised free and transverse slices (500–550-lm-thick)
were prepared from the ventral pole of the structure
extending between 1 and 4 mm from the end of the pole
using a McIlwain tissue chopper. We used slices from
the ventral part of the hippocampus solely because they
display greater likelihood of spontaneous generation of
SWRs compared with slices taken from the rest of the
structure (Papatheodoropoulos and Kostopoulos, 2002).
Slices were immediately transferred on the two
independent channels of an interface type recording
chamber and maintained at a constant temperature of
31.5 ± 0.5 °C. The slices were continuously humidified
with a mixed gas containing 95% O2 and 5% CO2 and
they were perfused with standard ACSF at a rate of about
1.0 ml/min.
Electrophysiology, types of recorded activity and
data analysis
Field spontaneous and evoked activity was recorded from
the pyramidal cell layer of the CA1b field using carbon
fiber electrodes with a diameter of 7 lm (Kation
Scientific, Minneapolis, USA). In some experiments
recordings from the CA3 field were also obtained (noted
in the text). Signal was amplified and filtered at 0.5 Hz–
2 kHz using a Neurolog system (Digitimer Limited, UK),
digitized at 5–10 kHz and stored on a computer for off-
line analysis using the CED 1401-plus interface and the
Signal and Spike2 software (Cambridge Electronic
Design, Cambridge, UK).
Spontaneous activity
Spontaneous activity was categorized as network and
single cell or unit activity. Network activity consisted of
synchronous slow field potentials associated with a
transient fast oscillation with frequency of $160 Hz and
a relatively intense multiunit activity (MUA) (Fig. 1A–B).
Previous in vitro studies (Papatheodoropoulos and
Kostopoulos, 2002; Kubota et al., 2003; Maier et al.,
2003; Behrens et al., 2005; Wu et al., 2005;
Papatheodoropoulos, 2010) have shown that this field
activity shares several features with in vivo sharp wave–
ripple complexes recorded from the hippocampus of
behaving rats (O’Keefe and Nadel, 1978; Buzsaki et al.,
1992). We will therefore refer to this synchronous activity
as sharp waves, ripples (or ripple oscillation) and MUA.
The complex co-occurrences of these components will
be called events of sharp waves–ripples (SWRs). In order
to measure the various parameters of this activity it was
decomposed to sharp waves by low-pass filtering original
signal at 40 Hz and to ripple oscillation by band-pass at
80–300 Hz, respectively (Fig. 1B). MUA accompanying
SWRs events was disclosed by filtering raw records at
400 Hz–2 kHz. Sharp waves were detected at filtered
records after setting a threshold at a level where all puta-
tive events were identified as verified by visual inspection.
Ripples and MUA were detected at filtered records after
setting a threshold at four times the standard deviation
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 27

3. of SWR-free baseline noise. Events were categorized as
ripples only when episodes of at least two consecutive
negative deflections were observed with delays between
them of at least 3 ms and no more than 11 ms.
Threshold was further verified by visual inspection.
SWRs occurred as either single events or in the form of
two or more consecutive events termed clusters or
sequences. The first SWR in a cluster is referred to as
the primary event, while the following are termed
secondary events. Clusters or sequences of events were
clearly distinguished from single events because the time
interval between consecutive events in a sequence was
short and strikingly stable ($100 ms) in a given slice
and between slices as compared with the interval
between discrete episodes (consisted of an isolated event
or a cluster). The existence of clusters could be revealed
in the distribution histogram of inter-event intervals (IEIs)
(Fig. 1C). Characteristically, these histograms showed
two clearly separated peaks of bimodal distribution.
From the distribution histogram of each slice we
determined the range of short and long intervals and we
used these measures in classifying activity into clusters
and isolated events. An additional criterion used in deter-
mining clusters was the usually gradual change in ampli-
tude of the events inside a cluster (evident in almost all
figures of SWRs). Events of SWRs were quantified by:
(1) the amplitude of the sharp wave determined as the
voltage difference between the positive peak and the
baseline. In clusters, primary and secondary events were
measured separately; (2) the IEI determined as the time
interval between successive individual SPWs regardless
of whether they occurred as isolated events or clusters;
(3) the intra-cluster interval (ICI) determined as the mean
value of the intervals between successive SWR events
inside a cluster; (4) the number of events of SWRs per
minute; (5) the probability of occurrence of clusters calcu-
lated by the number appearances divided by the total
number of episodes. Ripples were quantified by: (1) the
amplitude of the ripple event determined as the voltage
difference between the positive and negative maximum
A
B
F
C
G
D
E
Fig. 1. Types of spontaneous activity recorded from hippocampal slices. (A) Example of sharp wave–ripple activity recorded from the CA1 stratum
pyramidale of a slice taken from a 26-month-old rat. Note that SWRs occurred in episodes consisting of one or more events. (B) An episode
consisting of four SWR complexes is shown. Filtering the original record (top trace) at 80–300 Hz disclosed the high-frequency ($160 Hz) ripple
oscillation (middle trace), whereas filtering at 400 Hz-2 kHz revealed the multiunit activity (MUA) associated with SWRs (bottom trace). Record was
obtained from a 32-month-old rat. (C) Distribution histogram of inter-event intervals (IEI) illustrating the two peaks that corresponded to the short
intervals between successive events in sequences of SWRs and the long intervals between episodes of activity. Data were obtained from a 10-min-
long record collected from an old rat. (D) Record from an adult rat that illustrates the occurrence of a burst of complex spike cell (*) in isolation from
events of SWRs (top trace). The burst is shown in a faster speed on the bottom. (E) Scatter plot of instantaneous inter-event interval illustrating the
stability of spontaneous activity over time. Data were collected from a slice obtained from a 30-month-old rat. Recording started three hours after the
placement of the slice on the recording chamber. (F) Simultaneous recording of SWRs from the CA3b and CA1b subfields of a slice obtained from a
32-month-old rat exemplifying the leading role of CA3 field in generating SWRs. Dot line indicates the time-point that activity starts in CA3. (G)
Raster plot and distribution histogram of CA1b SWRs triggered by events in CA3b. Dots represent the automatically detected peaks that correspond
to sharp waves, in the low-pass filtered record. Data were obtained from an adult rat. Note that events at around 100 ms post-trigger correspond to
secondary events in sequences.
28 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

4. in each ripple event; (2) the duration of the ripple event;
(3) the number of ripple cycles in a ripple event calculated
as the number of negative deflections inside an event; (4)
the ripple frequency determined as the reciprocal of the
value of the mean inter-ripple interval. Measures of spon-
taneous potentials were made from recordings acquired
about three to five hours after the placement of the slices
in the recording chamber. In each slice or experimental
condition the measures of events of SWRs concerning
amplitude and intervals were made from a two-minute-
long record. The probability of clusters was calculated
from one-minute-long record. Measures of ripples were
made from about 30 primary events. MUA inside events
of SWRs was quantified by the intra-spike interval.
Unit activity organized in bursts that occurred in
isolation from SWR events was categorized as complex
spikes (CS), which have been previously observed and
described in the hippocampus in vivo (Ranck, 1973; Fox
and Ranck, 1975, 1981) (Fig. 1D). In accordance to the
previously described characteristics of CS the detection
and analysis of putative CS bursts in the present study
was performed by eye inspection in original records obey-
ing the following criteria: (a) the burst activity was
recorded from stratum pyramidale; (b) bursts consisted
of two to six spikes; (c) the amplitude of spikes inside
bursts most often declined from the first to the last spike;
(d) the inter-spike interval ranged between about 2 ms to
12 ms. The criteria of discrimination procedure used to
aggregate episodes of bursts of CS cells into discrete
groups (i.e., cells) included the shape of the first and the
following spikes, the amplitude of the spikes and the sta-
bility of the amplitude of the first spike from burst to burst.
The number of spikes was also taken into account since it
has been reported that the propensity of individual neu-
rons to fire a certain number of action potentials in a burst
is relatively stable over time (Suzuki and Smith, 1985). On
the other hand however, considering that bursts produced
by a given pyramidal cell may continually change over
time (Ranck, 1973) we used the criterion of spikes’ num-
ber with caution and only when activity satisfied the other
criteria. Whenever it was difficult to perform the segrega-
tion of bursts into different CS cells following the above
criteria we assumed that the different bursts were pro-
duced by a single CS cell. In addition, only bursts with
relatively large first spike amplitude (50–200 lV) were
selected for analysis. Measures of CS were made from
continuous records of at least 10 min. Quantification of
CS bursts included the number of spikes, the intervals
between consecutive spikes in the burst (inter-spike inter-
val, ISI) as well as the mean ISI in each cell.
Evoked activity
Evoked synaptic responses consisting of field excitatory
postsynaptic potentials (fEPSP) and population spikes
(PS) were recorded by delivering electrical pulses
(intensity 20–350 lA, duration 0.1 ms) every 30 s at the
Schaffer collaterals using a bipolar platinum/iridium wire
electrode (wire diameter of 25 lm, World Precision
Instruments, USA). PS was continuously monitored in
order to determine the stability of the response. Only
slices with a stable response for at least 10 min were
selected for further experimentation. In order to examine
the effects of aging and drugs on synaptic activity,
neuronal excitability and GABAergic inhibition we
constructed input/output curves of fEPSP and PS.
fEPSP was quantified by the maximal slope of its rising
phase and PS was quantified by its amplitude, measured
as the length of the projection of the negative peak on
the line connecting the two positive peaks of the PS
waveform. Synaptic effectiveness was quantified by
measuring the fEPSP at threshold stimulation strength
(fEPSPthr), the stimulation current intensity required to
produce half-maximal fEPSP (I50-fEPSP) and the maximal
fEPSP (fEPSPmax). Neuronal excitability was quantified
by measuring the stimulation current intensity required to
produce half-maximal PS (I50-PS), the maximal PS
(PSmax) the postsynaptic activation required to produce
half-maximal PS (fEPSP50), and the ratio between half-
maximal PS and the corresponding fEPSP (PS/fEPSP).
The strength of inhibition was quantified using the
protocol of paired-pulse stimulation consisting of two
consecutive stimuli of identical intensity at Schaffer
collaterals, separated by 10 ms. The first (conditioning)
stimulus of the pair (that evokes PS1) exerts a
depressive effect on the PS evoked by the second
stimulus (PS2, conditioned) by recurrently activating the
network of GABAergic interneurons. The depression is
expressed as a rightward and downward shift of the PS/I
input/output curve which therefore was taken as a
measure of the strength of recurrent inhibition in the
local network. The rightward shift was quantified by the
percent change in I50-PS (shift of I50-PS), the downward
shift was quantified by the ratio PS2/PS1 at stimulation
strength that evoked half-maximal PS1.
Drugs
The competitive antagonist of the NMDA receptors 3-((R)-
2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP,
10 lM, 35–40 min) and the blocker of L-type vdcc
nifedipine (20 lM, 65–70 min) were used. Drugs were
purchased from Tocris Cookson Ltd, UK. Both drugs
were first prepared as stock solutions. CPP was solved
in water while nifedipine was solved in dimethyl-
sulfoxide (DMSO). Drug solutions were prepared at the
desired concentration in the perfusion medium at the
time of the experiment. The percentage by volume of
DMSO in the final solution of nifedipine did not exceed
0.005%. Measures of the various variables of field
potentials were obtained from the last one to five
minutes or 10 min before and during drug application for
spontaneous or evoked potentials, respectively.
Statistical analysis
Values through the text are expressed as mean ± S.E.M.
or mean ± S.D. Measures performed in individual slices
obtained for a given animal were pooled together. In
average, from each adult and old animal we used 1.7
and 2.0 slices, respectively. In the pharmacological
experiments the number of slices equated the number
of animals. Statistical comparisons between the two age
groups were performed using the number of animals,
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 29

5. after calculating the mean value from all slices obtained
from a single animal. However, age-related comparisons
of complex spike bursting were made using the number
of cells. The nonparametric Mann–Whitney U test and
the Wilcoxon test as well as the two-tailed correlation
test were used for statistical evaluation of age-
dependent differences.
RESULTS
SWRs developed progressively in slices from both adult
and old rats during their maintenance in the recording
chamber. SWRs stabilized in amplitude and rate of
occurrence at about two to three hours after placing
slices in the recording chamber and remained
unchanged at least for the next three hours (Fig. 1E).
Previous in vivo (Buzsaki, 1986; Csicsvari et al., 2000)
and in vitro studies (Wu et al., 2005; Both et al., 2008)
have shown that SWRs in adult animals start in the CA3
and then spread to the CA1 field. We also examined the
relationship between the two hippocampal fields by
recording simultaneously from the CA3b and CA1b sub-
fields, with inter-electrode distance of 2.17 ± 0.08 mm
and 2.1 ± 0.13 in slices from adult and old rats, respec-
tively. In both age groups SWRs firstly occurred in the
CA3 field and then appeared in the CA1 with a delay of
7.3 ± 1.5 ms (n = 4) and 8.2 ± 0.7 ms (n = 3) in adult
and old slices, respectively (Fig. 1F, G).
Ripple oscillation
We made detailed measurements of amplitude, duration,
number and frequency of ripples in slices from adult
(n = 33) and old rats (n = 20). Fig. 2 shows that the
values of all the above parameters were lower in the
slices of aged animals. In particular, slices from aged
compared with adult rats showed a decreased number
of ripple troughs within individual ripple events
(3.96 ± 0.16 vs 4.64 ± 0.17, respectively; Mann–
Whitney U test, P < 0.01) and decreased mean
intra-ripple frequency (166.7 ± 2.9 vs 177.3 ± 2.3,
respectively; Mann–Whitney U test, P < 0.01).
Complex spike bursting
In vivo recordings from the CA1 have shown that
pyramidal cells are intensively activated during SWRs
(Chrobak and Buzsaki, 1996; Csicsvari et al., 1999a) typi-
cally firing short bursts of spikes (Buzsaki, 1986;
Kudrimoti et al., 1999). Because of this mode of firing
CA1 pyramidal cells are called ‘‘complex spike cells’’
(Ranck, 1973). In the present study putative complex
spike activity was indeed observed to occur during
SWRs (Fig. 3A). Additionally, scanning our long-lasting
recordings of spontaneous activity we have detected sin-
gle-cell activity occurring between SWR events in slices
from both age groups (Fig. 3B). This complex spike burst-
ing displayed several of the typical features of complex
spike activity recorded in vivo from CA1 pyramidal cells
(Ranck, 1973; Fox and Ranck, 1975, 1981;
McNaughton et al., 1983; Suzuki and Smith, 1985;
Harris et al., 2001) (see also Methods). We analyzed
activity of complex spike cells that occurred during non-
oscillatory periods, since identification of complex spike
bursts during SWRs was not possible. We analyzed a
total of 52 putative complex spike cells from 22 adult
and 30 aged cells. In keeping with previous reports
(Chrobak and Buzsaki, 1996; Csicsvari et al., 1999b) we
A B
C
Fig. 2. Ripple oscillation in adult and old animals. (A) Original records of SWRs from an adult and an old rat and the corresponding band-pass
filtered sweeps revealing the ripple oscillation (lower traces) are shown. (B) Examples of power spectrum graphs illustrating that ripple oscillation
displayed a lower frequency in slices from old compared with adult rats. (C) Comparative data of the various variables of the ripples oscillation
between adult and old animals. Asterisks denote statistically significant difference between adult and old values at P < 0.01 (Mann–Whitney U test).
30 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

6. observed that the peak of CS cell firing occurred during or
immediately after the negative peak of a ripple cycle
(Fig. 3C). Bursts of CS cells were typically composed of
two to five spikes (mean number of spikes 3.0 ± 0.11
spikes, 52 cells) and displayed an inter-spike interval
ranging from 2 to 12 ms (mean interval
7.42 ± 0.11 ms). Complex spike cell activity consisted
mainly of two consecutive spikes, whereas CS with a
higher number of spikes were observed less frequently
(Fig. 3D). However, CS of aged rats displayed similar
probabilities for having two or more spikes. Mean number
of spikes between the two age groups was significantly
higher in aged compared to adult rats (3.19 ± 0.14 vs
2.76 ± 0.16, respectively, Mann–Whitney U test and
ANOVA, F = 4.055, P < 0.05). Additionally the mean
inter-spike interval was significantly longer in aged
(8.1 ± 0.17 ms) compared with adult rats
(6.78 ± 0.14 ms) Mann–Whitney U test and ANOVA,
F = 29.9, P < 0.001) (Fig. 3E). CS from aged rats dis-
played a higher range of mean ISI (from 5.5 to 7.8 ms)
compared with adult rats (from 6.2 to 10.2 ms). Mean
value of each of the consecutive four ISI in complex spike
bursts was longer in cells from aged rats compared with
the adult animals (Fig. 3E).
MUA inside SWRs
MUA during SWRs events was analyzed in 12 adult and
11 old rats. As in the case of singe cell complex spike
bursting, MUA occurred at the negative peaks of the
ripple oscillation (Fig. 4B). Inter-spike intervals had short
duration, lower than 6–8 ms (Fig. 4C). The inter-spike
interval in MUA was similar between adult and old rats
(2.9 ± 0.135 vs 2.94 ± 0.1 ms) (Fig. 4D).
Events of SWRs
Events of SWRs in slices from both adult and aged
animals rats were organized either as single events or
in the form of sequences of two or more events (up to
seven) (Figs. 1A and 5A). The first event and the
following events in a single sequence were termed
‘‘primary’’ and ‘‘secondary’’ events, respectively. Both
primary and secondary events had similar amplitudes in
slices from adult and aged rats (73.5 ± 6.6 lV and
26.5 ± 1.8 lV vs 75.5 ± 9.6 lV and 27.7 ± 3.96 lV in
adult and old rats, respectively; Mann–Whitney test,
P > 0.05). We found no difference in the rate of
occurrence of SWR events expressed by the total
A B
C
E
D
Fig. 3. Comparison of complex spike bursts between adult and old animals. (A) Recording from the CA1 pyramidal cell layer of a slice from adult
animal showing a burst of complex spike cell occurring during the rising phase of a secondary SWR event (framed). This was a particularly rare
instance where we could easily disentangle CS from the rest of multiunit activity occurring during SWRs. (B) Examples of recordings showing the
occurrence of CS in isolation from SWRs, obtained from adult and old animals. Complex spike bursts are shown enlarged in lower traces. (C) The
top trace is a record from an old animal showing two SWRs and an isolated burst of complex spike cell (asterisk). The complex spike burst and the
corresponding ripple oscillation are shown enlarged in the lower left panel. The right panel shows one cycle of averaged ripple wave (top trace) and
the histogram of phase distribution of complex spikes relative to the negative of the ripple wave (diagram on the bottom). (D) Collective data of the
mean number of spikes in CS bursting (left diagram) and the incidence of burst with a given number of spikes (right diagram) in adult and old rats.
(E) Histogram of inter-spike intervals for all CS cells studied (left graph), the mean inter-spike interval in CS bursts (middle graph) and the 1st–5th
inter-spike interval (right graph) are shown. Asterisks denote statistically significant differences between the two ages at ⁄
P < 0.05; ⁄⁄
P < 0.005;
⁄⁄⁄
P < 0.001 (Mann–Whitney U test).
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 31

7. number of SWR events generated per minute
(167.0 ± 5.8 vs 183.0 ± 15.3 events in adult and old
rats, respectively; Mann–Whitney test, P > 0.05) and
the IEI of individual events (379.7 ± 10.6 ms vs
380.3 ± 26.1 ms in adult and old rats, respectively;
Mann–Whitney test, P > 0.05), between aged and adult
A B
C D
Fig. 4. Multiunit activity during SWRs in adult and old rats. (A) An event of SWR and the corresponding ripple and multiunit activity (MUA) revealed
by filtering the record of SWR are shown. (B) The average sweep of ripple oscillation and the histogram of phase distribution of MUA relative to the
negativities of the ripple oscillation are shown. Data were collected from a 15-min-long record. (C) Distribution histogram of inter-spike intervals in
MUA measured from a 15-min record. Note that most intervals fall below 10 milliseconds. (D) The mean inter-spike interval during MUA was similar
between adult and old animals.
A
B C
D
Fig. 5. Events of SWRs in adult and old animals. (A) Continuous records of SWRs from an adult and an old animal (traces on the left and right,
respectively). Note that the episodes of sharp waves in the old rat were organized in longer sequences than in the adult rat. (B) Cumulative data of
the amplitude, inter-event interval (IEI), rate of occurrence and intra-cluster interval (ICI) in adult and old animals. These measures were similar
between adult and old rats. For clarity reasons, error bars in the middle graph are now shown in S.D. (C) Collective distribution graphs of the inter-
event interval measured using an equal total recording time in the two age groups. Old rats displayed a very distinctive distribution peak at short
intervals (formed by the intra-cluster intervals) which was higher than that observed in adult animals. (D) The cumulative probability of occurrence of
sequences of SWRs (left graph) and the separate probabilities of occurrence of sequences with two, three, four of more events in adult and old
animals (right graph) are shown. Asterisks denote statistically significant difference between adult and old values at ⁄
P < 0.05, ⁄⁄
P < 0.01; (Mann–
Whitney U test).
32 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

8. rats (Fig. 5B). The interval between consecutive events
inside sequences (ICI was comparable between adult
(107.0 ± 2.9 ms, n = 35) and old rats (108.3 ± 5.2 ms,
n = 25, we observed an evident difference in the
pattern of SWRs’ generation between aged and adult
rats. Slices from aged rats displayed a statistically
significant higher propensity to generate events in the
form of sequences (Fig. 5A, C). (46.0 ± 3.1%, n = 25)
compared with adult rats (37.4 ± 2.6%, n = 38),
(Mann–Whitney U test, P < 0.05). Thus, the
probabilities of occurrence of clusters with three, four or
more events were all significantly higher in old than in
adult rats (Fig. 5C). The higher tendency of old rats to
display long sequences of SWRs might imply that it
represents a change in the old hippocampus in order to
counterbalance the impaired ripple oscillation. In order
to examine whether these two parameters are inversely
correlated between each other we compared the ability
of slices to generate clusters of SWRs with the number
and the frequency of ripples inside a given slice.
Comparing the cumulative probability of clusters, we
observed no correlation between the parameter Values.
However, we found a significant inverse correlation
between the frequency of the ripple oscillation and the
tendency of slices to organize clusters with three or
more events (r = 0.45, P < 0.05; one-tailed bivariate
correlation).
Drug effects on SWRs
It has been previously shown that NMDARs modulate the
amplitude of sharp waves (Colgin et al., 2005) and play an
important role in the organization of sequences or clusters
of SWR events (Papatheodoropoulos, 2010). Taking into
account that the function of NMDARs is altered in the
aged hippocampus (Serra et al., 1994; Magnusson
et al., 2010) and that clusters are longer in aged we asked
what is the involvement of NMDARs in the two age groups
Furthermore, in aging hippocampal pyramidal neurons
the expression and activity of L-type voltage-dependent
calcium channels is enhanced (L-vdcc) (Moyer and
Disterhoft, 1994; Kumar et al., 2009; Nunez-Santana
et al., 2013). Given that both NMDARs and L-VDCCs
are targets of the aging process we set out to assess their
involvement in the generation SWRs, using pharmaco-
logical blockers independently for NMDARs and L-
VDCCs and in combination as well. Fig. 6 shows that
the antagonist of NMDARs CPP applied to slices from
adult animals significantly enhanced the amplitude of pri-
mary SWR events (by 14.0 ± 3.5%) and reduced the fre-
quency of their occurrence (it increased IEI by
16.3 ± 2.8%). In addition, application of CPP reduced
the incidence of sequences of SWRs by 88 ± 1.7%.
Blockade of NMDARs in slices from aged rats produced
similar effects on the amplitude and IEI but the reduction
in the probability of sequences (55.6 ± 6.6%) was
significantly lower than the one seen in adult rats
(Mann–Whitney U test, P < 0.001). We observed that
CPP abolished the SWR sequences in three slices from
adult animals but in no one from aged rats. Given that
SWRs are initiated in the CA3, it is interesting to see
whether the effect of CPP that observed in the CA1 is
mediated through drug action in the CA3 or the CA1 cir-
cuitry. Thus, we examined the effect of CPP in the CA3
field of seven slices prepared from adult rats. As occurred
in the CA1, CPP robustly suppressed the occurrence of
sequences in the CA3 field by 93.2 ± 1.4% (P < 0.05).
CPP did not significantly altered ICI in any of the two
age groups. These data indicated that SWRs were less
sensitive to NMDAR’s blockade in aged than in adult ani-
mals. Application of nifedipine (20 lM) in the presence of
CPP produced an additional significant increase in the
amplitude of SWR events in both adult and aged rats
(by 14 ± 3% and 11 ± 2%, respectively) without affecting
any of the other parameters. When nifedipine was applied
alone, the only significant effect observed was the
increase in the amplitude of SWRs in adult but not aged
rats (by 18.7 ± 3.0%). None of the other parameters of
SWRs were significantly affected by nifedipine.
Application of CPP in the presence of nifedipine signifi-
cantly affected all parameters of sharp waves in both adult
and aged rats. These effects were similar to those
observed when CPP was applied alone (compare CPP
with NIF + CPP bars in Fig. 6B).
Blockade of NMDARs by CPP produced a modest yet
statistically significant enhancement in the ripple
oscillation. In particular, CPP in aged rats increased the
amplitude, duration, number and frequency of ripples by
7.3 ± 2.5%, 14.9 ± 7.0%, 11.9 ± 4.7% and
3.7 ± 1.4%, respectively (n = 13, P < 0.05, Wilcoxon
test). In the adult rats CPP significantly increased the
amplitude and the frequency of the ripple oscillation by
12.2 ± 4.1% and 4.1 ± 0.5%, respectively (n = 15,
P < 0.05, Wilcoxon test). CPP did not however
significantly affect any of the other ripple parameters in
the slices from adult rats. Nifedipine, applied in the
presence of CPP did not produce any further significant
effect in either adult or aged rats. When nifedipine was
applied alone, it significantly enhanced the amplitude of
ripples in adult (by 11.5 ± 3.7%) but not aged rats
(25.3 ± 17.6%) Nifedipine did not produce any
consistent effect on the other parameters of the ripple
oscillation. Interestingly however, nifedipine almost
completely occluded the action of CPP on ripples.
Evoked responses
In order to examine the effects of aging on the excitatory
and inhibitory synaptic properties, we recorded evoked
field potentials by stimulating the path of Schaffer
collaterals in 30 slices taken from 12 adult rats and in
26 slices obtained from 10 aged rats and constructing
input/output curves (Fig. 7A). The mean values for all
indexes are shown in Table 1. None of the indexes
quantifying synaptic effectiveness and neuronal
excitability differed between adult and aged rats.
However, slices from old rats displayed statistically
significant decreased strength of inhibition as compared
with adults. Specifically, paired-pulse stimulation
produced a significant rightward and downward shift in
the PS/I curve in adult animals (Fig. 7B) as measured
by the positive percent increase in the I50-PS and the
decrease in the PS2/PS1 ratio. In old rats paired-pulse
stimulation produced a negative percent change in the
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 33

9. I50-PS, i.e., it produced a leftward shift in the PS/I curve
and significantly increased the PS2/PS1 ratio.
In order to examine whether L-vdcc and NMDARs are
involved in the evoked responses we perfused slices with
nifedipine (40 lM) for 50 min and then we added CPP
(10 lM) for 30 min. We performed these experiments in
12 slices taken from 12 adult rats and in nine slices
obtained from 9 aged rats. As shown in Table 1 and
Fig. 7C, in slices from adult rats, nifedipine produced a
statistically significant decrease of fEPSP50 and
increase PS/fEPSP, thus leading to an increase in
postsynaptic excitability. Nifedipine did not significantly
change any of the other indexes in slices from adult rats
although a trend in increasing inhibition can be
observed (compare B with D in Fig. 7). In slices from
aged rats nifedipine produced no significant change in
A
B
Fig. 6. Effects of CPP and nifedipine on sharp waves in adult and old animals. (A) Examples of recordings of sharp waves from adult and old rats
before and during perfusion with the antagonist of NMDARs CPP and the blocker of L-vdcc nifedipine. (B) Plots of collective results showing the
effect of CPP and nifedipine on the various parameters of sharp waves in adult (plots on the left) and old rats (plots on the right). The four drug
conditions shown in each graph, and the number of adult and old animals used were: CPP (application of CPP alone, 16 adult and 14 old),
CPP + NIF (application of nifedipine in the presence of CPP, adult and 11 old), NIF (application of nifedipine alone, 21 adult and 11 old) and
NIF + CPP (application of CPP in the presence of nifedipine, 12 adult and seven old). Asterisks denote the statistically significant drug effects at
⁄
P < 0.05; ⁄⁄
P < 0.01; ⁄⁄⁄
P < 0.005. Wilcoxon test and Mann–Whitney U test we used for comparisons of drug effects inside an age group and
between the two age groups, respectively. It should be noted that the effects of combined application of the two drugs (i.e., CPP + NIF and
NIF + CPP) were statistically significant but only the significant further actions of the consecutively added drug are marked in the plots. The effects
of CPP on the distribution histograms of inter-event interval in the two age groups are shown at the bottom. Note that CPP completely suppressed
the early peak of the distribution in adult but not old rats.
34 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

10. A
B
C
D
Fig. 7. Comparisons of evoked potentials between adult and old animals. (A) The collective input/output curves fEPSP/I and PS/I are shown in the
left and middle plots for adult (19 slices/12 animals) and old (26 slices/10 animals) rats. The scatter plot on the right shows the relationship between
PS and fEPSP for all slices studied. (B) Input/output curves illustrating the depressing effect of paired-pulse stimulation on PS2 in slices from one
adult (left) and an old animal (right). Arrows indicate the values of I50-PS that correspond to the curves of PS1 and PS2 (arrow in dotted and solid line,
respectively). Examples of recordings are shown in the inserts. Calibration bars: 1 mV, 5 ms in adult and 0.5 mV and 5 ms in old animals. Note that
paired-pulse suppression of PS2, expressed by the rightward and downward shift of the corresponding curve, is absent in the slice from the old
animal. (C) Examples of input/output curves obtained from individual slices showing the effects of nifedipine and CPP on postsynaptic excitability in
adult and old animals. Arrows indicate the values of fEPSP50 for the three curves (the value in the control curve is indicated by the arrow in dotted
line). Note that nifedipine produced a leftward shift of EPSP50 (thus increasing postsynaptic excitability) in the slice from the adult but not the old
animal. (D) Examples of PS/I curves for the PS1 and PS2 showing the inhibitory effect of the paired-pulse stimulation before and during successive
application of nifedipine and CPP. Note that the depression of PS2 (arrows) was higher under CPP than under control conditions (filled and open
symbols, respectively) in both adult and old rat. Examples shown in (B), (C) and (D) were obtained from different experiments.
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 35

11. any of the indexes measured. Bath application of CPP
produced significant changes in the indexes quantifying
postsynaptic excitability and inhibition similarly in the
two age groups (Fig. 7D). In particular, CPP significantly
enhanced the PS/fEPSP ratio and also produced a
robust increase in the rightward shift of I50-PS, while it
decreased the ratio PS2/PS1. Interestingly, we found
that the drug-induced changes in the PS2/PS1 ratio and
the probability of SWR sequences were positively
correlated (two-tailed correlation test, r = 0.587,
P< 0.05). CPP also significantly reduced fEPSP50 in
the slices from aged but not adult rats.
DISCUSSION
The present study shows that hippocampal circuit in aged
rats displayed impaired ripples oscillation and increased
propensity to organize long sequences of SWRs. The
reduced frequency of ripples (i.e., increased intra-ripple
interval) in the slices of aged rats occurred in parallel
with increased inter-spike interval in complex spike
bursts implying that changes in the pyramidal cell firing
might contribute to the altered network oscillation.
Furthermore, the involvement of NMDARs on the
generation of SWR sequences was reduced in the
slices of aged rats.
Interpretation of age-related changes in spontaneous
activity
The finding of reduced ripples in old animals is in
agreement with previous observations showing reduced
energy or power of the ripple oscillation in aged mice
(Hermann et al., 2009; Kanak et al., 2013).
Furthermore, in keeping with previous in vivo observa-
tions (Gerrard et al., 2001) we found that the occurrence
and the amplitude of SWRs are similar between adult and
old animals. In addition, the present study demonstrates
for the first time that old rats display a significant tendency
to generate SWRs in the form of relatively long
sequences. Several lines of evidence suggest that aging
is accompanied by alterations in the balance between
excitation and inhibition in the hippocampal neuronal net-
work (Oh et al., 2010). These alterations might signifi-
cantly contribute to the impairment in the ripple
oscillation in old animals, given that ripple generation
requires an accurate balance between excitation and
inhibition in the local circuitry (Giannopoulos and
Papatheodoropoulos, 2013). Furthermore, the ability of
the local circuitry to generate sequences of SWRs may
also reside on changes in the basal excitability of the neu-
ronal network. The generation of sequences of SWR
events is favored by moderately lowering GABAergic
transmission (Papatheodoropoulos and Koniaris, 2011;
Giannopoulos and Papatheodoropoulos, 2013). More
specifically, the reduction in the activity of alpha5 sub-
unit-containing GABAA receptors facilitates the formation
of relatively long sequences of SWRs containing four or
more events (Papatheodoropoulos and Koniaris, 2011).
Consistently with the previous observation that the synap-
tic GABAergic inhibitory postsynaptic potentials in CA1
pyramidal neurons are smaller in aged compared to adult
Table1.Evokedpotentials
SynapticeffectivenessNeuronalexcitabilityInhibition
fEPSPthrI50-EPSPfEPSPmaxI50-PSPSmaxfEPSP50PS/fEPSPShiftofI50-PSPS2/PS1
AdultControl0.34±0.04(12)134.8±17.0(12)3.0±0.5(12)102.8±11.9(12)3.7±0.4(12)0.83±0.11(12)2.4±0.2(12)11.0±4.9%#
(12)0.88±0.06%(12)
Nifedipine
(12)
À9.7±5.4%3.8±3.3%À7.1±4.1%5.5±4.9%0.36±3.6%À8.0±2.1%⁄⁄
9.4±4.2%⁄
4.1±19.3%À5.8±3.2%
+CPP
(12)
7.5±10.3%1.3±1.8%6.5±7.8%À4.2±3.8%5.9±3.0%-3.5±3.1%10.9±2.9%⁄
138.3±29.3%⁄⁄
À18.3±3.7%⁄⁄
OldControl0.5±0.22(10)144.8±30.4(10)3.0±0.21(10)86.5±9.3(10)3.2±0.3(10)0.73±0.04(10)2.2±0.1(10)À6.6±4.0%⁄
(9)1.11±0.05%⁄
(9)
Nifedipine
(9)
4.4±13.1%0.06±3.9%À3.5±5.5%À0.24±3.9%11.4±7%À7.1±5.7%18.5±11.1%23.7±15.5%0.8±1.5%
+CPP(9)À0.7±8.4%1.1±1.5%À3.4±3.9%À0.52±2.1%1.1±2.8%À6.6±4.1%⁄
11.7±4.3%⁄
128.5±57.5%⁄⁄
À13.7±2.1%⁄⁄
Valuesintoparenthesisrepresentthenumberofanimals.
Asterisksappearingin‘‘Old-Control’’rowsdenotestatisticallysignificantdifferencesbetweenthetwoagegroupsusingtheMann–Whitneytest.
Asterisksappearingin‘‘Nifedipine’’and‘‘CPP’’rowsdenotesignificantdifferencesbetweenControlvsNifedipineandNifedipinevsCPPusingtheWilcoxontest.
Significantdifferenceswereobservedat⁄
P<0.05and⁄⁄
P<0.005,respectively.
#
DenotessignificanteffectofPPSonI50-PS(Wilcoxontest,P<0.05).
36 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

12. rats (Potier et al., 2006), the present results showed smal-
ler recurrent inhibition in the slices of old animals. Aging is
accompanied by a reduction of the number of bistratified
cells (Potier et al., 2006) which form synapses containing
a5GABAA receptors (Thomson et al., 2000) and the
expression of a5 subunit mRNA is robustly reduced in
the hippocampus of aged rats (see (Wilson et al.,
2006)). Thus, the increased ability of the aged hippocam-
pus to generate long sequences of SWRs may be attrib-
uted to reductions in GABAergic activity, especially that
involving alpha5-containing GABAA receptors.
In this study we detected and quantified the activity of
complex spike cells in adult and aged rats. To our
knowledge, this is the first in vitro study after that by
Bragin and Vinogradova (1983) in which un-induced
spontaneous complex spike bursting is detected and
quantified. We found that the CS bursts fired by old
CA1 pyramidal cells contained more spikes and had
longer mean inter-spike interval than those in adult cells.
Given that CS bursting occurred in correspondence with
ripple oscillation it could be argued that the interval
between individual spikes in CS bursting might contribute
in determining the interval between consecutive ripple
cycles. It is interesting that both measures displayed simi-
lar age-dependent alterations. In a previous in vivo study
the inter-spike interval was found similar between adult
and aged animals (Smith et al., 2000). The comparison
of inter-spike interval between ages in this study was
based on normalized distributions of inter-spike intervals
from each cell and then calculating the averaged dis-
tributions for each animal. Thus, methodological particu-
larities might contribute to the differences between
in vivo and in vitro observations. Accordingly, it should
be noted that the possibility that the methodological pro-
cedure of slice preparation would contribute to the
observed age-related differences in the present study
could not be excluded.
The increased calcium current in aging pyramidal
neurons (Landfield, 1987; Moyer and Disterhoft, 1994;
Thibault and Landfield, 1996; Foster and Norris, 1997;
Kumar et al., 2009) might provide a mechanism for the
alterations in complex spike activity observed in slices of
old animals. The inter-spike interval is mainly determined
by the fast after-hyperpolarization that follows the action
potential and is produced by the activation of calcium-
sensitive potassium current (Storm, 1990). Virtually,
increased calcium entry into the cell may enhance fast
after-hyperpolarization and prolong the inter-spike interval
(Su et al., 2001). We also found that the mean inter-spike
interval in MUA that occurs during SWRs was similar
between adult and aged rats. This might appear contradic-
tory to the observed age-dependent difference in inter-
spike interval in complex spike bursting. However, while
CS bursting is due to pyramidal cell activity only, spiking
activity during SWRs most probably involves firing from
local interneurons. Both, pyramidal cells and interneurons
increase their firing rate during SWRs (Csicsvari et al.,
1999a) contributing to the relatively short ISI. However,
in recordings made in vitro it is difficult to disentangle prin-
cipal cell from interneuron firing and conclude about their
relative involvement on the network activity of SWRs.
Interpretation of drug-induced effects on SWRs
The most conspicuous effect of blockade of NMDARs by
CPP was the reduction in the probability of sequences’
generation which is consistent with previous
observations (Papatheodoropoulos, 2010). This effect
was stronger in the slices from adult than aged rats.
NMDARs appear to follow functional alterations during
aging (for a review, see (Magnusson et al., 2010). For
instance, high levels of NMDARs in the hippocampus of
old animals have been observed to be associated with
compromised hippocampus-dependent learning and
memory (Nicolle et al., 1996; Topic et al., 2007) and unim-
paired animals displayed reduced binding to NMDARs (Le
Jeune et al., 1996). Accordingly, in the hippocampus of
Wistar rats a decline in the binding density to NMDARs
has been reported (Serra et al., 1994) although the
expression of one of the basic subunits of NMDARs,
namely GluN1, does not change with age (Adams et al.,
2001; Dyall et al., 2007). Interestingly, relatively high sus-
ceptibility to aging display those NMDARs that are located
toward the ventral part of the hippocampus (Magnusson
et al., 2006), the part of the structure where slices in the
present study were taken from. It is therefore reasonable
to suggest that the functional alterations of the NMDARs
contribute to the age-dependent different modulation of
SWRs by these receptors. It is interesting that the reduc-
tion in the sequences induced by CPP correlated with the
drug-induced increase in the inhibition, as evidenced by
its reducing effect on the PS2/PS1 ratio, supporting the
idea that relatively low levels of inhibitory actions might
contribute to the increased tendency for SWR sequences
in the old hippocampus.
Blockade of NMDARs also enhanced the amplitude of
SWRs and reduced their rate of occurrence. Sharp waves
in the CA1 correspond to GABAA receptor-mediated
synchronous inhibitory postsynaptic potentials
(Papatheodoropoulos and Kostopoulos, 2002; Wu et al.,
2002b; Maier et al., 2003; Papatheodoropoulos, 2010).
On the contrary, sharp waves in the CA3 correspond to
excitatory postsynaptic potentials (Wu et al., 2002a;
Colgin et al., 2004; Behrens et al., 2005). Although
SWRs can be generated by the CA1 circuit independently
of CA3 input as observed in CA1 mini-slices (Nimmrich
et al., 2005; Papatheodoropoulos, 2010; Maier et al.,
2011), previous (Csicsvari et al., 2000; Wu et al., 2005;
Both et al., 2008) and the present results show that in intact
hippocampal slices activity is most often initiated in the
CA3. Therefore, the amplitude of sharp waves recorded
in the CA1 of integral slices might be modulated by synap-
tic inhibition in the CA1 and/or excitation of the CA3 circuit.
Using evoked potentials, we found that CPP increases the
neuronal excitability and recurrent inhibition. Recent
reports have shown that absence of NMDARs enhances
the excitability of CA3 neurons (Fukushima et al., 2009).
Furthermore, it has been suggested that NMDARs can
serve to dampen the excitation of sharp waves generated
in the CA3 through the action of NMDAR-entering calcium
that activate calcium-dependent potassium channels,
which counteract depolarization in these cells (Colgin
et al., 2005). Hence, the increased amplitude of sharp
waves in the CA1 might result from an increase in the
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 37

13. activity of the CA3 circuit induced by CPP. The same
mechanism may also underlie the enhancing effect of
CPP on the ripple oscillation given that increase in the rip-
ple oscillation apparently requires a mild increase in princi-
pal cell activity (Csicsvari et al., 1999a; Koniaris et al.,
2011; Giannopoulos and Papatheodoropoulos, 2013).
It has been demonstrated that during aging L-vdcc
contribute to the increased calcium current into the CA1
pyramidal cells (Moyer and Disterhoft, 1994; Thibault
and Landfield, 1996; Foster and Norris, 1997; Kumar
et al., 2009). In the present study we examined for the first
time the possibility that L-vdcc are involved in the mod-
ulation of SWRs and that their involvement might differ
between adult and old animals. Surprisingly, we observed
that the most consistent effect of blockade of L-vdcc was
an increase in the amplitude of SWRs of adult rats only.
Taken into account that nifedipine enhanced the excitabil-
ity of the local network only in the slices of adult animals
and that the excitability of CA3 principal cells can regulate
the amplitude of sharp waves in the CA1, as discussed
above, the age-related differences in the effect of nifedip-
ine on the amplitude of SWRs may involve the action of
the drug on the CA3 neuronal network.
Implications of SWR changes for the memory
function in aging
Aging is associated with long-term rather than short-term
memory impairment (Balota et al., 2000; Beason-Held
and Horwitz, 2002). There is mounting evidence that
SWR activity plays important role in memory, namely in
the process of memory consolidation which consists of
a process of transformation of initially labile memory
traces into stable memories (Diekelmann and Born,
2010). It is thought that the implication of SWRs in the
process of memory consolidation lies in the coordination
of the repeated off-line reactivation of hippocampal mem-
ory traces (i.e., experience-related sequences of firing
patterns) which are subsequently integrated into cortical
circuits where memory content is long-term established
(O’Neill et al., 2010). There are a number of recent obser-
vations that directly connect the activity of SWRs with
memory processes and synaptic plasticity (Girardeau
et al., 2009; Ego-Stengel and Wilson, 2010). The altered
ripple oscillation observed in old rats could then imply
reduced mnemonic abilities in these rats.
One similarity between in vivo (Battaglia et al., 2004;
Klausberger et al., 2004; Ramadan et al., 2009) and
in vitro SWRs is the frequent generation of episodes of
SWRs under the form of groups of several events that
occur recurrently in sequences. It has been recently sug-
gested that a function of such sequences or clusters of
SWRs may be to keep in memory and express later pro-
longed experiences of several individual events that occur
in a path along the space (Davidson et al., 2009).
Alternatively, or in addition, sequences might represent
all physically available trajectories within the environment
including never experienced ones (Gupta et al., 2010). It
could be argued that the generation of sequences might
reflect an intrinsic pre-defined ability of the local circuitry
that help the hippocampus to incorporate the details of
actual (long) experiences (Papatheodoropoulos and
Koniaris, 2011). The incidence of sequences of SWRs
is increased in parallel with long-term synaptic potentia-
tion (Papatheodoropoulos, 2010) though the entire num-
ber of SWR events remains stable, indicating that
persistent plastic changes in the pattern of activation of
the hippocampal network are accompanied by homeo-
static mechanisms that tend to keep the total number of
circuit activations stable. It is interesting that the
increased propensity of old rats for generating long
sequences of SWR events occurred despite the fact that
the total number of events remained unchanged. This
propensity might represent the result of homeostatic
mechanisms that tend to compensate for the reduced
ability of the circuit to organize normal ripple oscillation
and concomitantly maintain the level of hippocampal cir-
cuit activation to normal levels. These compensatory
changes could virtually preserve an adequate level of
memory function. Compensatory changes have been
observed or have been suggested to occur at several
levels of organization in the old brain. For instance, an
age-related decrease in NMDAR function might represent
a compensation for the increased L-type calcium chan-
nels and calcium dysregulation associated with old age
(Kumar et al., 2009). Conversely, reduction in NMDAR-
dependent long-term synaptic potentiation (Rosenzweig
and Barnes, 2003) is apparently compensated by
increase in L-vdcc-dependent potentiation in the hip-
pocampus (Shankar et al., 1998; Boric et al., 2008).
Larger after hyperpolarizations in the old CA1 pyramidal
neurons (Oh et al., 2010) could be an adaptive change
to restrict hyperactivation of CA1 circuit by the high exci-
table old CA3 pyramidal cells (Wilson et al., 2005; Penner
and Barnes, 2007).
The present results demonstrate significant differences
in the SWR and principal cell activity between adult and old
age. Yet, given the differences between the in vitro and
in vivo conditions the observed age-related differences
should be interpreted with caution, awaiting the
confirmation from other future in vivo and in vitro studies.
CONCLUSION
The present findings show that the hippocampal slices of
aged compared with adult rats displays altered ripple
oscillation and increased tendency to organize SWRs in
long sequences displaying reduced dependence on
NMDARs. The altered characteristics of principal cells’
bursting activity might contribute to the impaired ripples
in aging. We propose that the decline in the ripple
oscillation observed during aging is accompanied by
changes in the pattern of generation of SWR events
resulting from the action of compensatory mechanisms
in the old hippocampus in order to maintain an efficient
behavioral outcome.
Acknowledgments—This research has been co-financed by the
European Union (European Social Fund – ESF) and Greek
national funds through the Operational Program ‘Education-
and-Lifelong-Learning’ of the National Strategic Reference
Framework (NSRF) – Research Funding Program: Thales.
Investing in knowledge society through the European Social
Fund. (MIS: 380342).
38 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

14. REFERENCES
Adams MM, Smith TD, Moga D, Gallagher M, Wang Y, Wolfe BB,
Rapp PR, Morrison JH (2001) Hippocampal dependent learning
ability correlates with N-methyl-D-aspartate (NMDA) receptor
levels in CA3 neurons of young and aged rats. J Comp Neurol
432:230–243.
Balota DA, Dolan PO, Cuchek JM (2000) Memory changes in healthy
older adults. In: Tulving E, Craik FIM, editors. The oxford
handbook of memory. Oxford: Oxford University Press. p.
395–409.
Battaglia FP, Sutherland GR, McNaughton BL (2004) Hippocampal
sharp wave bursts coincide with neocortical ‘‘up-state’’ transitions.
Learn Memory 11:697–704 (Cold Spring Harbor, NY).
Beason-Held LL, Horwitz B (2002) Aging brain. In: Ramachandran
VS, editor. Encyclopedia of the human brain. Elsevier. p. 43–57.
Behrens CJ, van den Boom LP, de Hoz L, Friedman A, Heinemann U
(2005) Induction of sharp wave–ripple complexes in vitro and
reorganization of hippocampal networks. Nat Neurosci
8:1560–1567.
Boric K, Munoz P, Gallagher M, Kirkwood A (2008) Potential adaptive
function for altered long-term potentiation mechanisms in aging
hippocampus. J Neurosci 28:8034–8039.
Both M, Bahner F, von Bohlen und Halbach O, Draguhn A (2008)
Propagation of specific network patterns through the mouse
hippocampus. Hippocampus 18:899–908.
Bragin AG, Vinogradova OS (1983) Comparison of neuronal activity
in septal and hippocampal grafts developing in the anterior eye
chamber of the rat. Brain Res 312:279–286.
Burger C (2010) Region-specific genetic alterations in the aging
hippocampus: implications for cognitive aging. Front Aging
Neurosci 2:140.
Burke DM, Mackay DG (1997) Memory, language, and ageing. Philos
Trans R Soc Lond B Biol Sci 352:1845–1856.
Burke SN, Barnes CA (2006) Neural plasticity in the ageing brain. Nat
Rev Neurosci 7:30–40.
Burke SN, Barnes CA (2010) Senescent synapses and hippocampal
circuit dynamics. Trends Neurosci 33:153–161.
Buzsaki G (1986) Hippocampal sharp waves: their origin and
significance. Brain Res 398:242–252.
Buzsaki G (1989) Two-stage model of memory trace formation: a role
for ‘‘noisy’’ brain states. Neuroscience 31:551–570.
Buzsaki G (1996) The hippocampo-neocortical dialogue. Cereb
Cortex 6:81–92.
Buzsaki G, Horvath Z, Urioste R, Hetke J, Wise K (1992) High-
frequency network oscillation in the hippocampus. Science
256:1025–1027.
Chrobak JJ, Buzsaki G (1996) High-frequency oscillations in the
output networks of the hippocampal-entorhinal axis of the freely
behaving rat. J Neurosci 16:3056–3066.
Colgin LL, Jia Y, Sabatier JM, Lynch G (2005) Blockade of NMDA
receptors enhances spontaneous sharp waves in rat hippocampal
slices. Neurosci Lett 385:46–51.
Colgin LL, Kubota D, Jia Y, Rex CS, Lynch G (2004) Long-term
potentiation is impaired in rat hippocampal slices that produce
spontaneous sharp waves. J Physiol 558:953–961.
Crook T, Bartus RT, Ferris SH, Whitehouse P, Cohen GD, Gershon S
(1986) Age-associated memory impairment: proposed diagnostic
criteria and measures of clinical change – report of a national
institute of mental health work group. Dev Neuropsychol
2:261–276.
Csicsvari J, Hirase H, Czurko A, Mamiya A, Buzsaki G (1999a) Fast
network oscillations in the hippocampal CA1 region of the
behaving rat. J Neurosci 19:RC20.
Csicsvari J, Hirase H, Czurko A, Mamiya A, Buzsaki G (1999b)
Oscillatory coupling of hippocampal pyramidal cells and
interneurons in the behaving Rat. J Neurosci 19:274–287.
Csicsvari J, Hirase H, Mamiya A, Buzsaki G (2000) Ensemble
patterns of hippocampal CA3-CA1 neurons during sharp wave-
associated population events. Neuron 28:585–594.
Davidson TJ, Kloosterman F, Wilson MA (2009) Hippocampal replay
of extended experience. Neuron 63:497–507.
de Jong GI, Naber PA, Van der Zee EA, Thompson LT, Disterhoft JF,
Luiten PG (1996) Age-related loss of calcium binding proteins in
rabbit hippocampus. Neurobiol Aging 17:459–465.
Diekelmann S, Born J (2010) The memory function of sleep. Nat Rev
Neurosci 11:114–126.
Dyall SC, Michael GJ, Whelpton R, Scott AG, Michael-Titus AT
(2007) Dietary enrichment with omega-3 polyunsaturated fatty
acids reverses age-related decreases in the GluR2 and NR2B
glutamate receptor subunits in rat forebrain. Neurobiol Aging
28:424–439.
Ego-Stengel V, Wilson MA (2010) Disruption of ripple-associated
hippocampal activity during rest impairs spatial learning in the rat.
Hippocampus 20:1–10.
Eschenko O, Ramadan W, Molle M, Born J, Sara SJ (2008)
Sustained increase in hippocampal sharp-wave ripple activity
during slow-wave sleep after learning. Learn Mem 15:222–228.
Foster TC, Norris CM (1997) Age-associated changes in Ca(2+)-
dependent processes: relation to hippocampal synaptic plasticity.
Hippocampus 7:602–612.
Fox SE, Ranck Jr JB (1975) Localization and anatomical identification
of theta and complex spike cells in dorsal hippocampal formation
of rats. Exp Neurol 49:299–313.
Fox SE, Ranck Jr JB (1981) Electrophysiological characteristics of
hippocampal complex-spike cells and theta cells. Exp Brain Res
41:399–410.
Fukushima F, Nakao K, Shinoe T, Fukaya M, Muramatsu S,
Sakimura K, Kataoka H, Mori H, Watanabe M, Manabe T,
Mishina M (2009) Ablation of NMDA receptors enhances the
excitability of hippocampal CA3 neurons. PLoS ONE 4:e3993.
Gallagher M, Rapp PR (1997) The use of animal models to study the
effects of aging on cognition. Annu Rev Psychol 48:339–370.
Gerrard JL, Kudrimoti H, McNaughton BL, Barnes CA (2001)
Reactivation of hippocampal ensemble activity patterns in the
aging rat. Behav Neurosci 115:1180–1192.
Giannopoulos P, Papatheodoropoulos C (2013) Effects of mu-opioid
receptor modulation on the hippocampal network activity of sharp
wave and ripples. Br J Pharmacol 168:1146–1164.
Girardeau G, Benchenane K, Wiener SI, Buzsaki G, Zugaro MB
(2009) Selective suppression of hippocampal ripples impairs
spatial memory. Nat Neurosci 12:1222–1223.
Gupta AS, van der Meer MA, Touretzky DS, Redish AD (2010)
Hippocampal replay is not a simple function of experience.
Neuron 65:695–705.
Harris KD, Hirase H, Leinekugel X, Henze DA, Buzsaki G (2001)
Temporal interaction between single spikes and complex spike
bursts in hippocampal pyramidal cells. Neuron 32:141–149.
Hermann D, Both M, Ebert U, Gross G, Schoemaker H, Draguhn A,
Wicke K, Nimmrich V (2009) Synaptic transmission is impaired
prior to plaque formation in amyloid precursor protein-
overexpressing mice without altering behaviorally-correlated
sharp wave–ripple complexes. Neuroscience 162:1081–1090.
Inostroza M, Born J (2013) Sleep for preserving and transforming
episodic memory. Annu Rev Neurosci 36:79–102.
Kanak DJ, Rose GM, Zaveri HP, Patrylo PR (2013) Altered network
timing in the CA3-CA1 circuit of hippocampal slices from aged
mice. PLoS ONE 8:e61364.
Kelly KM, Nadon NL, Morrison JH, Thibault O, Barnes CA, Blalock
EM (2006) Neurobiology of aging. Epilepsy Res 68(Suppl.
1):S5–20.
Kirkwood TB, Boys RJ, Gillespie CS, Proctor CJ, Shanley DP,
Wilkinson DJ (2003) Towards an e-biology of ageing: integrating
theory and data. Nat Rev Mol Cell Biol 4:243–249.
Klausberger T, Marton LF, Baude A, Roberts JD, Magill PJ, Somogyi
P (2004) Spike timing of dendrite-targeting bistratified cells during
hippocampal network oscillations in vivo. Nat Neurosci 7:41–47.
Klausberger T, Somogyi P (2008) Neuronal diversity and temporal
dynamics: the unity of hippocampal circuit operations. Science
321:53–57.
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 39

15. Koniaris E, Drimala P, Sotiriou E, Papatheodoropoulos C (2011)
Different effects of zolpidem and diazepam on hippocampal sharp
wave–ripple activity in vitro. Neuroscience 175:224–234.
Kubota D, Colgin LL, Casale M, Brucher FA, Lynch G (2003)
Endogenous waves in hippocampal slices. J Neurophysiol
89:81–89.
Kudrimoti HS, Barnes CA, McNaughton BL (1999) Reactivation of
hippocampal cell assemblies: effects of behavioral state,
experience, and EEG dynamics. J Neurosci 19:4090–4101.
Kumar A, Bodhinathan K, Foster TC (2009) Susceptibility to calcium
dysregulation during brain aging. Front Aging Neurosci 1:2.
Landfield PW (1987) ‘Increased calcium-current’ hypothesis of brain
aging. Neurobiol Aging 8:346–347.
Le Jeune H, Cecyre D, Rowe W, Meaney MJ, Quirion R (1996)
Ionotropic glutamate receptor subtypes in the aged memory-
impaired and unimpaired Long-Evans rat. Neuroscience
74:349–363.
Magnusson KR, Brim BL, Das SR (2010) Selective vulnerabilities of
N-methyl-D-aspartate (NMDA) receptors during brain aging. Front
Aging Neurosci 2:11.
Magnusson KR, Kresge D, Supon J (2006) Differential effects of
aging on NMDA receptors in the intermediate versus the dorsal
hippocampus. Neurobiol Aging 27:324–333.
Maier N, Nimmrich V, Draguhn A (2003) Cellular and network
mechanisms underlying spontaneous sharp wave–ripple
complexes in mouse hippocampal slices. J Physiol 550:873–887.
Maier N, Tejero-Cantero A, Dorrn AL, Winterer J, Beed PS, Morris G,
Kempter R, Poulet JF, Leibold C, Schmitz D (2011) Coherent
phasic excitation during hippocampal ripples. Neuron 72:137–152.
Markowska AL, Stone WS, Ingram DK, Reynolds J, Gold PE, Conti
LH, Pontecorvo MJ, Wenk GL, Olton DS (1989) Individual
differences in aging: behavioral and neurobiological correlates.
Neurobiol Aging 10:31–43.
McNaughton BL, Barnes CA, O’Keefe J (1983) The contributions of
position, direction, and velocity to single unit activity in the
hippocampus of freely-moving rats. Exp Brain Res 52:41–49.
Monacelli AM, Cushman LA, Kavcic V, Duffy CJ (2003) Spatial
disorientation in Alzheimer’s disease: the remembrance of things
passed. Neurology 61:1491–1497.
Moradi-Chameh H, Peng J, Wu C, Zhang L (2014) Intracellular
activities related to in vitro hippocampal sharp waves are altered
in CA3 pyramidal neurons of aged mice. Neuroscience
277:474–485.
Moyer Jr JR, Disterhoft JF (1994) Nimodipine decreases calcium
action potentials in rabbit hippocampal CA1 neurons in an age-
dependent and concentration-dependent manner. Hippocampus
4:11–17.
Nicolle MM, Bizon JL, Gallagher M (1996) In vitro autoradiography of
ionotropic glutamate receptors in hippocampus and striatum of
aged Long-Evans rats: relationship to spatial learning.
Neuroscience 74:741–756.
Nimmrich V, Maier N, Schmitz D, Draguhn A (2005) Induced sharp
wave–ripple complexes in the absence of synaptic inhibition in
mouse hippocampal slices. J Physiol 563:663–670.
Nunez-Santana FL, Oh MM, Antion MD, Lee A, Hell JW, Disterhoft JF
(2013) Surface L-type Ca channel expression levels are
increased in aged hippocampus. Aging Cell.
O’Keefe J, Nadel L (1978) The hippocampus as a cognitive
map. Oxford: Clarendon Press.
O’Neill J, Pleydell-Bouverie B, Dupret D, Csicsvari J (2010) Play it
again: reactivation of waking experience and memory. Trends
Neurosci 33:220–229.
Oh MM, Oliveira FA, Disterhoft JF (2010) Learning and aging related
changes in intrinsic neuronal excitability. Front Aging Neurosci
2:2.
Papatheodoropoulos C (2010) Patterned activation of hippocampal
network (approximately 10 Hz) during in vitro sharp wave–ripples.
Neuroscience 168:429–442.
Papatheodoropoulos C, Koniaris E (2011) Alpha5GABAA receptors
regulate hippocampal sharp wave–ripple activity in vitro.
Neuropharmacology 60:662–673.
Papatheodoropoulos C, Kostopoulos G (2002) Spontaneous
GABA(A)-dependent synchronous periodic activity in adult rat
ventral hippocampal slices. Neurosci Lett 319:17–20.
Penner MR, Barnes CA (2007) Memory changes with age:
neurobiological correlates. In: Kesner RP, Martinez JL,
editors. Neurobiology of Learning and Memory. Elsevier. p.
483–518.
Potier B, Jouvenceau A, Epelbaum J, Dutar P (2006) Age-related
alterations of GABAergic input to CA1 pyramidal neurons and its
control by nicotinic acetylcholine receptors in rat hippocampus.
Neuroscience 142:187–201.
Ramadan W, Eschenko O, Sara SJ (2009) Hippocampal sharp wave/
ripples during sleep for consolidation of associative memory.
PLoS ONE 4:e6697.
Ranck Jr JB (1973) Studies on single neurons in dorsal hippocampal
formation and septum in unrestrained rats. I. Behavioral
correlates and firing repertoires. Exp Neurol 41:461–531.
Rosenzweig ES, Barnes CA (2003) Impact of aging on hippocampal
function: plasticity, network dynamics, and cognition. Prog
Neurobiol 69:143–179.
Serra M, Ghiani CA, Foddi MC, Motzo C, Biggio G (1994) NMDA
receptor function is enhanced in the hippocampus of aged rats.
Neurochem Res 19:483–487.
Shankar S, Teyler TJ, Robbins N (1998) Aging differentially alters
forms of long-term potentiation in rat hippocampal area CA1. J
Neurophysiol 79:334–341.
Siapas AG, Wilson MA (1998) Coordinated interactions between
hippocampal ripples and cortical spindles during slow-wave sleep.
Neuron 21:1123–1128.
Singer AC, Frank LM (2009) Rewarded outcomes enhance
reactivation of experience in the hippocampus. Neuron
64:910–921.
Sirota A, Csicsvari J, Buhl D, Buzsaki G (2003) Communication
between neocortex and hippocampus during sleep in rodents.
Proc Natl Acad Sci U S A 100:2065–2069.
Smith AC, Gerrard JL, Barnes CA, McNaughton BL (2000) Effect of
age on burst firing characteristics of rat hippocampal pyramidal
cells. NeuroReport 11:3865–3871.
Stanley DP, Shetty AK (2004) Aging in the rat hippocampus is
associated with widespread reductions in the number of
glutamate decarboxylase-67 positive interneurons but not
interneuron degeneration. J Neurochem 89:204–216.
Storm JF (1990) Potassium currents in hippocampal pyramidal cells.
Prog Brain Res 83:161–187.
Su H, Alroy G, Kirson ED, Yaari Y (2001) Extracellular calcium
modulates persistent sodium current-dependent burst-firing in
hippocampal pyramidal neurons. J Neurosci 21:4173–4182.
Suzuki SS, Smith GK (1985) Burst characteristics of
hippocampal complex spike cells in the awake rat. Exp Neurol
89:90–95.
Thibault O, Landfield PW (1996) Increase in single L-type calcium
channels in hippocampal neurons during aging. Science
272:1017–1020.
Thomson AM, Bannister AP, Hughes DI, Pawelzik H (2000)
Differential sensitivity to Zolpidem of IPSPs activated by
morphologically identified CA1 interneurons in slices of rat
hippocampus. Eur J Neurosci 12:425–436.
Topic B, Willuhn I, Palomero-Gallagher N, Zilles K, Huston JP,
Hasenohrl RU (2007) Impaired maze performance in aged rats
is accompanied by increased density of NMDA, 5-HT1A, and
alpha-adrenoceptor binding in hippocampus. Hippocampus
17:68–77.
Wang SH, Morris RG (2010) Hippocampal-neocortical interactions in
memory formation, consolidation, and reconsolidation. Annu Rev
Psychol 61(49–79):C41–44.
Wierzynski CM, Lubenov EV, Gu M, Siapas AG (2009) State-
dependent spike-timing relationships between hippocampal and
prefrontal circuits during sleep. Neuron 61:587–596.
Wilson IA, Gallagher M, Eichenbaum H, Tanila H (2006)
Neurocognitive aging: prior memories hinder new hippocampal
encoding. Trends Neurosci 29:662–670.
40 S. Kouvaros et al. / Neuroscience 298 (2015) 26–41

16. Wilson IA, Ikonen S, Gallagher M, Eichenbaum H, Tanila H (2005)
Age-associated alterations of hippocampal place cells are
subregion specific. J Neurosci 25:6877–6886.
Wu C, Asl MN, Gillis J, Skinner FK, Zhang L (2005) An in vitro model
of hippocampal sharp waves: regional initiation and intracellular
correlates. J Neurophysiol 94:741–753.
Wu C, Shen H, Luk WP, Zhang L (2002a) A fundamental
oscillatory state of isolated rodent hippocampus. J Physiol
540:509–527.
Wu C, Shen H, Luk WP, Zhang L (2002b) A fundamental
oscillatory state of isolated rodent hippocampus. J Physiol
540:509–527.
(Accepted 6 April 2015)
(Available online 11 April 2015)
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 41