HIPPOCAMPAL SHARP WAVES AND RIPPLES: EFFECTS OF AGING AND MODULATION BY NMDA RECEPTORS AND L-TYPE CA 2+ 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: [email protected](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
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Neuroscience 298 (2015) 26–41
HIPPOCAMPAL SHARP WAVES AND RIPPLES: EFFECTS OF AGINGAND MODULATION BY NMDA RECEPTORS AND L-TYPE CA2+ CHANNELS
S. KOUVAROS, D. KOTZADIMITRIOU � ANDC. 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
http://dx.doi.org/10.1016/j.neuroscience.2015.04.0120306-4522/� 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Laboratory of Physiology, MedicalSchool, University of Patras, 26 500 Rio, Patras, Greece. Tel: +30-2610-969117; fax: +30-2610-997215.
E-mail address: [email protected] (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 postsynapticpotentials; 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.
26
alterations in the firing mode of pyramidal cells might under-
lie to some extent the changes in ripples that occur in old
animals. � 2015 IBRO. Published by Elsevier Ltd. All rights
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
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
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
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 29
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)-
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
B
WRs from an adult and an old rat and the corresponding band-pass
Examples of power spectrum graphs illustrating that ripple oscillation
) Comparative data of the various variables of the ripples oscillation
nce between adult and old values at P< 0.01 (Mann–Whitney U test).
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
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
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 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
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
S. Kouvaros et al. / Neuroscience 298 (2015) 26–41 33
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
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
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
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
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