Purinergic P2X, P2Y and adenosine receptors differentially modulate hippocampal gamma oscillations.

Abstract

The present study was designed to investigate the role of extracellular ATP and its receptors on neuronal network activity. Gamma oscillations (30-50 Hz) were induced in the CA3 region of acute rat hippocampal slices by either acetylcholine (ACh) or kainic acid (KA). ATP reduced the power of KA-induced gamma oscillations exclusively by activation of adenosine receptors after its degradation to adenosine. In contrast, ATP suppressed ACh-induced oscillations through both adenosine and ATP receptors. Activation of adenosine receptors accounts for about 55%, activation of P2 receptors for ∼45% of suppression. Monitoring the ATP degradation by ATP biosensors revealed that bath-applied ATP reaches ∼300 times lower concentrations within the slice. P2 receptors were also activated by endogenous ATP since inhibition of ATP-hydrolyzing enzymes had an inhibitory effect on ACh-induced gamma oscillations. More specific antagonists revealed that ionotropic P2X2 and/or P2X4 receptors reduced the power of ACh-induced gamma oscillations whereas metabotropic P2Y(1) receptor increased it. Intracellular recordings from CA3 pyramidal cells suggest that adenosine receptors reduce the spiking rate and the synchrony of action potentials during gamma oscillations whereas P2 receptors only modulate the firing rate of the cells. In conclusion, our results suggest that endogenously released ATP differentially modulates the power of ACh- or KA-induced gamma oscillations in the CA3 region of the hippocampus by interacting with P2X, P2Y and adenosine receptors. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

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Purinergic P2X, P2Yand adenosine receptors differentially modulate hippocampal
gamma oscillations
Steffen B. Schulza,b,1, Zin-Juan Klafta,1, Anton R. Röslera, Uwe Heinemanna,b, Zoltan Gerevicha,*
aInstitute of Neurophysiology, Charité-Universitätsmedizin Berlin, Oudenarder Str. 16, D-13347 Berlin, Germany
bNeuroCure Research Centre, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany
a r t i c l e i n f o
Article history:
Received 14 June 2011
Received in revised form
20 September 2011
Accepted 22 September 2011
Keywords:
Adenosine receptors
Acetylcholine
Gamma oscillations
P2X receptors
P2Y receptors
ATP
a b s t r a c t
The present study was designed to investigate the role of extracellular ATP and its receptors on neuronal
network activity. Gamma oscillations (30e50 Hz) were induced in the CA3 region of acute rat hippocampal
slices by either acetylcholine (ACh) or kainic acid (KA). ATP reduced the power of KA-induced gamma
oscillations exclusively by activation of adenosine receptors after its degradation to adenosine. In contrast,
ATP suppressed ACh-induced oscillations through both adenosine and ATP receptors. Activation of aden-
osine receptors accounts for about 55%, activation of P2 receptors for w45% of suppression. Monitoring the
ATP degradation by ATP biosensors revealed that bath-applied ATP reaches w300 times lower concentra-
tions within the slice. P2 receptors were also activated by endogenous ATP since inhibition of ATP-
hydrolyzing enzymes had an inhibitory effect on ACh-induced gamma oscillations. More specific antago-
nists revealed that ionotropic P2X2 and/or P2X4 receptors reduced the power of ACh-induced gamma
oscillations whereas metabotropic P2Y1receptor increased it. Intracellular recordings from CA3 pyramidal
cells suggest thatadenosine receptors reduce the spikingrate and the synchronyof actionpotentials during
gammaoscillationswhereasP2receptorsonlymodulatethefiringrateofthecells.Inconclusion,ourresults
suggest that endogenously released ATP differentially modulates the power of ACh- or KA-induced gamma
oscillations in the CA3 region of the hippocampus by interacting with P2X, P2Yand adenosine receptors.
This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.
? 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Synchronized fast neural network oscillations in the gamma
frequency band (w30e90 Hz) can be found in a variety of brain
regions and are associated with various cognitive functions,
including sensory processing, selective attention, learning and
memory (Bartos et al., 2007; Buzsáki and Draguhn, 2004).
Conversely, impairment of gamma oscillations might underlie
cognitive dysfunction in diseases such as schizophrenia and Alz-
heimer’s disease (Nakazawa et al., 2011; Palop and Mucke, 2010).
Gamma oscillations can be evoked in the hippocampus in vitro for
example by bath application of muscarinic acetylcholine receptor
(mAChR) agonists (mimicking cholinergic input from the septum,
Fisahn et al., 1998) or by glutamate via activation of metabotropic
glutamate or kainate receptors (KARs) (Fisahn et al., 2004; Hájos
et al., 2000). These models have different properties and may
differ in their reliance on excitation and inhibition (Bartos et al.,
2007).
Adenosine 50-triphosphate (ATP) is an extracellular signaling
molecule released e.g. by neurons and astrocytes (Abbracchio et al.,
2009). Once released it acts on ionotropic P2X and metabotropic
P2Y receptors and, after metabolism to adenosine, on metabotropic
adenosine receptors. Functional P2Y1receptors have been found on
interneurons in the CA3 stratum oriens close to the pyramidal cell
layer, but not on pyramidal cells. These interneurons are suggested
to be O-LM or basket cells, and calretinin-positive interneurons in
the CA1 stratum radiatum and lacunosum-moleculare. In response
to P2Y1activation, interneurons fire more action potentials and
release more GABA onto their postsynaptic targets (Bowser and
Khakh, 2004; Kawamura et al., 2004). The expression of func-
tional P2X receptors in the hippocampus seems to be restricted to
axons and axon terminals of pyramidal cells of both CA1 and CA3
pyramidal neurons (Khakh et al., 2003; Rodrigues et al., 2005).
Abbreviations: ACh, acetylcholine; AP, action potential; ATP, Adenosine
50-triphosphate; FP, field potential; KA, kainic acid; KAR, kainate receptor; mAChR,
muscarinic acetylcholine receptor; Physo, physostigmine; PPADS, pyridoxal phos-
phate-6-azo(benzene-2,4-disulfonic acid; str. pyr., stratum pyramidale; TNP-ATP,
20,30-O-(2,4,6-trinitrophenyl)adenosine-50-triphosphate.
* Corresponding author. Tel.: þ49 30 450528155; fax: þ49 30 450528962.
E-mail address: zoltan.gerevich@charite.de (Z. Gerevich).
1These authors contributed equally to this work.
Contents lists available at SciVerse ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ e see front matter ? 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2011.09.024
Neuropharmacology 62 (2012) 914e924

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A1and A2Areceptors are the predominant adenosine receptors
in the hippocampus. A1receptors are localized mainly presynap-
tically on axons and axon terminals and inhibit the release of
glutamate (Sasaki et al., 2011; Schubert et al., 1986) but are also
present postsynaptically on pyramidal cells and on CA1 interneu-
rons where they reduce neuronal excitability (Greene and Haas,
1991; Li and Henry, 2000). In contrast, A2Areceptors have lower
affinity for adenosine and are expressed at much lower levels in the
hippocampus, mainly postsynaptically on pyramidal cells and
enhance excitability and synaptic plasticity (Rebola et al., 2008).
The less densely distributed presynaptic A2Areceptors have been
found to stimulate glutamate (Cunha et al.,1994) and inhibit GABA
release (Cunha and Ribeiro, 2000) from hippocampal nerve
terminals. The net effect of ATP on the hippocampal network
activity may be determined by the local release of ATP, its catabo-
lism byectonucleotidases, the cellular and subcellular expression of
purinergic ATP and adenosine receptors and the affinity of the
receptors for the different ATP metabolites (Cunha et al., 1998).
In the present study we investigated in the CA3 subfield of the
hippocampus whether ATP by activation of purinergic receptors
modulates gamma oscillations induced either by ACh or KA.
2. Materials and methods
2.1. Slice preparation
Hippocampal slices were prepared from Wistar ratsof either sex at anageof 5e7
weeks (150e200 g). Animal procedures were conducted in accordance with the
guidelines of the European Communities Council and the institutional guidelines
approved by the Berlin Animal Ethics Committee (Landesamt für Gesundheit und
Soziales Berlin, T0096/02). Animals were anesthetized with isoflurane and then
decapitated. Their brains were rapidly removed and washed with ice-cold ACSF
containing (in mM): NaCl,129; KCl, 3; NaH2PO4,1.25; NaHCO3, 21; CaCl2,1.6; MgSO4,
1.8; D-glucose,10, saturated with carbogen (95% O2/5% CO2). For recordings with the
ATP sensors, 2 mM glycerol was added. The brain was cut into 400 mm thick hori-
zontal hippocampal slices with a vibratome (DSK microslicer DTK-1000, Dosaka,
Japan). Slices were immediately transferred to an interface-type recording chamber
perfused with warm and carbogenated ACSF (36?C, flow rate 1.6e1.7 ml/min,
pH 7.4). Slices were left for recovery for at least 1 h before commencing with the
experiments.
2.2. Extracellular recordings
Extracellular field potentials (FPs) were recorded from stratum pyramidale (str.
pyr.) of area CA3b with glass pipettes filled with ACSF (resistance < 3 MU) and
placed 80e120 mm below the cut surface of the slice. Recordings were amplified by
a custom-made amplifier, low-pass filtered at 1 kHz and sampled at 5 kHz by a CED
1401 interface (Cambridge Electronic Design, Cambridge, UK). Gamma oscillations
were induced by bath perfusion of either 10 mM acetylcholine (ACh) and 2 mM
physostigmine (Physo) or 100 nM kainic acid (KA). ACh/Physo and KAwere perfused
for 90 and 50 min, respectively, to allow stabilization of gamma oscillations before
drug application. Note that gamma oscillations were evoked in an interface-type
chamber known for slower equilibration of slices with a given drug than in
submerged chambers (Hájos et al., 2009).
2.3. Intracellular recordings
Intracellular recordings were made from CA3b pyramidal cells with sharp glass
microelectrodes filled with 2 M Kþ-acetate (resistance 60e100 MU). Intracellular
signals were amplified by a SEC-05 LX amplifier (npi electronics, Tamm, Germany),
low-pass filtered at 2 kHz and sampled at 10 kHz using the CED 1401 interface.
Recordings were done in bridge mode. Cells were penetrated during the induction of
gamma oscillations. The measurements were started after the stabilization of
gamma oscillations but at least 20 min after penetration. Only cells were accepted
which showed stable overshooting action potentials (APs) over the full period of the
experiment. The mean membrane potentials of pyramidal cells were ?50.2 ? 1.0
(n ¼ 9) and ?50.4 ? 1.6 mV (n ¼ 14) after the stabilization of KA- and ACh-induced
gamma oscillations, respectively.
2.4. Measurement of changes in the extracellular ATP concentration
Changes in the extracellular ATP concentration were detected using micro-
electrode electrochemical biosensors (Sarissa Biomedical, Coventry, UK) which were
used inparallelwith FP recordings in CA3b. The function of the sensor is described in
detail elsewhere (Llaudet et al., 2005). In short, the sensor consists of a platinum (Pt)
wire coated with the enzymes glycerol kinase and glycerol-3-phosphatase oxidase.
These enzymes degrade ATP by producing H2O2which gets subsequently oxidized
on the Pt wire, finally resulting in two free electrons per ATP molecule. For the
oxidation, a potential of 500 mV had to be supplied permanently to the Pt wire. Both
the ATP and a reference sensor, which lacks the specific enzyme layer to detect ATP
(subsequently called null sensor), were connected to a potentiostat (Duostat
ME200þ, Sarissa Biomedical, Coventry, UK) where the signals were low-pass filtered
at 0.1 Hz before they were sampled by the CED 1401 interface at 1 kHz. To estimate
the ATP concentration on basis of the measured current, a one-point-calibration
with 10 mM ATP was done before and after each experiment (Frenguelli et al.,
2007). The ATP sensor and the null sensor were inserted over the full length
(500 mm, angle of the sensor to the vertical >37?) in stratum pyramidale at least
45 min before the beginning of the experiments to exclude detection of ATP signals
potentially produced by tissue damage.
2.5. Drugs
All drugs were purchased from SigmaeAldrich (ACh 10 mM; ATP 0.1 mM e
3 mM; CGS-15943 10 mM; PPADS (pyridoxal phosphate-6-azo(benzene-2,4-
disulfonic acid)) 30 mM; physostigmine 2 mM) or Tocris (A 740003 10 mM; ARL
67156 50 mM; KA 100 nM; MRS 2179 30 mM; 2meS-ADP 30 mM; MRS 2211 30 mM;
MRS 2578 5 mM; NF 110 1 mM; NF 279 1 mM; NF 340 10 mM; NF 449 10 mM; RO-3
10 mM; TNP-ATP (20,30-O-(2,4,6-trinitrophenyl)adenosine-50-triphosphate) 10 mM)
and dissolved in ACSF except A 740003, CGS-15943, MRS 2578, and RO-3 which
were first dissolved in DMSO and then further diluted in ACSF with a final [DMSO]
of 0.2& (v/v).
2.6. Data analysis and statistics
For analysis of oscillations in FP, power spectra were calculated for 2-min
periods, and peak power, peak frequency and spectral gamma area of each power
spectrum were determined by using a custom-made script for the Spike2 software
(Cambridge Electronic Design, Cambridge, UK). Because of the large variability in
absolute power of the oscillations, the oscillation power was normalized to a 10-min
period prior to drug application or the corresponding time in control. In control
experiments, slices received only ACh/Physo or KA during the whole recording.
Phase histograms of APs from intracellular recordings and the corresponding FP
waveform averages were calculated by the Spike2 software over time windows
covering 1000 APs each. Thereby 0?represents the trough of the FP gamma cycles.
Occurrence of fast components at the negative peak of gamma oscillations (most
probably spikes in pyramidal cells adjacent to the electrode tip) made a low-pass
filtering (100 Hz) of the data necessary (Fisahn et al., 1998). The filtering of the
recordings caused a w25?shift inphase of APs of pyramidal cells, since without low-
pass filtering of our registrations the phase of the APs was found to be w0?(as
published bye.g. Hájos et al., 2004; Gulyáset al., 2010). This applies to all tested drug
conditions. Nevertheless we used the filtered data, because for the analysis of the
accuracy of spike timing it was more important to obtain spike-corrected field
troughs rather than a precise phase preservation. The resulting phase shift is
a systematic error which basically does not bias the calculated changes in phase
accuracy or preferred phase induced by purinergic ligands. The mean vector for each
cell was calculated and the resulting mean phases F and vector lengths r were used
to calculate the mean vectors and the circular standard deviations for the cell
populations of different drug conditions (Batschelet, 1981; Mardia and Jupp, 1972).
Time-frequency-analysis of FPs was done using the Morletwavelet transform (Farge,
1992). The so called mother wavelet is constructed in such a way that it has zero
mean and is localized in both time and frequency space. The family of the Morlet
wavelets ja,b(t) was then generated by ja;bðtÞ ¼ ð1=
J0is the nondimensional angular frequency set to 6 to satisfy the admissibility
condition, b is the translation variable and a the scale variable. The normalization
1=
a
assures equal energy for all scaled wavelets. The continuous wavelet trans-
formation Wða;bÞ ¼
a Morlet wavelet ja;bðtÞ. The Morlet power spectrum, defined as jWða;bÞj2, has the
disadvantage of the slant phenomenon where equal amplitude Fourier components
with different frequencies show different Morlet power. Therefore we used
a modified equal amplitude wavelet power spectrum (mMPS) (Shyu and Sun, 2002)
which corresponds more closely to power spectra obtained with the discrete Fourier
transform: mMPS ¼ ð2=a
were scale summed, averaged for each 5 s and normalized to the control period of
5 min before drug administration. All Morlet wavelet calculations were done by
usage of a custom-made script for MATLAB software (MathWorks, Natick, MA).
Data were represented as mean ? s.e.m unless otherwise denoted. Statis-
tical comparisons between the drug-induced changes and the time-matched
changes in control experiments were made using one-way ANOVA, unpaired
or, where appropriate, paired Student’s t-test. Each drug effect was compared to
its matched control group. For circular data, Rayleigh-test, Moore’s test,
WatsoneWilliams test, and, where appropriate, Hotelling test for paired
samples were performed (Batschelet, 1981; Zar, 2010). Significance level was set
at p < 0.05.
ffiffiffi
a
p
ÞRþN
?Nj0ðt ? b=aÞdt, where
ffiffiffip
RþN
?NxðtÞja;b*ðtÞdt is the convolution of a signal xðtÞ with
ffiffiffiffi
p
p
Þ jWða;bÞj2. To calculate the power sum, the mMPS
S.B. Schulz et al. / Neuropharmacology 62 (2012) 914e924
915

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3. Results
3.1. Kainate-induced gamma oscillations are affected by adenosine
receptors but not by purinergic type 2 (P2) receptors
Bath application of 100 nM kainate (KA) for 50 min reliably
induced persistent gamma oscillations in the str. pyr. of area CA3
with an average frequency of 46.7 ? 2.2 Hz (n ¼ 13) and a gamma
peak power of 1913 ? 1308 mV2. Bath application of ATP (300 mM)
inhibited the peak power of gamma oscillations to 57.2 ? 7.1%
(n ¼ 9; p < 0.05 compared to control; Fig.1B, E), whereas it did not
change the peak frequency (99.0 ? 3.6% of control, p > 0.05). To
investigate if P2 receptors are involved in this effect of ATP, we
applied PPADS (30 mM; Wirkner et al., 2004), a broad spectrum P2
receptorantagonist which blocks most P2Yand P2X receptors, prior
to ATP and found that it did not antagonize the inhibitory effect of
ATP (peak gamma power: 52.6 ? 16.0% of control, n ¼ 6; p > 0.05
compared to 300 mM ATP alone; Fig.1D, E). However, ATP is known
to be metabolized by ectonucleotidases to adenosine which was
recently found to modulate KA-induced gamma oscillations in the
hippocampus by A1 and A2A receptors (Pietersen et al., 2009).
Therefore, we next blocked A1 and A2A adenosine receptors by
CGS-15943 (10 mM; Heidemann et al., 2005). While the blockade of
the adenosine receptors itself increased the oscillations (peak
power: 155.2 ? 16.7% of control, n ¼ 5, p < 0.05) which is in line
with previous findings of Pietersen et al. (2009), it subsequently
abolished the effect of ATP (peak gamma power: 103.5 ? 4.0% of
control period before ATP application, n ¼ 5; p < 0.05 compared to
300 mM ATP alone; Fig. 1C, E). These data indicate that ATP by the
activation of P2 receptors does not modulate KA-induced gamma
oscillations in the hippocampus but does so after degradation to
adenosine by the activation of adenosine receptors, as described by
Pietersen et al. (2009).
3.2. Determination of ATP levels within the slices during bath
application of ATP
To investigate which concentration of ATP is reached within the
slice during its application, we next bath-applied ATP at different
concentrations and simultaneously measured the concentrations
reaching a biosensor positioned in the str. pyr. as described in
Section 2.4. We registered much lower concentrations than those
which were applied (Fig. 2). During application of 300 mM ATP,
a concentration producing w50% inhibition of the gamma power,
only 1.06 ? 0.56 mM ATP (geometric mean ? s.e.m.) was measured
in the slice (n ¼ 11; Fig. 2B).
Fig. 1. Kainate-(KA)-induced gamma oscillations in the CA3 area of the hippocampus are inhibited by adenosine but not by ATP receptors. (A) Gamma oscillations induced by bath
application of KA (100 nM), recorded extracellularly in the CA3 pyramidal cell layer from a rat hippocampal slice. Top: field recording before, during and after the application of ATP
(300 mM). Middle: wavelet spectrum showing the oscillation frequency over time (warmer colors indicate higher power, with color scaled linearly between zero and maximum
value). Bottom: change of the total power over all gamma frequencies shown in the wavelet spectrum (30e60 Hz) normalized to the 5-min period before ATP application. (BeD)
Left: field recordings before and during the application of 300 mM ATP under certain conditions. Right: corresponding power spectra of oscillations induced by KA (plus purine
receptor antagonist where applicable, black) and following the application of ATP (300 mM, gray or colored, resp.). (B) Effect of ATP on KA-induced gamma oscillations. (C) Effect of
ATP on KA-induced oscillations in the presence of the adenosine A1/A2Areceptor antagonist CGS-15943 (10 mM) (D) Effect of ATP on KA-induced gamma oscillations in the presence
of the broad spectrum P2 receptor antagonist PPADS (30 mM). (E) Quantification of the experiments shown in BeD. Bars represent the change of the peak power of KA-induced
gamma oscillations normalized to the baseline directly before the wash-in of ATP (n ¼ 5e13). *p < 0.05 compared to time-matched controls. (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this article.)
S.B. Schulz et al. / Neuropharmacology 62 (2012) 914e924
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3.3. Acetylcholine-induced gamma oscillations are inhibited
by activation of P2 and adenosine receptors
To investigate whether ATP has an impact on cholinergically
induced gamma oscillations, we applied 10 mM ACh and 2 mM
physostigmine for 90 min in order to evoke persistent gamma
oscillations with a peak power of 2074 ? 972 mV2(n ¼ 8) and a peak
frequency of 38.6 ? 2.9 Hz. Application of ATP (300 mM) reduced
the peak power to 48.1 ?8.4% (n ¼ 4; p < 0.05 compared to control;
Fig. 3A, E), whereas the peak frequency was not changed
(96.7 ? 5.7% of control; p > 0.05; Fig. 3A). In contrast to KA-induced
gamma oscillations, CGS-15943 alone did not alter this oscillation
(peak power: 96.5 ? 9.4% of control, frequency: 104.6 ? 1.5% of
control, n ¼ 8, p > 0.05 each) but significantly inhibited the effect of
ATP (peak gamma power: 81.7 ? 5.6% of control, n ¼ 8; p < 0.05
compared to both ATP and control; Fig. 3C, E). Addition of PPADS to
the CGS-15943-containing bath solution, completely abolished the
inhibitory effect of ATP (106.8 ? 5.3% of control, n ¼ 8; p > 0.05
compared to control; Fig. 3E) indicating that in contrast to
KA-induced gamma oscillations, ACh-induced gamma oscillations
are modulated by both adenosine and P2 receptors.
The reduction of the ACh-induced gamma power by ATP was
found to be concentration-dependent. By plotting the measured
concentration during bath application of ATP against its inhibitory
effect, the half maximum effect (IC50) in the presence of CGS-15943
was 1.93 ? 0.16 mM (bath-applied: 733 ? 108 mM) (n ¼ 5e8) with
a Hill slope of 2.32 ? 0.38. In comparison, the IC50value and the Hill
slope for ATP alone were found to be 0.96 ? 0.51 mM (bath-applied:
312 ? 107 mM) and 1.06 ? 0.46 (n ¼ 4), respectively (Fig. 3D). These
data suggest that net ATP concentrations at the low micromolar
range are able to considerably affect gamma oscillations.
3.4. Acetylcholine-induced gamma oscillations are affected by
endogenously released ATP
To test if endogenously released ATP and the following activa-
tion of ATP receptors is also able to affect gamma oscillations we
applied the selective ecto-ATPase inhibitor ARL-67156 (50 mM) to
increase extracellular ATP levels in the slice (Bowser and Khakh,
2004). The peak power of the ACh-induced oscillations was
reduced to56.1 ?16.9% (n ¼ 3, p < 0.05 compared to control; Fig. 4),
suggesting that ATP released from the slice itself is able toattenuate
gamma oscillations. The peak frequency did not change signifi-
cantly after application of ARL-67156 (101.9 ? 2.7% of control;
p > 0.05).
Next we applied ATP at a low concentration (30 mM) in the
presence of ARL-67156 to investigate whether it is more effective in
the presence of the ATPase inhibitor. In addition to the inhibitory
effect of ARL-67156, ATP further reduced the gamma peak power to
38.6 ? 10.5% of control period prior to ATP application (n ¼ 6) and
much stronger than without ARL-67156 (to 87.4 ? 2.6% of control,
n ¼ 4, p < 0.05; Figs. 3 and 4D). These data indicate that bath-
applied ATP is rapidly degraded in the slice by ATPases reaching
lower concentrations and weaker effects on purinergic receptors.
Since endogenous ATP and the subsequent activation of P2
receptors are able to inhibit ACh-induced gamma oscillations, we
aimed to examine if ATP is released during gamma oscillations and
if the activated P2 receptors control their power. We applied the
broad spectrum P2 receptor antagonist PPADS (30 mM) during
ACh-induced gamma oscillations and found that the peak power
dramatically increased to 233.8 ? 48.2% after a 60-min wash in
period (n ¼ 6, p < 0.05 compared to control; Fig. 5A and C). As it was
the case with other manipulations, the peak frequency of the
oscillations was not affected by PPADS (96.2 ? 1.9% of control;
p > 0.05). We also found that inhibition of P2 receptors by PPADS
attenuated the decay of gamma oscillations after the washout of
ACh/Physo (Fig. 5Ac). The peak power of ACh-induced gamma
oscillations declined 30 min after the washout of ACh/Physo to
17.9 ? 3.6% (n ¼ 11). When PPADS was present in the bath, the
decay of the gamma oscillations was slower, the power was still
41.9 ? 7.2% after 30 min (n ¼ 5, p < 0.05), suggesting that P2
receptors may temporarily limit oscillation periods.
In comparison, KA-induced gamma oscillations were not
affected by PPADS (peak power: 106.6 ? 17.6% of control, n ¼ 8,
p > 0.05 compared to control; peak frequency: 90.9 ? 2.3% of
control, p > 0.05; Fig. 5B and C), confirming again that P2 receptors
are not involved in the modulation of kainate induced gamma
oscillations and that PPADS did not affect ACh-induced gamma
oscillations via a non-ATP receptor mediated mechanism. In
summary, these data suggest that ATP is present in the extracellular
spacewhen ACh is used toinduce gamma oscillations and limits the
power of gamma oscillations.
3.5. P2Y and P2X receptors differentially modulate ACh-induced
gamma oscillations
In the next series of experiments we aimed to examine which P2
receptor subtypes are responsible for the effect of ATP on
ACh-induced gamma oscillations. In the hippocampus, functional
P2Y1 and P2X receptors are expressed on interneurons and
Fig. 2. Bath-applied ATP reaches a much lower concentration in the hippocampal slice
than its actual concentration in the bath medium. (A) Recordings of ATP biosensor, null
biosensor and the calculated net ATP signal in CA3 stratum pyramidale after bath
application of different concentrations of ATP. (B) Concentration-response curve
for measured ATP concentration in the slice after application of ATP (geometric
mean ? s.e.m.).
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principal cells, respectively (Bowser and Khakh, 2004; Kawamura
et al., 2004; Khakh et al., 2003). Therefore, we first applied MRS
2179 (30 mM), a specific antagonist for P2Y1receptors (Gerevich
et al., 2004; Guzman et al., 2010). As seen on Fig. 6A, 30 mM MRS
2179 reduced the peak power of oscillations to 75.5 ?14.4% (n ¼ 5,
p < 0.05 compared to control). In contrast, MRS 2179 did not
influence the peak frequency of the oscillations (106.8 ? 2.0% of
control; p > 0.05). To explore whether additional P2Y receptors are
also able to modulate gamma oscillations we applied specific
antagonists for other P2Y subtypes (Fig. 6C). Neither MRS 2578
(5 mM; Mamedova et al., 2004; peak power: 91.6 ? 11.0% of control,
n ¼ 9), NF 340 (10 mM; Meis et al., 2010; 97.7 ? 10.8%, n ¼ 8) nor
MRS 2211 (30 mM, Ortega et al., 2011; 84.7 ? 6.1%, n ¼ 8), specific
antagonists for the P2Y6, P2Y11and P2Y13receptors, respectively,
affected the power of gamma oscillations (p > 0.05 each, compared
to control).
Next we investigated whether P2X receptors also have modu-
latory effects on gamma oscillations and applied TNP-ATP (10 mM),
a selective antagonist for P2X1, P2X2, P2X3 and P2X4 receptors
(Lorca et al., 2011). We found that TNP-ATP enhanced the peak
power of gamma oscillations to 236.7 ? 27.8% (n ¼ 7, p < 0.05
compared to control; Fig. 6B) and reduced the peak frequency of
the oscillations to 90.0 ? 3.5% (p < 0.05). In contrast to this, neither
the P2X1 selective antagonists NF 279 (1 mM; Rettinger et al., 2000;
91.4 ?12.0% of control, n ¼ 4) and NF 449 (10 mM; Hausmann et al.,
2006; 104.8 ? 3.6%, n ¼ 3), the P2X3 and P2X2/3 selective NF 110
(1 mM; Hausmann et al., 2006; 134.0 ? 26.4, n ¼ 8), the P2X3
selective RO-3 (10 mM; Geveret al., 2006; 83.6 ? 20.1, n ¼ 6) nor the
P2X7 selective A 740003 (10 mM; Honore et al., 2006; 105.8 ? 34.7,
n ¼ 6) changed significantly the power of gamma oscillations
(p > 0.05, compared to control; Fig. 6D). Together, these experi-
ments suggest that P2Y1receptors enhance ACh-induced gamma
oscillations whereas P2X2 or P2X4 receptors inhibit it.
3.6. P2 and adenosine receptors differentially alter spiking rate
and spike timing of CA3 pyramidal cells
Changes in FP power can be due to alterations in the firing rate
of neurons or in the synchrony of action potentials between cells. In
order to get some insight into the underlying mechanisms, we
recorded intracellularly from CA3b pyramidal cells during gamma
oscillations induced either by KA or ACh/Physo. All recorded cells
Fig. 3. Acetylcholine-(ACh)-induced gamma oscillations in the CA3 area of the hippocampus are inhibited by both adenosine and ATP receptors. (A) Gamma oscillations induced by
bath application of 10 mM ACh and 2 mM physostigmine, recorded extracellularly in the CA3 pyramidal cell layer from a rat hippocampal slice. For further explanation see the legend
of Fig.1. (B) Top: field recording before, during and after the application of ATP (300 mM) in the presence of CGS-15943 (10 mM) to block A1and A2Aadenosine receptors. The wavelet
spectra underneath show the oscillation frequency over time for 1-s periods before (a) and during (b) the application of ATP. Warmer colors indicate higher power, with color scaled
linearly between zero and the overall maximum value of both spectra. (C) Comparison of the oscillation properties induced by ACh/Physo in the presence of CGS-15943 (10 mM,
black line) and following application of 300 mM ATP (gray line) using power spectra of representative time windows. (D) Concentration-response curve for inhibition of ACh-induced
gamma oscillations by ATP in the absence (n ¼ 4, open circles) and presence of CGS-15943 (10 mM; n ¼ 5e8, gray circles). The x-axis represents local ATP concentrations in str. pyr.
measured after the application of ATP with the sensors as shown in Fig. 2 (geometric means ? s.e.m.). (E) Bars represent the change of the peak power of ACh-induced gamma
oscillations after application of ATP (300 mM) in the absence (open, n ¼ 8) and presence of the antagonists (gray, n ¼ 4e8) in comparison to time-matched controls (black, n ¼ 8).
*p < 0.05 compared to time-matched controls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Endogenously released ATP inhibits gamma oscillations induced by ACh in the CA3 area of the hippocampus. (A) Field recordings after induction of gamma oscillations by
ACh before (top) and during (bottom) the application of the selective ecto-ATPase inhibitor ARL-67156 (50 mM) to increase extracellular ATP levels. (B) Power spectrum of
ACh-induced gamma oscillations from the experiment shown in A before (black) and after (gray) the application of ARL-67156. (C) Bars represent the change of the peak power of
ACh-induced gamma oscillations after application of ARL-67156 (gray, n ¼ 3) compared to the time-matched controls (black, n ¼ 8). (D) Subsequent application of ATP (30 mM)
further reduces the peak power of gamma oscillations (gray bar, normalized to control period prior to ATP application, n ¼ 6) with higher effectiveness than ATP alone (black, n ¼ 4).
*p < 0.05.
Fig. 5. P2 receptors modulate ACh- but not KA-induced gamma oscillations. (A) ACh-induced gamma oscillations are enhanced by the application of PPADS. (Aa) Field recordings
before (top) and during the application of PPADS (30 mM; bottom). (Ab) Power spectrum of ACh-induced gamma oscillations from the experiment shown in Aa before (black) and
after (gray) the application of PPADS. (Ac) Inhibition of P2 receptors by PPADS attenuates the decay of gamma oscillations after the washout of ACh. Normalized peak power of
gamma oscillations over time after washout of ACh in the presence (black circles, n ¼ 5) and absence (open circles, n ¼ 11) of PPADS. (B) KA-induced gamma oscillations are not
affected by the application of the broad-spectrum P2 receptor antagonist PPADS. (Ba) Field recordings before (top) and during the application of PPADS (30 mM; bottom). (Bb) Power
spectrum of KA-induced gamma oscillations from the experiment shown in Ba before (black) and after (gray) the application of PPADS. (C) Bars represent the change of the peak
power of ACh-induced (black, n ¼ 8) and of KA-induced (gray, n ¼ 8) gamma oscillations after application of PPADS (30 mM).
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(n ¼ 23) in all tested drug conditions as well as the cells as pop-
ulations showed firing behavior phase-locked to the FP of the
gamma oscillation (Rayleigh test, p < 0.001 for each cell and
condition and Moore’s test, p < 0.005 for each population and
condition, respectively, Figs. 7A and 8C).
Analysis of circular data (as is the case here due to the FP
oscillation) provides two key parameters: i) the mean phase F,
calculated by averaging the phases of all individual APs of a cell,
states the phase within an oscillation cycle when the average AP
occurs; and ii) the mean vector length r, which indicates how
accurate the neuron fires within the cycle (in a case of r ¼ 1, the cell
would fire all APs at the very same phase with maximal accuracy; if
all APs were equally distributed over the oscillation cycle, vector
length would be r ¼ 0). Given that the same number of APs for each
cell is used, F and r of a cell population can be obtained by second
order statistics. These parameters are compared here between
different pharmacological conditionsbyappropriatestatistical tests
(Batschelet, 1981; Zar, 2010).
Phase analysis of APs related to the gamma cycle (trough ¼ 0?)
during KA-induced gamma oscillations revealed a mean phase F
of 22.8?and a vector length r ¼ 0.863 (n ¼ 9) after low-pass
filtering (see Section 2.6). Since P2 receptor activation didn’t show
any effect on KA-induced gamma oscillations, to activate adeno-
sine receptors we applied ATP as the physiological source of
adenosine. Application of 300 mM ATP significantly altered spike
timing (F ¼ 21.0?, r ¼ 0.774, Hotelling test for paired samples,
p < 0.05, Fig. 7A) by reducing the accuracy of spiking (r; paired
t-test, p < 0.01, Fig. 7A, C) whereas the preferred phase (F) was not
affected (p > 0.05). In addition, the spike rate significantly
decreased in response to ATP (ATP: 5.58 ? 0.77 s?1, control:
8.31 ? 0.88 s?1, p < 0.05), an effect that was completely reversed
after ATP was washed out (7.70 ? 0.82 s?1, p > 0.05 compared to
control, Fig. 7B). We found weak correlations between the
reduction in FP power by ATP and the reduction in the spike rate
and accuracy of spiking among cells, respectively (r ¼ 0.626,
p < 0.07 and r ¼ ?0.670, p < 0.048, resp.; Fig. 7D, E). Thus, these
data suggest that ATP inhibits KA-induced gamma oscillations via
activation of adenosine receptors after its degradation to adeno-
sine by means of reducing spiking rate and synchrony of action
potentials of CA3 pyramidal neurons.
Fig. 6. P2Y1receptors potentiate and P2X1e4 receptors inhibit ACh-induced gamma oscillations in the CA3 area of rat hippocampal slices. (A) Selective inhibition of P2Y1receptors
by MRS 2179 inhibits the power of ACh-induced gamma oscillations. (Aa) Field recordings before (top) and during the application of MRS 2179 (30 mM; bottom). (Ab) Properties of
the oscillations induced by ACh and physostigmine (black) and following application of MRS 2179 (gray) compared by using power spectra of representative time windows.
(B) Inhibition of the P2X1-4 receptors by TNP-ATP enhances the power of ACh-induced gamma oscillations. (Ba) Field recordings before (top) and during the application of TNP-ATP
(10 mM; bottom). (Bb) Properties of the ACh-induced oscillations before (black) and following application of TNP-ATP (gray) compared by using power spectra of representative time
windows. (C, D) Bars show normalized peak power of ACh-induced gamma oscillations after application of P2Y (C, gray) and P2X (D, black) receptor antagonists (n ¼ 3e9) selective
for the indicated receptor subunits.*p < 0.05 compared to time-matched controls.
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Fig. 7. ATP reduces the spiking rate and alters spike timing of CA3 pyramidal cells during KA-induced gamma oscillation. (A) From top to bottom: intracellular recording from a CA3
pyramidal cell and the corresponding field recording of CA3 str. pyr. after induction of gamma oscillations by KA; waveform average of the corresponding oscillatory FP cycles and
phase histograms of action potentials (APs) of the shown cell (bin size: 10?); mean vectors of each recorded cell (gray arrows, n ¼ 9) and the mean vector ? circular standard
deviation of the whole population (black, bold arrow). For each cell, 1000 APs were analysed before (left column) and during ATP application (300 mM, right column). 0?represents
the troughs of the gamma cycles after low-pass filtering. Arrow lengths represent mean vector lengths r (circle radius ¼ 1). (B) Normalized peak power of KA-induced gamma
oscillations (upper trace) and spike rate of the corresponding recordings from CA3 pyramidal cells (lower trace) over time before, during and after the application of 300 mM ATP.
Bars summarize the effect of ATP on the spike rate of 10 min periods before (black), during (gray) and after (white) ATP application. (C) Changes in mean vector length r of each
recorded cell after ATP application (300 mM) (D) Scatter plot showing the reduction in mean vector length r and FP peak power during ATP application normalized to the phase
before application for each cell-FP recording pair. (E) Scatter plot showing spike rate and FP peak power during ATP application relative to the phase before application for each
cell-FP recording pair. *p < 0.05 compared to control period.
Fig. 8. Blockade of P2 receptors increased the spiking rate but did not affect spike timing of CA3 pyramidal cells during ACh-induced gamma oscillation. (A) Intracellular recording
from a CA3 pyramidal cell and the corresponding field recording in CA3 str. pyr. (B) Bars summarize the effects of PPADS (30 mM) and CGS-15943 (10 mM) on the firing rate. (C) Mean
vectors of each recorded cell (gray arrows) and the mean vector ? circular standard deviation of the whole population (black, bold arrow). 1000 APs are analyzed in the absence
(control) and presence of drugs. 0?represents the troughs of the gamma cycles after low-pass filtering. Arrow lengths represent mean vector lengths r (circle radius ¼ 1). *p < 0.05
compared to control period.
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During ACh-induced gamma oscillations, CA3 pyramidal cells
fired at a mean phase of F ¼ 26.1?(r ¼ 0.741, n ¼ 7) after low-pass
filtering (see Section 2.6), which was not altered after blockade of
P2 and adenosine receptors by PPADS and CGS-15943, respectively
(PPADS: F ¼ 24.9, r ¼ 0.680, CGS-15943: F ¼ 15.9?, r ¼ 0.823; n ¼ 7;
Hotelling test for paired samples and WatsoneWilliams test, resp.,
p > 0.05, Fig. 8C). However, while the blockade of adenosine
receptors by CGS-15943 did not alter the firing rate of the neurons
(5.32 ? 1.06 s?1; p > 0.05, compared to control: 6.04 ? 1.71 s?1),
PPADS significantly increased the firing rate compared to the
preceding control phase (PPADS: 7.85 ? 2.09 s?1, p < 0.05, Fig. 8B).
These data suggest that activation of ATP receptors by endogenous
ATP inhibits ACh-induced gamma oscillations byreducing the firing
rate in CA3 pyramidal neurons and not by changing the synchrony
of action potentials.
4. Discussion
The main finding of our data is that extracellular ATP modulates
the power of both KA- and ACh-induced hippocampal gamma
oscillations. While ATP reduces the power of KA-induced gamma
oscillations exclusively byactivation of adenosine receptors after its
degradation, we show here for the first time that ACh-induced
gamma oscillations are inhibited by both adenosine and ATP
sensitive P2 receptors. Under our experimental conditions, the
inhibitory effect of ATP comprises an adenosine receptor compo-
nent of w55% and a P2 receptor component of w45% (Fig. 3D, E).
4.1. Effect of P2Y and P2X receptors on gamma oscillations
Analyzing the P2 receptor component of the inhibition in more
details we found two opposing effects: activation of P2Y1receptors
by ATP enhanced gamma oscillations whereas P2X receptors
reduced them. Among the P2Y receptor antagonists only MRS 2179,
a selective inhibitorof P2Y1receptors, was able tomodulate gamma
oscillations. These receptors are functionally expressed on CA3
interneurons but not on pyramidal cells (Bowser and Khakh, 2004;
Kawamura et al., 2004). Their stimulation may increase the
precisely timed GABA release from perisomatic-targeting inter-
neurons which may lead to increased synchronization of pyramidal
cell firing and subsequently to enhanced oscillation power (Mann
and Paulsen, 2007).
Among the P2X receptors, P2X2, P2X4 and P2X6 receptors are
known to be expressed in the hippocampus (Kanjhan et al., 1999;
Rubio and Soto, 2001). We found that only TNP-ATP but not other
applied P2X receptor antagonists affected gamma oscillations.
TNP-ATP blocks P2X1, P2X2, P2X3 and at a less extent P2X4
receptors (Jarvis and Khakh, 2009). Since antagonists of P2X1 and
P2X3 receptors did not modulate gamma oscillations, it can be
suggested that P2X2 and/or P2X4 receptors are the predominant
P2X subunits involved in the suppression of gamma oscillations.
Further studies e.g. with KO mice are needed to prove the exact P2X
receptor subtype due to the lack of more specific antagonists. P2X
channels seem to be restricted to the axons of CA3 pyramidal cells
(Khakh et al., 2003; Khakh, 2009; Rodrigues et al., 2005). Ionotropic
receptors expressed on axon terminals (Schicker et al., 2008) or
along the axons of CA3 pyramidal cells (Sasaki et al., 2011) can
modify network activity by altering synaptic transmission or AP
conduction. P2X receptors seem to have similar effects (Khakh
et al., 2003; Rodrigues et al., 2005). Since generation of ectopic
spikes in CA3 pyramidal cell axons may be essential for the
development of gamma oscillations (Traub et al., 2000, 2004), their
inhibition by P2X receptors can explain the inhibitory effect of P2X
receptors on gamma oscillations. Support for this hypothesis are
our findings showing that a blockade of P2X channels increases the
firing rate of pyramidal cells, possibly due to higher phasic excit-
atory input via the recurrent collaterals in the CA3 region.
4.2. Effect of adenosine receptors on gamma oscillations
Ithas beenpreviously shownthatadenosine inhibits
KA-induced and spontaneous gamma oscillations, particularly via
the activation of A1receptors (Pietersen et al., 2009). Herewefound
that both KA- and ACh-induced gamma oscillations were inhibited
by adenosine receptors. Hippocampal adenosine A1receptors are
known to reduce transmitter release and excitability of pyramidal
cells and interneurons (Li and Henry, 2000; Schubert et al., 1986).
Moreover, axonal A1 receptors on CA3 pyramidal cells were
described to decrease AP conduction and synaptic efficacy (Sasaki
et al., 2011). These effects may account for the inhibition of
gamma oscillations by adenosine and are in accordance with
our results showing that adenosine receptor activation during
KA-induced gamma oscillations reduced both the spiking rate of
CA3 pyramidal cells and the synchrony of action potentials in these
neurons. Selective activation of A2A receptors enhances gamma
oscillations (Pietersen et al., 2009). However, the expression of
these receptors (Fredholm et al., 2005) and their affinity for aden-
osine are lower (Ciruela et al., 2006) making A1receptors to be the
functionally dominating adenosine receptor subtype in the
hippocampus.
4.3. Extracellular ATP levels
We found that ATP concentrations reach w300 times lower
levels in the slice after bath application. This has been observed also
byothers (Frenguelli et al., 2007) and maybe explained bythe rapid
degradation of ATP. The equilibration time course of ATP and the
development of its effects were found to be much faster than that
observed for other drugs in interface chambers (Decker et al., 2009;
Wójtowicz et al., 2009). An explanation might be that ATP induces
waves of astrocyte activation mediated by P2Y receptors and
subsequent ATP release from astrocytes resulting in a rapid equil-
ibration of the slice but at much lower ATP concentrations (Bowser
and Khakh, 2004; Guthrie et al., 1999).
Our results indicate that the strength of cholinergically induced
gamma oscillations is also controlled by ambient extracellular ATP
since both blocking the degradation of ATP to adenosine and
application of purinergic antagonists alone were able to modulate
gamma oscillations. Thus, endogenous ATP at concentrations in the
range of a few hundred nanomolar to few micromolar can finely tilt
the balance between excitation and inhibition in the hippocampal
network via the activation of P2Y, P2X and adenosine receptors.
Since the net impact of exogenously applied ATP is an inhibition of
the gamma oscillations, the inhibitory effect of P2X and adenosine
receptors seems to dominate this effect.
4.4. Modulation of KA-induced and cholinergic gamma oscillations
by neuromodulators
A number of neurotransmitters and neuropeptides have been
shown to modulate gamma oscillations in the hippocampus such as
dopamine (Weiss et al., 2003; Wójtowicz et al., 2009), 5-HT (Krause
and Jia, 2005; Wójtowicz et al., 2009), noradrenaline (Hajós et al.,
2003; Wójtowicz et al., 2009), cannabinoids (Hájos et al., 2000),
C-type natriuretic peptide (Decker et al., 2009), histamine (Andersson
et al., 2010) and the aforementioned adenosine (Pietersen et al.,
2009). Among them, monoamines such as dopamine and 5-HT
seem to inhibit both ACh- and KA-induced gamma oscillations
(Weiss et al., 2003; Wójtowicz et al., 2009), whereas histamine was
found to decrease only KA- but not carbachol-induced gamma
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oscillations in the hippocampus (Andersson et al., 2010). The selective
modulatory effect of P2 receptors for ACh-induced gamma oscilla-
tions presented in our study further confirms that cholinergic and
KA-evoked gamma oscillations involve different neuronal pathways,
can be differentially modulated and may have distinct physiological
functions. Besides gamma oscillations,activation of KARs induces also
epileptiform bursts and kainate application has long been used as an
animal model for epileptogenesis (Ben-Ari and Cossart, 2000; Nadler,
1981). Our findings that ATP and its receptors do not modulate
KA-induced network activity are in line with reports about the
missing or very limited effects of P2 receptors on epileptiform activity
(Lopatá? r et al., 2011; Ross et al., 1998).
4.5. Functional relevance
We preferred using an interface-type recording chamber to
submerged conditions because gamma oscillations require a high
oxygen supply (Huchzermeyer et al., 2008) guaranteed only in an
interface-type chamber. Hypoxia causes gamma oscillations to
collapse within minutes (Fano et al., 2007; Huchzermeyer et al.,
2008) associated with elevated extracellular ATP levels (Dale and
Frenguelli, 2009; Frenguelli et al., 2007). It can be suggested that
the hypoxia-induced collapse of gamma oscillations is caused by
the increased ATP levels and the subsequent inhibition of gamma
oscillations by activation of P2X and adenosine receptors. This
would bean energy conserving
consumption seems to be highest during gamma network activity
(Huchzermeyer et al., 2008).
ATP is, among glutamate and D-serine, one of the most impor-
tant gliotransmitters (Haas et al., 2006; Halassa et al., 2009) known
to be released from astrocytes enabling these cells to modulate
neuronal processes such as synaptic transmission (Pascual et al.,
2005). Our findings suggest that astrocytes, by releasing ATP and
the subsequent activation of purinergic receptors, may also be able
to control neuronal gamma oscillations.
The power and the synchrony of gamma oscillations have been
found to be reduced in schizophrenia and the degree of this
reduction seems to correlate with the severity of negative symp-
toms (Lee et al., 2003). Thus, drugs which selectively activate fast
spiking interneurons could provide an effective approach in
schizophrenia to increase the synchronization of pyramidal cell
firing at gamma frequencies and consequently improving the
symptoms of the disease (Gandal et al., 2011). Our results suggest
that the selective activation of P2Y1receptors or antagonism of P2X
receptors may provide a possible approach to reverse the loss of
gamma power and the subsequent cognitive impairments in
schizophrenia.
mechanismsince oxygen
Acknowledgements
ThisworkwassupportedbytheDeutscheForschungsgemeinschaft
(He1173/17-1)andbytheHertiefoundation.S.B.S.wasastipendiaryof
NeuroCure (DFG Exc 257) and Z.-J.K. was supported by the Gra-
duiertenkolleg 1123 (Learning & Memory). The authors thank Dr.
Arpad Mike for helpful discussion.
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