JOURNAL OF NEUROCHEMISTRY

| 2014

doi: 10.1111/jnc.12693

, ,

*Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany †Bernstein Center for Computational Neuroscience (BCCN) Heidelberg/Mannheim, Heidelberg, Germany

Abstract The cholinergic system is critically involved in the modulation of cognitive functions, including learning and memory. Acetylcholine acts through muscarinic (mAChRs) and nicotinic receptors (nAChRs), which are both abundantly expressed in the hippocampus. Previous evidence indicates that choline, the precursor and degradation product of Acetylcholine, can itself activate nAChRs and thereby affects intrinsic and synaptic neuronal functions. Here, we asked whether the cellular actions of choline directly affect hippocampal network activity. Using mouse hippocampal slices we found that choline efficiently suppresses spontaneously occurring sharp wave–ripple complexes (SPW-R) and can induce gamma oscillations. In addition, choline reduces synaptic transmission

between hippocampal subfields CA3 and CA1. Surprisingly, these effects are mediated by activation of both mAChRs and a7-containing nAChRs. Most nicotinic effects became only apparent after local, fast application of choline, indicating rapid desensitization kinetics of nAChRs. Effects were still present following block of choline uptake and are, therefore, likely because of direct actions of choline at the respective receptors. Together, choline turns out to be a potent regulator of patterned network activity within the hippocampus. These actions may be of importance for understanding state transitions in normal and pathologically altered neuronal networks. Keywords: choline, hippocampal slice, muscarinic, nicotinic, sharp wave–ripple complexes, synaptic transmission. J. Neurochem. (2014) 10.1111/jnc.12693

Local networks within the hippocampal formation express a variety of state-dependent network oscillations which are believed to organize neuronal activity and synaptic plasticity during memory formation. During active wakefulness neurons are entrained by theta (4–10 Hz) and gamma (30–80 Hz) oscillations (O’Keefe 1993; Bragin et al. 1995), while phases of quiet wakefulness and slow-wave sleep are characterized by propagating sharp waves, which are superimposed by high-frequency ripples at ~200 Hz (Buzs
aki 1989; Buzs
aki et al. 1992; Chrobak and Buzs
aki 1996). Coherent discharges of place-encoding neurons during sharp wave–ripple complexes (SPW-R) have been suggested to define memory-forming neuronal assemblies (Buzs
aki 1989, 2006; Buzs
aki and Silva 2012). Consequently, propagation of SPW-R along the hippocampal output loop (Chrobak et al. 2000) may support memory consolidation in the neocortex (Buzs
aki 1986; Ji and Wilson 2007). This widely accepted framework of assembly formation and memory consolidation requires switches between

well-defined functional states of the network (Buzs
aki and Draguhn 2004) and, at the cellular level, state-dependent modulation of synaptic plasticity (Huerta and Lisman 1996; Hasselmo 2005). The cholinergic system is crucially involved in these processes, in line with the important role of this transmitter in cognitive processes including learning Received November 26, 2013; revised manuscript received February 14, 2014; accepted February 18, 2014. Address correspondence and reprint requests to Dr. Alexei V. Egorov, Institute of Physiology and Pathophysiology, Heidelberg University, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany. E-mail: [email protected] Abbreviations used: ACh, acetylcholine; ACSF, artificial CSF; BPF, bandpass filtered; CSF, cerebrospinal fluid; fEPSPs, field excitatory postsynaptic potentials; GABA, c-Aminobutyric acid; HC-3, hemicholinium-3; LFP, local field potentials; mAChRs, muscarinic acetylcholine receptors; MEC, mecamylamine; MLA, methyllycaconitine; MTL, the medial temporal lobe; nAChRs, nicotinic acetylcholine receptors; NMDA, N-methyl-d-aspartate; SLM, stratum lacunosum-moleculare; SP, stratum pyramidale; SPW-R, sharp wave–ripple complexes; SR, stratum radiatum.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

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and memory (Hasselmo and Giocomo 2006). Acetylcholine (ACh) exerts its effects via activation of metabotropic muscarinic and ionotropic nicotinic receptors, which are both abundantly expressed in the hippocampus (Drever et al. 2011). ACh has profound effects on intrinsic firing properties of neurons, synaptic transmission, plasticity, and oscillatory network activity (Hasselmo 2006; Drever et al. 2011). Despite its heterogeneous actions at different receptors, cell types, and pre- or post-synaptic locations, the overall effects of ACh support a facilitating role for memory formation in hippocampal networks. Acetylcholine is synthesized in the pre-synaptic compartment and, following release, degraded into choline and acetate by cholinesterase in the extracellular space. Subsequently, choline is transported back into the terminal for further synthesis of ACh (Sarter and Parikh 2005). Several lines of evidence suggest that concentrations of choline are normally subsaturating, such that cholinergic transmission can be modulated by increasing levels of the precursor. Indeed, choline affects intrinsic properties of neurons and hippocampal synaptic transmission (Alkondon et al. 1997; Alkondon and Albuquerque 2001; Mielke et al. 2011). It is likely that direct effects of choline include actions at cholinergic receptors. Choline selectively activates a7containing nicotinic acetylcholine receptors (nAChRs) in Xenopus oocytes (Papke et al. 1996) and hippocampal neurons (Alkondon et al. 1997, 1999). This effect underlies choline-mediated modulation of inhibition in hippocampal networks, which shows preference for interneurons in stratum lacunosum-moleculare (SLM; Alkondon and Albuquerque 2001). Importantly, evoked field excitatory postsynaptic potentials (fEPSPs) are suppressed by choline following the facilitation of inhibition via nicotinic receptors (Mielke et al. 2011). It is likely that the extracellular space contains relevant levels of choline. Low micromolar concentrations have been found in cerebrospinal fluid (CSF; Klein et al. 1992), but local concentrations following synaptic release may be significantly higher. Moreover, elevated choline levels in the brain have been reported in various pathological conditions, including traumatic brain injury, ischemic strokes, and brain tumors (Kinoshita and Yokota 1997; Friedman et al. 1998; Karaszewski et al. 2010). We therefore asked whether the cellular actions of choline directly affect hippocampal network states. Using an in vitro model of sharp wave–ripple oscillations in mouse hippocampal slices we found that choline is able to suppress this ‘non-cholinergic’ network state, to induce gamma oscillations, and to reduce synaptic transmission between hippocampal subfields. The effects of choline are mediated by direct activation of cholinergic pathways and involve both muscarinic and nicotinic receptors. Together, choline turns out to be a potent regulator of patterned activity within hippocampal networks.

Materials and methods Preparation of mouse brain slices Horizontal brain slices (450 lm thick) were obtained from male C57BL6 mice aged 4–8 weeks using standard techniques (B€ahner et al. 2011). Mice were purchased from Charles River Laboratories (Sulzfeld, Germany). All experimental protocols were conducted in compliance with German law and with the approval of the state government of Baden-W€ urttemberg. Mice were killed under deep CO2-induced anesthesia. After decapitation, brains were rapidly removed and placed in a cold (1–4°C) oxygenated artificial CSF (ACSF) containing (in mM): 124 NaCl, 3 KCl, 1.8 MgSO4, 1.6 CaCl2, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, saturated with 95% O2, and 5% CO2 (pH 7.4 at 37°C; no buffer correction for temperature effects). Brain slices were cut using a Leica Vibratome (VT1000S, Nussloch, Germany), then transferred into a recording Haas-type interface chamber, and superfused with same oxygenated ACSF solution at a rate of 1.5–2 mL/min, and maintained at 34  1°C. Prior to electrophysiological recordings, slices were allowed to recover for at least 2 h. Electrophysiological recordings Local field potentials (LFP) were amplified 100x with an EXT 102F amplifier (npi electronics, Tamm, Germany). Signals were lowpass filtered at 2 kHz and high-pass filtered at 0.3 Hz, digitized at 20 kHz with an analog-to-digital converter [Cambridge Electronic Design MICRO 1401 mkll, Cambridge, UK] and saved on a computer using Spike2 software (Cambridge Electronic Design, Cambridge, UK) for off-line analysis. LFP recordings were obtained with ACSF filled borosilicate glass electrodes (tip diameter of 3–5 lm). After recovery, acute hippocampal slices displayed spontaneously occurring SPW-R (Maier et al. 2003), which could be monitored in the LFP up to ~10 h. For each experiment, baseline activity was recorded for at least 10 min prior to drug application. Bath applications of substances were performed for at least 30 min. Local applications were performed via glass pipettes (see below). Electrically evoked field potential responses were generated by square voltage pulses (0.1 ms) from an isolated stimulator through a bipolar platinum/iridium electrode (75 lm tip separation, impedance 0.1 MO, MicroProbes, Gaithersburg, MD, USA). Pulses were delivered every 60 s with an individually calibrated strength, evoking 50% of the maximal fEPSP amplitude in stratum radiatum (SR). To prevent the simultaneous occurrence of spontaneous network events (SPW-R) and evoked fEPSPs, pulses were emitted 200 ms after SPW-R events, which were detected by threshold algorithm in Spike2. Field EPSPs in CA1 were elicited via stimulation of the Schaffer collaterals, and fEPSPs in CA3 were elicited by stimulation of commissural/associational fibers. Slices were not further analyzed if the fEPSP amplitude was not stable during the control recording period (< 10% of slices with systematic drift or deviations of > 10%). Parts of stimulation artifacts were eliminated in the represented traces. Chemicals Choline (0.05–8 mM), atropine (10 lM), pirenzepine (1 lM), hemicholinium-3 (HC-3, 100 lM), and methyllycaconitine (MLA, 1 lM) were obtained from Sigma-Aldrich (Taufkirchen, Germany). VU 0255035 (5 lM) and mecamylamine (MEC, 10 lM) were ordered from Tocris (Biozol, Eching, Germany). These drugs were

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

Choline modulates sharp wave–ripples

bath applied by continuous perfusion. Choline was dissolved freshly in ACSF on each experimental day. VU 0255035 was applied from stock solutions made in dimethylsulfoxide (DMSO). The final concentration of DMSO in ACSF was 0.025%. Control experiments revealed no measurable effects of DMSO on SPW-R (n = 4). Other drugs were applied at the desired concentrations from stock solutions in distilled water. In some experiments choline (8 mM) and nicotine (300 lM, Sigma-Aldrich, Taufkirchen, Germany) were applied locally using glass electrodes (tip diameter ~10–15 lm). Local application electrodes were gently placed on the surface of the slices in SR and removed immediately after visible delivery of the substance. Control experiments revealed that local application of ACSF induced no measurable changes in network activity (n = 4). Previous studies from our group showed that this method is appropriate to apply drugs in a restricted area of hippocampal slices (~300 lm in diameter; see B€ahner et al. 2011). Data analysis Data were analyzed off-line using custom-written routines in Matlab (MathWorks) or Spike2 software (Cambridge Electronic Design, Cambridge, UK). Sharp waves were detected from low-pass filtered raw data (20 or 60 Hz) by identification of local maxima amplitude ≥ 0.01 mV within 30 ms time windows. A corner frequency of 20 Hz was chosen to exclude potential contamination with gamma oscillations. The threshold exceeds baseline noise by four standard deviations, yielding reliable detection of SPW-R, as confirmed by visual inspection of traces. High-frequency ripple oscillations were analyzed with continuous wavelet transform (complex Morlet wavelet), starting 33 ms before and ending 67 ms after the peak of each SPW-R (Both et al. 2008). From the resulting wavelet spectrogram, we extracted the leading ripple frequency (which is the most prominent frequency > 140 Hz) and the ripple energy [defined as the integral of the spectrogram at this particular frequency with borders at two times the standard deviation (SD) of event-free baseline noise]. The numbers of ripple cycles, represented by the number of ripple troughs that occur during a SPW-R event, were detected by band-pass filtering at 150–300 Hz. The limits for this computation were set to three times the SD of event-free baseline. Sharp waves were classified as ‘sharp waves with ripples’ if at least 3 ripple cycles were present during the event. Under control conditions, the vast majority of sharp waves were indeed superimposed by at least 3 ripple cycles (median 95%, P25 = 89.5%, and P75 = 98%; n = 68 slices). Amplitude of fEPSPs was calculated as the difference between baseline and peak of the field potential transient. Slopes of the initial phase of the fEPSPs were determined by fitting a linear function between two manually set borders within the early descending part of the fEPSPs which could be clearly discerned from the earlier negative peak of the afferent fiber volley. Bar diagrams of electrophysiological parameters represent data from 10 min of recording for SPW-R and from at least 3 min of recording for fEPSPs. To visualize the full time course of experiments, parameters were normalized to control conditions and plotted over time. SPW-R data were binned for 2.5 min intervals, whereas the fEPSP values were presented for each point of stimulation. Statistical analysis Quantitative data from multiple slices are given as mean  SEM or as median and the first and third quartiles (P25 and P75). Underlying

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parameters within individual slices were calculated as mean or median, respectively, depending on normality. Statistical analysis was performed using Matlab (MathWorks) or GraphPad (InStat, San Diego, CA, USA) software. For statistical evaluation of drug effects, baseline values were compared to the latest phase of the interval with drugs present (at least 30 min, where steady-state effects were reached). These values were compared by a one-way repeatedmeasures ANOVA followed by appropriate post hoc tests, depending on parametric or non-parametric data distribution. For local drug applications, the maximum of the transient effect was directly compared to baseline values by paired two-tailed Student’s t-test or Wilcoxon’s matched-pairs signed-ranks test. Effects of different choline concentrations on SPW-R parameters (concentration– response curves) were measured by perfusing slices with stepwise increasing concentrations of the agent. To control for timedependent changes in activity we compared effects of each concentration to corresponding values from control slices which had been perfused with ACSF for the same duration. Relative changes between both groups of slices (choline vs. ACSF) were compared using unpaired two-tailed Student’s t-test or Mann– Whitney test. A p < 0.05 was regarded as significant. For all data: *p < 0.05, **p < 0.01, ***p < 0.001, NS, not significant.

Results Choline suppresses SPW-R activity in CA1 To assess the effects of choline on hippocampal network activity, we recorded local field potentials from the pyramidal layer of mouse hippocampal slices. Spontaneous network activity presented as propagating sharp waves, which were generated in CA3 and were superimposed by high-frequency oscillations (Maier et al. 2003). In CA1, these sharp wave– ripple complexes (SPW-R) occurred at 2.7  0.1 Hz (n = 105) and had a mean amplitude of 0.26  0.01 mV (n = 102). Superimposed ripples had a leading frequency of 264 Hz (median, P25 = 246 Hz and P75 = 283 Hz; n = 101). The number of ripple cycles per sharp wave was 5 (median, P25 = 5 and P75 = 6; n = 75) and ripple energy was 0.4  0.02 lV*ms (n = 94). Following stable baseline recordings for at least 10 min, slices were superfused with 2 mM choline. Choline strongly suppressed SPW-R activity (Fig. 1a and c), reducing the frequency of SPW-R in CA1 to 7.6  3.1% of baseline values (n = 11, p < 0.001, ANOVA, Dunn’s post hoc). Further waveform parameters were only assessed for 8/11 slices, omitting three slices with complete block of SPW-R activity. In the remaining experiments, amplitude of SPW-R was reduced to 67.6  5.4% of control (n = 8, p < 0.05, ANOVA, Student–Newman–Keuls post hoc). The number of ripple cycles per sharp wave was 4.42  0.4 (n = 7) under control conditions and decreased to 2.85  0.5 in the presence of choline (64.5  10%; p < 0.05, ANOVA, Bonferroni’s post hoc). The leading ripple frequency reduced to 219  14.1 Hz (82.6  5.3% of control values, n = 7, p < 0.05, ANOVA, Bonferroni’s post hoc). In some slices, bath application of choline was

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Fig. 1 Bath-applied choline suppresses sharp wave–ripple complexes (SPW-R) and fEPSPs in CA1 by activation of muscarinic receptors. (a) Spontaneously occurring SPW-R recorded in CA1 stratum pyramidale (SP) under artificial CSF (ACSF), in the presence of choline (2 mM) and after adding atropine (10 lM). Events marked by horizontal bars are shown below at an expanded time scale in unfiltered (middle) and 100–500 Hz band pass-filtered (BPF; bottom) form. (b) Gamma oscillatory activity in CA1 SP induced by 5 mM choline. A part of the trace (noted by horizontal bar) is shown below at an expanded time scale. Power spectrum of the respective gamma activity indicated on the left. (c) Normalized time course of choline-induced reduction in SPW-R frequency (top), amplitude (middle), and number of ripple cycles per sharp wave (bottom). Note reversal of effects by atropine. (d) Representative fEPSPs in CA1 stratum radiatum (SR) evoked by stimulation of the Schaffer collaterals (top; indicated by arrowhead). Bar diagrams show mean fEPSP amplitude (middle) and slope (bottom) under control conditions (ACSF), in the presence of 2 mM choline and after adding atropine (10 lM). Error bars indicate SEM. ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

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followed by continuous oscillatory activity in the gamma frequency range (Fig. 1b). Such slices were excluded from further analysis. Effects of bath-applied choline were reversible after washout of the drug (Fig. 1b; tested in nine slices). Moreover, the effects of choline on hippocampal SPW-R were highly sensitive to the muscarinic acetylcholine receptor (mAChR) antagonist atropine (10 lM). Thus, SPWR frequency in CA1 recovered from 7.6  3.1% to 72.6  13% of baseline values after adding atropine to the perfusion solution (n = 11, p < 0.05). The apparent remaining difference toward baseline value (100%) was not significant, and the slightly lower numerical value may be because of some rundown of activity during long-term recordings. Amplitude of SPW-R recovered completely to 99.4  14.7% of control values after adding atropine (n = 8, p < 0.05 toward choline). The ripple frequency recovered to 102  7% (n = 7, n < 0.01 toward choline, NS compared to baseline). Comparable effects were observed for the number of ripple cycles per sharp wave, which recovered to 81.4  11% under atropine (n = 7, NS compared to both choline and baseline values). As a control, we examined whether atropine itself affects SPW-R oscillations, which has

been suggested by previous observations in vivo (Buzs
aki 1986). Under our conditions, however, atropine had no obvious effect on SPW-R parameters (% of control: SPW-R amplitude 103.3  9%; SPW-R frequency 91  7%; number of ripple cycles per sharp wave 100  4%; ripple energy 97  3.5%; ripple frequency 99  1%; n = 6, NS, t-test for all values). The above-described effects were measured at 2 mM of choline, which appears to be a quite high concentration. Therefore, we applied lower concentrations of choline (stepwise increasing from 100 to 1000 lM; n = 12) and measured the effects on SPW-R (Fig. 2a). At the end of recordings atropine (10 lM) was applied to test for recovery. During prolonged recordings, SPW-R parameters may change. We therefore analyzed choline-induced changes relative to time-matched control recordings from slices perfused with ACSF (n = 11). Application of drugs was restricted to 30 min for each concentration to minimize drift of parameters. In these experiments, changes in SPW-R waveform became visible at 100 lM choline (p < 0.05 for the fraction of sharp waves carrying detectable ripples, Mann–Whitney test). At higher concentrations, choline

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

Choline modulates sharp wave–ripples

induced robust effects on several parameters of SPW-R (Fig. 2a). In interface recording chambers, equilibrium concentration of drugs within brain slices are reached after long times of ~1 h (M€ uller et al. 1988), which might be critical in experiments using minimal effective drug concentrations. Therefore, we additionally applied low concentrations of choline (50 lM) for 1 h (n = 12). We found significant suppression of ripple energy and of the fraction of sharp waves carrying superimposed ripples (Fig. 2a, right), indicating that low choline concentrations preferentially affect the high-frequency component of SPW-R. To reduce variance of effects, subsequent experiments using bath application of choline were performed at 2 mM. We further investigated which subtype of muscarinic receptors mediates the effects of choline on SPW-R. We found that the suppressive effects of 2 mM choline were highly sensitive to the M1-muscarinic acetylcholine receptor antagonists pirenzepine (1 lM, n = 12) or VU 0255035 (5 lM, n = 12) (Fig. 2b). Together, our results suggest that bath-applied choline suppresses spontaneous SPW-R activity in CA1 by activation of M1-muscarinic receptors.

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Choline suppresses synaptic transmission between CA3 and CA1 Effects of drugs on network activity may be caused by alterations of synaptic transmission. Recent reports from rat hippocampal slices show that choline suppresses synaptic transmission from CA3 to CA1 via the Schaffer collaterals (Mielke et al. 2011). Importantly, these effects were sensitive to nicotinic receptor antagonists. We therefore tested the effects of choline on amplitude and slope of stimulationevoked fEPSP in CA1 (Fig. 1d). In our preparation, choline (2 mM) decreased fEPSP amplitude to 85.9  1.5% of control, whereas the slope was lowered to 82.3  3% of baseline values. These effects were statistically significant (n = 6, p < 0.05 for both parameters, ANOVA, Bonferroni’s post hoc test for fEPSP amplitude, and Dunn’s post hoc test for slope), but not as strong as reported for rat slices (Mielke et al. 2011). To identify the receptor mediating this effect, we added atropine to the bath solution containing choline. As indicated in Fig. 1d, the muscarinic receptor antagonist returned transmission back to baseline conditions. Amplitude of

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Fig. 2 Concentration dependence and receptor specificity of choline effects on sharp wave–ripple complexes (SPW-R) activity in CA1. (a) Effects of bath-applied choline (100–1000 lM; 30 min per concentration) on SPW-R values. Grey bars show parameters in the presence of choline compared to time-matched control recordings in artificial CSF (ACSF) (black bars). Each experiment was finished with adding atropine (10 lM). Drug concentrations and corresponding time of recording are indicated at the bottom. Right panel shows the effects of 50 lM choline on SPW-R parameters following prolonged bath application. Note suppression of SPW-R activity by 50–250 lM of choline (unpaired t-test or Mann–Whitney test). (b) Bath-applied choline suppresses SPW-R by activation of M1-muscarinic receptors. Bar diagrams representing normalized changes in SPW-R values under control conditions (ACSF), in the presence of choline (2 mM) and after adding the M1-AChR antagonists pirenzepine (1 lM, left panels) or VU 0255035 (5 lM, right panels). ANOVA, followed by Bonferroni’s or Dunn’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant. All values are normalized to control. Data are given as mean  SEM.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

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fEPSPs recovered to 103  2.6%, and fEPSP slope reached 107  5% compared to baseline values (n = 6). The apparent slight increase in CA1 fEPSP slopes in the presence of atropine has been reported previously (Sokolov and Kleschevnikov 1995). These data suggest that choline-mediated suppression of fEPSPs in CA1 of mouse hippocampal slices is mediated by muscarinic receptor activation. To investigate potential nicotinic components in the choline-mediated suppression of fEPSPs we also added the non-selective nicotinic receptor antagonist MEC (10 lM). In the presence of choline (2–6 mM), this compound did not rescue synaptic transmission. Mean fEPSP amplitude was 74.8  3.6% of baseline following application of choline, and remained at 65.5  6.2% after further addition of MEC. Likewise, fEPSP slope was 62.8  10% of baseline during choline application alone versus 70.7  5.5% during choline and MEC (n = 5, NS for both parameters, ANOVA, Bonferroni’s post hoc). In summary, we could not identify a nicotinic component in suppression of fEPSPs by bath application of choline. Dual contribution of muscarinic and nicotinic receptors to effects of choline The apparent difference between results from rat slices (Mielke et al. 2011) and our present findings may hint toward heterogeneous local and temporal mechanisms. For example, a7 subunit-containing nAChRs desensitize upon activation by choline (Alkondon et al. 1997), which may emphasize muscarinic receptor-mediated effects during slow bath application of the common agonist (Wang and Sun 2005). We therefore tested effects of choline following short-time application through a local pipette containing 8 mM choline (note that the effective concentration within the tissue depends on diffusion time and distance to the pipette). As shown in Fig. 3, local application of choline in proximal SR efficiently suppressed SPW-R activity in CA1. Frequency of the events reached 60  7.5% of control, SPW-R amplitude was reduced to 63.3  4.8%, and ripple energy to 73.7  3.7% after choline application (n = 6, p < 0.01 for all parameters, t-test). Blocking muscarinic receptors with atropine previous to choline application prevented the effect partially but not completely. In the presence of atropine, SPW-R frequency was reduced to 81.1  6.7% of control, amplitude to 89.6  3%, and ripple energy to 90.1  4% after local choline applications (n = 6, NS for SPW-R frequency and ripple energy and p < 0.05 for SPW-R amplitude, t-test for all parameters). We therefore asked whether the remaining effects may be because of activation of a7-nAChR. Indeed, when the preferential a7nicotinic receptor antagonist MLA (1 lM) was present together with atropine, choline did not affect SPW-R at all (Fig. 3a and b; n = 6, NS for all parameters, t-test for SPWR frequency and amplitude, Wilcoxon test for ripple energy).

After washout of both antagonists, choline did again evoke effects comparable to the above-described local applications in drug-free control solution [SPW-R frequency 54.9  12.7% (p < 0.05); SPW-R amplitude 72  6% (p < 0.01); ripple energy 70.9  5.2% (p < 0.05) of control; n = 6 for all parameters, t-test for SPW-R frequency and amplitude, Wilcoxon test for ripple energy]. Simultaneously performed recordings of Schaffer collateral-evoked fEPSPs in CA1 revealed that local application of choline affects synaptic transmission through both muscarinic and nicotinic components (Fig. 3a and c). Choline decreased fEPSP amplitudes to 55.7  8.4% of control and slopes to 47.9  9.8% (n = 7, p < 0.05 for both parameters, t-test for amplitude, and Wilcoxon test for slope). In the presence of atropine, choline induced significant, but less pronounced reductions (amplitude 79.2  9%; slope 72.5  10% of baseline; n = 7, p < 0.05 for both, Wilcoxon test for amplitude, and t-test for slope). Further addition of MLA completely blocked the choline-mediated suppression of fEPSPs (amplitude 104.4  4.4%; slope 105.8  9.2% of baseline; n = 7, NS, t-test for both). After washout of the antagonists, local application of choline had again the initial effects on fEPSPs (amplitude decreased to 59.5  6.6%; slope to 57  8%; n = 7, p < 0.01 for both, t-test for both; Fig. 3c). To isolate the nAChR-mediated component of the effects we then locally applied nicotine (300 lM) in CA1. As illustrated in Fig. 4, nicotine clearly suppressed fEPSPs and had slight effects on SPW-R. Amplitude of fEPSP was reduced to 70.8  7.8% of baseline, and fEPSP slope decreased to 63.2  9.6% (n = 6, p < 0.05 for both parameters, t-test for amplitude, and Wilcoxon test for slope). In presence of the nAChR-antagonist MEC (10 lM) local application of nicotine had no effects on evoked fEPSPs (n = 6; Fig 4a and c). The effect of MEC was reversible after washout of the antagonist, i.e. local application of nicotine showed a comparable effect as initially. We then quantified effects of nicotine on SPW-R activity and found an apparent slight reduction in SPW-R frequency, which did not reach significance (92  6% of control, n = 6, NS, t-test). However, SPW-R amplitude (87.1  2.11%) and ripple energy (88.8  2.2%) were significantly reduced immediately after application of nicotine (n = 6, p < 0.01, t-test for both parameters), and this effect was again abolished by adding MEC (Fig. 4a and b). In summary, local, rapid application of choline reveals both nicotinic and muscarinic effects on hippocampal networks. Block of choline uptake does not affect choline-mediated suppression of SPW-R Choline is taken up by neurons and used as a precursor of acetylcholine. Therefore, adding choline to brain slices may exert indirect effects by increasing pre-synaptic release of ACh. To distinguish between direct and indirect effects of

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

Choline modulates sharp wave–ripples

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Fig. 3 Local application of choline affects sharp wave–ripple complexes (SPW-R) and fEPSPs in CA1 by activation of both muscarinic and nicotinic cholinergic receptors. (a) Sample traces showing SPW-R recorded in stratum pyramidale (SP) (top) and evoked fEPSPs recorded in stratum radiatum (SR) (bottom; each panel showing response before/after local application of choline). (b) Representative time–response plots of the normalized effects of local choline application (indicated by arrow) on SPW-R frequency (top), amplitude (middle), and ripple energy (bottom). Top bars indicate recording time

in control conditions, in the presence of atropine (10 lM) and in the additional presence of the preferential a7-nAChR antagonist methyllycaconitine (MLA) (1 lM). (c) Time course of amplitude (top) and slope (bottom) of evoked fEPSPs. Bars indicate pharmacological conditions similar to (b). (d) Schematic representation of the hippocampal slice with position of recording, stimulation, and application electrodes. All data are given as mean  SEM. Paired t-test or Wilcoxon signed-ranks test, *p < 0.05, **p < 0.01, ns: not significant.

choline we next performed experiments in the presence of the choline reuptake blocker HC-3. We first established whether bath application of HC-3 (100 lM) has effects on spontaneous SPW-R oscillations in CA1 (Fig. 5a). In the presence of HC-3, SPW-R frequency decreased to 59.8  11.3% and SPW-R amplitude reached 85.5  8% of baseline (n = 8, NS for both parameters, ANOVA, Bonferroni’s post hoc). While the apparent reduction in frequency and amplitude was not significant, the data still indicate some potential effect of HC-3 on spontaneous network activity. Adding choline in the presence of HC-3, however, suppressed SPW-R oscillations further (Fig. 5a and c). SPW-R frequency decreased to 36.3  12.5% (NS against HC-3) and SPW-R amplitude to 63.3  8.9% of baseline (n = 8, p < 0.05 against HC-3, both changes significant against control values). In an alternative approach, we applied choline prior to HC-3. When applied in this order, block of choline reuptake did not significantly alter prior choline-mediated suppression of SPW-R (Fig. 5b and c; n = 6 and 5 for SPW-R frequency and amplitude, respectively, NS for choline vs. choline + HC-3, ANOVA, Bonferroni’s post hoc). These findings

suggest that the effect of exogenously applied choline is largely independent from its role for acetylcholine production, but does likely reflect direct activation of muscarinic and nicotinic receptors. Effects of choline on SPW-R and synaptic transmission in CA3 Our data show a strong suppressive effect of choline on SPW-R in CA1. This pattern of activity is usually generated within area CA3 and then propagates through CA1 to the subiculum and the entorhinal cortex. We therefore looked for potential effects of choline within CA3. We applied choline globally through the bath solution and recorded field potentials in SR and stratum pyramidale. As illustrated in Fig. 6a and b, SPW-R frequency in CA3 was decreased to 23.1  9% of control values (p < 0.001), and this effect was not diminished by the nAChR-antagonist MEC (10 lM, 22.1  7.6%, n = 6, NS vs. application of choline alone). In contrast, further addition of atropine did significantly increase the frequency of SPW-R to 71.9  11.2% (n = 6, p < 0.01 for choline + atropine vs. choline + atropine + MEC, ANOVA, Bonferroni’s post hoc).

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(a)

(b)

(c)

Fig. 4 Local application of nicotine suppresses sharp wave–ripple complexes (SPW-R) and fEPSPs in CA1. (a) Sample traces showing effects of nicotine on SPW-R in stratum pyramidale (SP) (top) and evoked fEPSPs in stratum radiatum (SR) (bottom; each panel showing response before/after local application of nicotine). (b, c) Pooled data

(a)

(b)

show normalized changes (mean  SEM) in SPW-R amplitude, ripple energy, and fEPSP amplitude and slope after local nicotine application (indicated by arrow). Top bar shows application time of the nAChRantagonist mecamylamine (MEC) (10 lM). Paired t-test or Wilcoxon signed-ranks test *p < 0.05, **p < 0.01, ns: not significant.

(c)

Fig. 5 Blockade of choline reuptake does not prevent choline-mediated suppression of sharp wave–ripple complexes (SPW-R) in CA1. (a) Sample traces showing SPW-R in CA1 stratum pyramidale (SP) under artificial CSF (ACSF), in the presence of the choline reuptake blocker hemicholinium-3 (HC-3) (100 lM), after adding choline (2 mM), and after washout of both drugs (ACSF, bottom). (b) SPW-R

recorded in CA1 SP under control conditions (ACSF), in the presence of choline (2 mM), after adding HC-3 (100 lM), and after washout of both drugs. (c) Bar diagrams representing mean SPW-R frequency (top) and amplitude (bottom) in control conditions and after application of drugs. Error bars indicate SEM. ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

Similarly, amplitude of SPW-R in CA3 dropped to 51.6  4% in the presence of choline (n = 5, p < 0.01), did not change during additional MEC administration (n = 5, NS), and recov-

ANOVA,

ered to 84  6.7% after addition of atropine (n = 5, p < 0.05, Bonferroni’s post hoc). Comparable effects were seen for the high-frequency component of SPW-R, where the

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

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number of ripple cycles dropped to 43.3  7.6% of baseline values (p < 0.05), was unaffected by MEC, but returned almost to baseline values after adding atropine (88  8%; n = 5, p < 0.05 for choline + atropine vs. choline + atropine + MEC, ANOVA on ranks, Student–Newman–Keuls post hoc). Similar effects were observed for ripple energy (Fig. 6b, ANOVA, Tukey–Kramer’s post hoc) and ripple frequency. The ripple frequency in CA3 was 242.6  8.8 Hz under control conditions, decreased to 217.6  4.5 Hz in the presence of choline (90.8  2.3% of control values, p < 0.01), was unaffected by MEC (93  2.1%, NS against choline), and recovered to 232.4  8.5 Hz after further adding atropine (96  0.76%, NS against control, n = 5, ANOVA, Bonferroni’s post hoc). These data show that bath-applied choline strongly suppresses spontaneously generated network activity in CA3 through muscarinic receptor-mediated mechanisms. In addition to the suppression of SPW-R, in about half of recordings bath application of choline (2–5 mM) induced atropine-sensitive gamma oscillations in CA3 (averaged

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frequency 34  1.7 Hz, n = 6). These slices were not included in analysis of SPW-R. In contrast to effects on network activity, choline-mediated suppression of evoked post-synaptic potentials in CA3 was regulated by both muscarinic and nicotinic receptor activations (Fig. 6a and c). Stimulation of commissural/associational fibers elicited fEPSPs in SR. In the presence of choline, amplitude of these potentials decreased to 82  3.6% and slope was reduced to 81.3  5% respectively (n = 7, p < 0.001 for amplitude and p < 0.01 for slope). Adding MEC induced a recovery of the fEPSP amplitude up to 92.4  2.2% of baseline values (p < 0.05 vs. choline, NS vs. control ACSF). Further addition of atropine enhanced the fEPSP amplitude to 138.6  9% of control (n = 7, p < 0.001 vs. choline + MEC, ANOVA, Bonferroni’s post hoc). Slopes of fEPSPs displayed comparable behavior, increasing from 81.3  5% of control to 97.3  2.9% after blocking nicotinic receptors by MEC, and reaching 142.1  7.1% after additional application of atropine (Fig. 6c, ANOVA, Bonferroni’s post hoc). Control

(a)

(b)

Fig. 6 Bath-applied choline suppresses sharp wave–ripple complexes (SPW-R) and synaptic transmission in CA3. (a) SPW-R recorded in CA3 stratum pyramidale (SP) (top) and fEPSPs in CA3 stratum radiatum (SR) (bottom, arrowhead indicates stimulation of the commissural/ associational fibers) under artificial CSF (ACSF), in the presence of choline (2 mM), after adding mecamylamine (MEC) (10 lM), and after further adding atropine (10 lM). (b) Pooled data showing normalized changes in SPW-R values during bath applications of drugs as indicated by the bars at the top of each panel. (c) Bar diagrams representing mean fEPSP amplitude (top) and slope (bottom) under control conditions (ACSF) and in the presence of drugs as indicated. (d) Scatter plot comparing percentual reduction in SPW-R amplitude versus reduction in fEPSP amplitude in CA1 (filled cycles) and CA3 (triangles) following bath application of choline. (e) Schematic representation of the hippocampal slice showing position of recording and stimulation electrodes within CA3. All data are given as mean  SEM. ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

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(c)

(d)

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experiments with exclusive application of atropine revealed an increase in fEPSP amplitude to 112  3.5% of control (n = 6, p < 0.05, t-test), while fEPSP slope was not significantly changed (NS, t-test). Likewise, SPW-R in CA3 were not visibly altered by atropine (% of control: SPW-R amplitude 99  7%; SPW-R frequency 103  12%; number of ripple cycles per sharp wave 92  4%; ripple energy 91  7%; ripple frequency 98  2%; n = 6, NS, Wilcoxon test for ripple frequency, t-test for all other values). Together, our data suggest that effects of choline on evoked fEPSPs in CA3 are mediated by both muscarinic and nicotinic receptors. Generation of sharp wave–ripple complexes in the CA3 network is strongly suppressed by choline, mainly using muscarinic signalling pathways. Figure 6d shows a comparison of choline-mediated effects (bath application) on amplitude of field EPSPs and SPW-R in CA1 and CA3. Obviously, reduction in the SPW-R network pattern is stronger as compared to the reduction in evoked synaptic potentials. These data illustrate that complex network patterns are more strongly affected by choline than monosynaptic excitatory transmission, suggesting that additional mechanisms contribute to the suppression of SPW-R.

Discussion We investigated effects of choline on spontaneously occurring sharp wave–ripple complexes (SPW-R) and on synaptic transmission in the mouse hippocampus in vitro. We report that choline suppresses SPW-R and reduces fEPSPs by activation of both muscarinic and nicotinic cholinergic receptors. Moreover, the mode of drug application (bath perfusion vs. local application) influences the contribution of different types of receptors, probably because of rapid desensitization of nAChR. It has been previously reported that choline acts as an efficient agonist of a7-containing nAChRs (Papke et al. 1996; Alkondon et al. 1997, 1999), which are widely expressed by interneurons in the hippocampus. Accordingly, activation of a7-nAChRs results in significant inhibition of both hippocampal pyramidal neurons as well as interneurons (Buhler and Dunwiddie 2002). The action of choline differs between subtypes of hippocampal neurons, with strongest effects on stratum lacunosum-moleculare cells (Alkondon and Albuquerque 2001). In addition, Mielke et al. (2011) have described that choline suppresses excitatory synaptic transmission in CA1 following the facilitation of inhibition via nicotinic receptors. An important novel finding of this study is that choline does also activate muscarinic cholinergic receptors in hippocampal networks. Using bath application of the drug we found a pronounced atropine-sensitive suppression of SPW-R in areas CA1 and CA3, as well as reduction in evoked fEPSPs in CA1. Moreover, suppressive effects of choline on SPW-R were highly sensitive to M1-muscarinic

receptor antagonists (tested in area CA1). In these experiments, the nicotinic antagonist MEC was without effect, suggesting a merely muscarinic receptor-mediated action of choline. We note, however, that fast, local applications revealed a clear contribution by nAChR (see below). Our experiments further suggest that choline acts by direct activation of cholinergic receptors, as block of choline reuptake with HC-3 did not prevent the effects. We can, therefore, exclude that choline is first transported into synaptic terminals where it is converted into acetylcholine and then released into the extracellular space. Cholinergic muscarinic effects on intrinsic and synaptic properties of neurons have been shown to be vital to memory processes (Hasselmo 1999, 2005). In the hippocampus, activation of mAChRs excites hippocampal pyramidal neurons by modulating several ionic conductances, including leak K+ currents, M currents, Ca2+ -dependent K+ currents, and NMDA receptor-mediated conductances (Benardo and Prince 1982; Halliwell and Adams 1982; Cole and Nicoll 1983; Markram and Segal 1992; Egorov and M€ uller 1999; Egorov et al. 1999). At the same time, activation of mAChRs suppresses excitatory synaptic transmission (Hasselmo and Schnell 1994; Egorov et al. 1996). Activation of mAChRs also excites GABAergic interneurons in CA1, but inhibits GABA release (Behrends and ten Bruggencate 2003). Sharp wave–ripples are a complex pattern of propagating network activity with superimposed fast oscillations. While the underlying mechanisms are not fully understood, they clearly involve highly synchronous fast synaptic inhibition, precisely timed synaptic excitation, increased activity of pyramidal cells and selected subtypes of interneurons, and electrical coupling (Traub et al. 2004; Ellender et al. 2010; Buzs
aki and Silva 2012). Acetylcholine interferes with several of these mechanisms, and it is therefore not surprising that it can suppress the finely tuned spatiotemporal pattern of SPW-R. Indeed, it has been recently shown that activation of muscarinic receptors by pilocarpine disrupts hippocampal SPW-R in vivo and in vitro, the latter being mediated by M1 mAChRs (Norimoto et al. 2012). Measurements of postsynaptic currents indicated that the underlying mechanisms involve a relative increase in inhibition over excitation. It is well feasible that the present results are caused by similar effects of choline, including a choline-mediated suppression of fEPSPs, measured as field potentials. However, quantitative analysis of SPW-R and fEPSPs showed a much stronger effect of choline at the network level as compared to excitatory synaptic transmission (Fig. 6d). In addition, synaptic effects of choline in CA3 were highly sensitive to nicotinic antagonists, whereas the suppression of SPW-R remained. It is, therefore, likely, that suppression of SPW-R involves mechanisms beyond the reduction in excitatory transmission in hippocampal pathways. As a result, choline induces muscarinic modulation of various ionic currents, mimicking a transition from ‘sleep-related’ SPW-R

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Choline modulates sharp wave–ripples

oscillatory patterns to states resembling wakefulness or rapid eye movement sleep. This notion is corroborated by the observed increase in baseline noise under choline (Fig. 5b) and the occurrence of gamma oscillations (Fig. 1b). Similar transition to gamma activity has been reported in vitro for ACh or its stable analog, carbachol (Fisahn et al. 1998; Kann et al. 2011). Our results are also in line with previous reports showing that choline suppresses synaptic transmission by activation of nAChRs (Mielke et al. 2011). It has been suggested that this effect is linked to activation of nicotinic receptors on GABAergic interneurons (Alkondon et al. 1999; Alkondon and Albuquerque 2001; Giocomo and Hasselmo 2005). Besides the potentiation of GABA release, nicotinic activation could contribute to the release of various other neurotransmitters (McKay et al. 2007). In our experiments, the nicotinic component of choline effects induced suppression of fEPSPs. Interestingly, in CA1 this effect was only observed following local drug application but not during constant bath perfusion with choline. In CA3, nicotinic and muscarinic components of choline-mediated effects were more obvious, even upon bath application. It is likely that the discrepancy between local and global application of choline in CA1 is caused by nAChR desensitization upon sustained agonist exposure. Fast desensitization of a7-nAChRs by nicotine or choline administration has indeed been reported (Alkondon and Albuquerque 1993; Alkondon et al. 1997). Again, nicotinic effects had strong effects at the network level. Local application of choline decreased SPW-R and fEPSPs values to ~60% in CA1. These effects were only partially blocked by atropine, whereas a mixture of both muscarinic and nicotinic receptor antagonists exerted a full block. Importantly, we used MLA as a nAChR antagonist with preference for a7 subunit-containing receptors. The observed block of choline effects is in line with reports showing that choline acts as a specific a7 agonist (Alkondon et al. 1997; Vogt and Regehr 2001). Choline does not affect a4b2-nAChR, another subtype expressed in the hippocampus (Papke et al. 1996; Alkondon et al. 1999). Together with the blocking effect of MLA this provides evidence for a key role of a7-containing receptors. Recently, it has been shown in vitro that nicotine dose dependently transformed stimulus-induced SPW-R in CA3 area into recurrent epileptiform discharges, going along with decreased inhibition (Liotta et al. 2011). Moreover, low concentrations of bath-applied nicotine enhance excitatory synaptic transmission in the hippocampus (Gray et al. 1996; Chiodini et al. 1999). We observed inhibitory effects of nicotine on spontaneously occurring SPW-R and fEPSPs in CA1 (and in CA3; unpublished observations) without any signs of enhanced excitation. Again, these discrepancies may be explained by differences in receptor expression between rats and mice, differences in kinetics of drug exposure (local application vs. perfusion), or different

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mechanisms underlying spontaneously occurring and stimulus-induced SPW-R. Choline has been used as a compound to adjust osmolarity of extracellular solutions. Here we have shown that choline has strong effects on particular network patterns and on synaptic transmission. Moreover, adding choline to the bath solution suppressed slow-wave oscillations in the entorhinal cortex of juvenile mice via activation of muscarinic receptors (our unpublished observations). Slow-wave activity, as well as SPW-R, represents a pattern observed during sleep and anesthesia (Steriade 2006). Thus, adjusting osmolarity with choline might have direct impacts on sleep-related oscillatory patterns and consequently strongly affect experimental results. It remains to be investigated whether endogenous levels of choline can influence cholinergic activation. Basal concentrations of choline in the CSF are relatively stable at 4–12 lM (Klein et al. 1992). The concentration of choline in prefrontal cortex reaches its maximum around 1–2 lM during cue detection (Parikh et al. 2007). In the hippocampus, peak concentration of choline during theta oscillations was detected at about 0.1 lM (Zhang et al. 2010). Thus, ambient choline is clearly below the concentrations used in this study and is unlikely to cause a strong activation of cholinergic receptors under resting conditions (Sarter and Parikh 2005). However, depending on the activity of cholinergic fibers, choline might reach far higher concentrations in the synaptic microenvironment. Pathologically increased electrical activity in the brain, however, might raise concentrations of acetylcholine and choline far above baseline values. In animal models of status epilepticus choline has been found to reach ~300% of control values in the cerebral cortex and hippocampus of rats (Jope et al. 1987; Jope and Gu 1991). Under such conditions, extracellular concentrations of choline may well match the effective concentrations found in our experiments, i.e. 50 lM. Increased concentrations of choline have also been found in human patients following ischemic lesions where the increase was correlated with the extent of the insult (Karaszewski et al. 2010). Similar increases in choline in white and grey matter have been reported following traumatic brain injury, with a positive correlation with the severity of damage (Ricci et al. 1997; Friedman et al. 1998; Garnett et al. 2000). Moreover, choline-containing compounds strongly increase in various human brain tumors (Kinoshita and Yokota 1997). It should be kept in mind that estimates of choline in the human brain in vivo are based on relative, rather than absolute measurements of concentrations. It is likely, however, that the consistent effect of choline at concentrations of 50 lM or above is in a relevant, although possibly not physiological range. Interestingly, nutrient choline modulates different levels of neuronal activity in vivo and in vitro. Thus, prenatal choline supplementation in rodents accelerates hippocampal

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maturation and increases the power of gamma oscillations (Mellott et al. 2004; Cheng et al. 2008). Supplementation of choline during the prenatal period improves later ability for learning and memory formation, including performance in hippocampus-dependent tasks (Meck and Williams 2003; Glenn et al. 2008; Wong-Goodrich et al. 2008). While such effects may be linked to enhanced long-term potentiation of adult hippocampal synapses following increased gestational availability of choline (Pyapali et al. 2013), our present results suggest that choline-induced modulation of network oscillations may also play a role in these processes. In humans, choline levels in the CSF are decreased in Parkinson’s and Huntington’s diseases (Manyam et al. 1990a,b) and increased in patients with dementia of the Alzheimer type (Elble et al. 1989). Higher dietary choline intake is associated with an improved cognitive performance (Poly et al. 2011; Zeisel 2012). Thus, choline might serve as an instrument for modulation of normal and pathological network activity in humans, especially in situations of disturbed cholinergic activity.

Acknowledgements and conflict of interest disclosure This work was supported by BMBF (01GQ1003A, BCCN Heidelberg/Mannheim, B3) and by IB BMBF (01DJ12061, RUS 11/015). We thank Dr. Papageorgiou and Dr. Kunz for helpful advice on this manuscript. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

References Alkondon M. and Albuquerque E. X. (1993) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes. J. Pharmacol. Exp. Ther. 265, 1455–1473. Alkondon M. and Albuquerque E. X. (2001) Nicotinic acetylcholine receptor alpha7 and alpha4beta2 subtypes differentially control GABAergic input to CA1 neurons in rat hippocampus. J. Neurophysiol. 86, 3043–3055. Alkondon M., Pereira E. F., Cortes W. S., Maelicke A. and Albuquerque E. X. (1997) Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur. J. Neurosci. 9, 2734–2742. Alkondon M., Pereira E. F. R., Eisenberg H. M. and Albuquerque E. X. (1999) Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices. J. Neuosci. 19, 2693– 2705. B€ahner F., Weiss E. K., Birke G., Maier N., Schmitz D., Rudolph U., Frotscher M., Traub R. D., Both M. and Draguhn A. (2011) Cellular correlate of assembly formation in oscillating hippocampal networks in vitro. Proc. Natl Acad. Sci. USA 108, E607–E616. Behrends J. C. and ten Bruggencate G. (2003) Cholinergic modulation of synaptic inhibition in the guinea pig hippocampus in vitro:

excitation of GABAergic interneurons and inhibition of GABArelease. J. Neurophysiol. 69, 626–629. Benardo L. S. and Prince D. A. (1982) Ionic mechanisms of cholinergic excitation in mammalian hippocampal pyramidal cells. Brain Res. 249, 333–344. Both M., B€ahner F., von Bohlen und Halbach O. and Draguhn A. (2008) Propagation of specific network patterns through the mouse hippocampus. Hippocampus 18, 899–908. Bragin A., Jando G., Nadasdy Z., Hetke J., Wise K. and Buzs
aki G. (1995) Gamma (40–100 Hz) oscillation in the hippocampus of the behaving rat. J. Neurosci. 15, 47–60. Buhler A. V. and Dunwiddie T. V. (2002) alpha7 nicotinic acetylcholine receptors on GABAergic interneurons evoke dendritic and somatic inhibition of hippocampal neurons. J. Neurophysiol. 87, 548–557. Buzs
aki G. (1986) Hippocampal sharp waves: their origin and significance. Brain Res. 398, 242–252. Buzs
aki G. (1989) Two-stage model of memory trace formation: a role for “Noisy” brain states. Neuroscience 31, 551–570. Buzs
aki G. (2006) Rhythms of the Brain, Oxford University Press, New York. Buzs
aki G. and Draguhn A. (2004) Neuronal oscillations in cortical networks. Science 304, 1926–1929. Buzs
aki G. and Silva F. L. (2012) High frequency oscillations in the intact brain. Prog. Neurobiol. 98, 241–249. Buzs
aki G., Horv
ath Z., Urioste R., Hetke J. and Wise K. (1992) Highfrequency network oscillation in the hippocampus. Science 256, 1025–1027. Cheng R. K., Williams C. L. and Meck W. H. (2008) Oscillatory bands, neuronal synchrony and hippocampal function: implications of the effects of prenatal choline supplementation for sleep-dependent memory consolidation. Brain Res. 1237, 176– 194. Chiodini F. C., Tassonyi E., Hulo S., Bertrand D. and Muller D. (1999) Modulation of synaptic transmission by nicotine and nicotinic antagonists in hippocampus. Brain Res. Bull. 48, 623–628. Chrobak J. J. and Buzs
aki G. (1996) High-frequency oscillations in the output networks of the hippocampal-entorhinal axis of the freely behaving rat. J. Neurosci. 16, 3056–3066. Chrobak J. J., L€orincz A. and Buzs
aki G. (2000) Physiological patterns in the hippocampo-entorhinal cortex system. Hippocampus 10, 457–465. Cole A. and Nicoll R. (1983) Acetylcholine mediates a slow synaptic potential in hippocampal pyramidal cells. Science 221, 1299– 1301. Drever B. D., Riedel G. and Platt B. (2011) The cholinergic system and hippocampal plasticity. Behav. Brain Res. 221, 505–514. Egorov A. V. and M€uller W. (1999) Subcellular muscarinic enhancement of excitability and Ca2 + -signals in CA1-dendrites in rat hippocampal slice. Neurosci. Lett. 261, 77–80. Egorov A. V., Heinemann U. and M€uller W. (1996) Muscarinic activation reduces changes in [Ca2+]o evoked by stimulation of Schaffer collaterals during blocked synaptic transmission in rat hippocampal slices. Neurosci. Lett. 214, 187–190. Egorov A. V., Gloveli T. and M€uller W. (1999) Muscarinic control of dendritic excitability and Ca2+ signaling in CA1 pyramidal neurons in rat hippocampal slice. J. Neurophysiol. 82, 1909–1915. Elble R., Giacobini E. and Higgins C. (1989) Choline levels are increased in cerebrospinal fluid of Alzheimer patients. Neurobiol. Aging 10, 45–50. Ellender T. J., Nissen W., Colgin L. L., Mann E. O. and Paulsen O. (2010) Priming of hippocampal population bursts by individual perisomatic-targeting interneurons. J. Neurosci. 30, 5979–5991.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

Choline modulates sharp wave–ripples

Fisahn A., Pike F. G., Buhl E. H. and Paulsen O. (1998) Cholinergic induction of network oscillations at 40 Hz in the hippocampus in vitro. Nature 394, 186–189. Friedman S. D., Brooks W. M., Jung R. E., Hart B. L. and Yeo R. A. (1998) Proton MR spectroscopic findings correspond to neuropsychological function in traumatic brain injury. Am. J. Neuroradiol. 19, 1879–1885. Garnett M. R., Blamire A. M., Rajagopalan B., Styles P. and CadouxHudson T. A. (2000) Evidence for cellular damage in normal-appearing white matter correlates with injury severity in patients following traumatic brain injury: a magnetic resonance spectroscopy study. Brain 123, 1403–1409. Giocomo L. M. and Hasselmo M. E. (2005) Nicotinic modulation of glutamatergic synaptic transmission in region CA3 of the hippocampus. Eur. J. Neurosci. 22, 1349–1356. Glenn M. J., Kirby E. D., Gibson E. M., Wong-Goodrich S. J., Mellott T. J., Blusztajn J. K. and Williams C. L. (2008) Age-related declines in exploratory behavior and markers of hippocampal plasticity are attenuated by prenatal choline supplementation in rats. Brain Res. 1237, 110–123. Gray R., Rajan A. S., Radcliffe K. A., Yakehiro M. and Dani J. A. (1996) Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 383, 713–716. Halliwell J. V. and Adams P. R. (1982) Voltage-clamp analysis of muscarinic excitation in hippocampal neurons. Brain Res. 250, 71–92. Hasselmo M. E. (1999) Neuromodulation: acetylcholine and memory consolidation. Trends Cogn. Sci. 3, 351–359. Hasselmo M. E. (2005) What is the function of hippocampal theta rhythm?–Linking behavioral data to phasic properties of field potential and unit recording data. Hippocampus 15, 936–949. Hasselmo M. E. (2006) The role of acetylcholine in learning and memory. Curr. Opin. Neurobiol. 16, 710–715. Hasselmo M. E. and Giocomo L. M. (2006) Cholinergic modulation of cortical function. J. Mol. Neurosci. 30, 2005–2007. Hasselmo M. E. and Schnell E. (1994) Laminar selectivity of the cholinergic suppression of synaptic transmission in rat hippocampal region CA1: computational modeling and brain slice physiology. J. Neurosci. 14, 3898–3914. Huerta P. T. and Lisman J. E. (1996) Synaptic plasticity during the cholinergic theta-frequency oscillation in vitro. Hippocampus 6, 58–61. Ji D. and Wilson M. A. (2007) Coordinated memory replay in the visual cortex and hippocampus during sleep. Nat. Neurosci. 10, 100–107. Jope R. S. and Gu X. (1991) Seizures increase acetylcholine and choline concentrations in rat brain regions. Neurochem. Res. 16, 1219–1226. Jope R. S., Simonato M. and Lally K. (1987) Acetylcholine content in rat brain is elevated by status epilepticus induced by lithium and pilocarpine. J. Neurochem. 49, 944–951. Kann O., Huchzermeyer C., Kov
acs R., Wirtz S. and Schuelke M. (2011) Gamma oscillations in the hippocampus require high complex I gene expression and strong functional performance of mitochondria. Brain 134, 345–358. Karaszewski B., Thomas R. G., Chappell F. M., Armitage P. A., Carpenter T. K., Lymer G. K., Dennis M. S., Marshall I. and Wardlaw J. M. (2010) Brain choline concentration. Early quantitative marker of ischemia and infarct expansion? Neurology 75, 850–856. Kinoshita Y. and Yokota A. (1997) Absolute concentrations of metabolites in human brain tumors using in vitro proton magnetic resonance spectroscopy. NMR Biomed. 10, 2–12. Klein J., K€ oppen A., L€offelholz K. and Schmitthenner J. (1992) Uptake and metabolism of choline by rat brain after acute choline administration. J. Neurochem. 58, 870–876.

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Liotta A., Caliskan G., ul Haq R., Hollnagel J. O., R€osler A., Heinemann U. and Behrens C. J. (2011) Partial disinhibition is required for transition of stimulus-induced sharp wave-ripple complexes into recurrent epileptiform discharges in rat hippocampal slices. J. Neurophysiol. 105, 172–187. Maier N., Nimmrich V. and Draguhn A. (2003) Cellular and network mechanisms underlying spontaneous sharp wave-ripple complexes in mouse hippocampal slices. J. Physiol. (Lond.) 550, 873–887. Manyam B. V., Giacobini E. and Colliver J. A. (1990a) Cerebrospinal fluid acetylcholinesterase and choline measurements in Huntington’s disease. J. Neurol. 237, 281–284. Manyam B. V., Giacobini E. and Colliver J. A. (1990b) Cerebrospinal fluid choline levels are decreased in Parkinson’s disease. Ann. Neurol. 27, 683–685. Markram H. and Segal M. (1992) The inositol 1,4,5-trisphosphate pathway mediates cholinergic potentiation of rat hippocampal neuronal responses to NMDA. J. Physiol. 447, 513–533. McKay B. E., Placzek A. N. and Dani J. A. (2007) Regulation of synaptic transmission and plasticity by neuronal nicotinic acetylcholine receptors. Biochem. Pharmacol. 74, 1120–1133. Meck W. H. and Williams C. L. (2003) Metabolic imprinting of choline by its availability during gestation: implications for memory and attentional processing across the lifespan. Neurosci. Biobehav. Rev. 27, 385–399. Mellott T. J., Williams C. L., Meck W. H. and Blusztajn J. K. (2004) Prenatal choline supplementation advances hippocampal development and enhances MAPK and CREB activation. FASEB J. 18, 545–547. Mielke J. G., Ahuja T. K., Comas T. and Mealing G. A. (2011) Cholinemediated depression of hippocampal synaptic transmission. Nutr. Neurosci. 14, 186–194. M€uller W., Misgeld U. and Heinemann U. (1988) Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration. Exp. Brain Res. 72, 287–298. Norimoto H., Mizunuma M., Ishikawa D., Matsuki N. and Ikegaya Y. (2012) Muscarinic receptor activation disrupts hippocampal sharp wave-ripples. Brain Res. 1461, 1–9. O’Keefe J. (1993) Hippocampus, theta, and spatial memory. Curr. Opin. Neurobiol. 3, 917–924. Papke R., Bencherif M. and Lippiello P. (1996) An evaulation of neuronal nicotinic acetylcholine receptor activation by quarternary nitrogen compounds indicates that choline is selective for the alpha-7 subtype. Neurosci. Lett. 213, 201–204. Parikh V., Kozak R., Martinez V. and Sarter M. (2007) Prefrontal acetylcholine release controls cue detection on multiple timescales. Neuron 56, 141–154. Poly C., Massaro J. M., Seshadri S., Wolf P. A., Cho E., Krall E., Jacques P. F. and Au R. (2011) The relation of dietary choline to cognitive performance and white-matter hyperintensity in the Framingham Offspring Cohort. Am. J. Clin. Nutr. 94, 1584–1591. Pyapali G. K., Turner D. A., Williams C. L., Meck W. H. and Scott H. (2013) Prenatal dietary choline supplementation decreases the threshold for induction of long-term potentiation in young adult rats. J. Neurophysiol. 79, 1790–1796. Ricci R., Barbarella G., Musi P., Boldrini P., Trevisan C. and Basaglia N. (1997) Localised proton MR spectroscopy of brain metabolism changes in vegetative patients. Neuroradiology 39, 313–319. Sarter M. and Parikh V. (2005) Choline transporters, cholinergic transmission and cognition. Nat. Rev. Neurosci. 6, 48–56. Sokolov M. V. and Kleschevnikov A. M. (1995) Atropine suppresses associative LTP in the CA1 region of rat hippocampal slices. Brain Res. 672, 281–284. Steriade M. (2006) Grouping of brain rhythms in corticothalamic systems. Neuroscience 137, 1087–1106.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693

14

V. Fischer et al.

Traub R. D., Bibbig A., LeBeau F. E., Buhl E. H. and Whittington M. A. (2004) Cellular mechanisms of neuronal population oscillations in the hippocampus in vitro. Annu. Rev. Neurosci. 27, 247–278. Vogt K. E. and Regehr W. G. (2001) Cholinergic modulation of excitatory synaptic transmission in the CA3 area of the hippocampus. J. Neurosci. 21, 75–83. Wang H. and Sun X. (2005) Desensitized nicotinic receptors in brain. Brain Res. Rev. 48, 420–437.

Wong-Goodrich S.J.E.,GlennM. J.,MellottT.J.,Blusztajn J.K.,Meck W.H. and Williams C. L. (2008) Spatial memory and hippocampal plasticity are differentially sensitive to the availability of choline in adulthood as a function of choline supply in utero. Brain Res. 1237, 153–166. Zeisel S. H. (2012) A brief history of choline. Ann. Nutr. Metab. 61, 254–258. Zhang H., Lin S. C. and Nicolelis M. A. (2010) Spatiotemporal coupling between hippocampal acetylcholine release and theta oscillations in vivo. J. Neurosci. 30, 13431–13440.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12693