Y Audrey Hay

Przemyslaw Jarzebowski

Yu Zhang

Richard Digby

Viktoria Brendel

Ole Paulsen

Vincent Magloire
email: v.magloire@ucl.ac.uk

Cholinergic modulation of Up-Down states in the mouse medial entorhinal cortex in vitro

Keywords:

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Introduction

Acetylcholine (ACh) is a key neuromodulator that strongly influences cortical network activity.

The cortical ACh concentration is high during wake and rapid eye movement (REM) sleep and lower during slow wave sleep (SWS), when basal forebrain cholinergic neurons still discharge at around 1 Hz, maintaining a low cholinergic tone [START_REF] Kametani | Alterations in acetylcholine release in the rat hippocampus during sleep-wakefulness detected by intracerebral dialysis[END_REF][START_REF] Marrosu | Microdialysis measurement of cortical and hippocampal acetylcholine release during sleep-wake cycle in freely moving cats[END_REF][START_REF] Lee | Cholinergic basal forebrain neurons burst with theta during waking and paradoxical sleep[END_REF][START_REF] Mena-Segovia | Cholinergic brainstem neurons modulate cortical gamma activity during slow oscillations[END_REF]. SWS is characterised by a cortical slow oscillation (<1 Hz) consisting of synchronous membrane potential fluctuations between a depolarized ('Up') and hyperpolarized ('Down') state [START_REF] Steriade | A novel slow (< 1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components[END_REF].

Neocortical Up states in anaesthetised mice are dependent on muscarinic ACh receptors [START_REF] Lőrincz | A distinct class of slow (∼0.2-2 Hz) intrinsically bursting layer 5 pyramidal neurons determines UP/DOWN state dynamics in the neocortex[END_REF], and pharmacological reinstatement of a low cholinergic tone in neocortical slices induces rhythmic Up and Down states with properties similar to those observed during the slow oscillation in vivo, consistent with a low cholinergic tone during SWS [START_REF] Carracedo | A neocortical delta rhythm facilitates reciprocal interlaminar interactions via nested theta rhythms[END_REF][START_REF] Lőrincz | A distinct class of slow (∼0.2-2 Hz) intrinsically bursting layer 5 pyramidal neurons determines UP/DOWN state dynamics in the neocortex[END_REF].

The medial entorhinal cortex (mEC) is the main gateway between the neocortex and the hippocampus [START_REF] Strien | The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network[END_REF]. During SWS, the mEC is suggested to modulate corticohippocampal interactions because mEC Up states outlast and bridge between consecutive neocortical Up states [START_REF] Hahn | Spontaneous persistent activity in entorhinal cortex modulates corticohippocampal interaction in vivo[END_REF]. Slices from the mEC show spontaneous longduration Up states [START_REF] Mann | Distinct roles of GABA A and GABA B receptors in balancing and terminating persistent cortical activity[END_REF], reminiscent of those seen in vivo, mediated by kainate receptors [START_REF] Digby | Distinct mechanisms of Up state maintenance in the medial entorhinal cortex and neocortex[END_REF]. The mEC is highly sensitive to cholinergic modulation [START_REF] Klink | Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons[END_REF][START_REF] Egorov | Graded persistent activity in entorhinal cortex neurons[END_REF][START_REF] Steriade | Acetylcholine systems and rhythmic activities during the wakingsleep cycle[END_REF][START_REF] Fransén | Mechanism of graded persistent cellular activity of entorhinal cortex layer V neurons[END_REF][START_REF] Zhang | TRPC channels underlie cholinergic plateau potentials and persistent activity in entorhinal cortex[END_REF][START_REF] Jochems | Cholinergic receptor activation supports persistent firing in layer III neurons in the medial entorhinal cortex[END_REF], which raises the question of how a low cholinergic tone modulates the spontaneous slow oscillation in the mEC.

We addressed this question using a slice preparation of the juvenile mouse mEC, which shows spontaneous Up-Down states when superfused with a solution containing 1 mM Mg 2+ and 1.2 mM Ca 2+ [START_REF] Mann | Distinct roles of GABA A and GABA B receptors in balancing and terminating persistent cortical activity[END_REF]. We recorded Up and Down state activity using dual intra-and extracellular electrodes and two-photon calcium imaging of population activity.

Using whole-cell recording, we show that a low physiological concentration of ACh (100-500 nM; [START_REF] Kametani | Alterations in acetylcholine release in the rat hippocampus during sleep-wakefulness detected by intracerebral dialysis[END_REF][START_REF] Marrosu | Microdialysis measurement of cortical and hippocampal acetylcholine release during sleep-wake cycle in freely moving cats[END_REF] increases Up state duration and incidence via muscarinic receptor activation, while maintaining the balance of excitatory and inhibitory recurrent synaptic transmission. Using calcium imaging, we find that ACh increases the duration, frequency, and rhythmic occurrence of population-wide Up states and that ACh modulates the participation of neurons in these Up-state events. These results suggest an important role for cholinergic input in modulating Up-Down states in the medial entorhinal cortex.

Materials and Methods:

Animals: All experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act (1986) following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). All animal procedures were authorised under Personal and Project licenses held by the authors. Animals were housed in conventional cages, on a 12h/12h light/dark cycle, with ad libitum access to food and water.

Electrophysiological experiments were performed on C57BL/6 (Harlan, Bicester, UK) mice of both sexes. For calcium imaging recordings, we used either heterozygous Thy1-GCaMP6f transgenic mice (Jax: 024276; [START_REF] Dana | Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo[END_REF] or homozygous GCaMP6f-floxed mice (Jax: 024105; [START_REF] Madisen | Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance[END_REF] crossed with heterozygous Necab1-cre mice, which express the Cre recombinase in the thalamus as well as in excitatory neurons in the cortex [START_REF] Phillips | A repeated molecular architecture across thalamic pathways[END_REF]. Necab1-cre mice were made in the Janelia Transgenic Mouse Core (https://www.janelia.org/support-team/gene-targeting-and-transgenics) and provided prepublication by the Hantman and Dudman laboratories.

Slice preparation: Horizontal slices (400 µm) through the hippocampus-medial entorhinal cortex were prepared from both sexes on postnatal day (P)14 to P19 between one and three hours into the light phase of the light/dark cycle as previously described [START_REF] Mann | Distinct roles of GABA A and GABA B receptors in balancing and terminating persistent cortical activity[END_REF][START_REF] Craig | Distinct roles of GABA B1a -and GABA B1b -containing GABA B receptors in spontaneous and evoked termination of persistent cortical activity[END_REF]. Briefly, decapitation was performed under deep isoflurane-induced anesthesia and, after removal of the skull, the brain was submerged in ice-cold (≤4 ⁰C) standard artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl (126), KCl (3.5),

NaH 2 PO 4 (1.25), MgSO 4 (2), CaCl 2 (2), NaHCO 3 (26), and glucose (10), pH 7.2-7.4 for 1-2 min. Slices were then cut using a vibrating blade microtome (Leica, VT 1200 S, Germany) and transferred to an interface chamber between humidified carbogen gas (95% O 2 , 5% CO 2 ) and modified artificial CSF (aCSF) with reduced MgSO 4 (1 mM) and CaCl 2 (1.2 mM). Slices were maintained in interface condition prior to recordings.

Electrophysiology: Slices were mounted on coverslips coated with 0.1% poly-L-lysine in ultrapure H 2 O (Sigma-Aldrich) and transferred to a submerged-style recording chamber perfused with modified aCSF at 4-5 mL . min -1 at 30-32 ⁰C to promote spontaneous persistent network activity [START_REF] Hájos | Maintaining network activity in submerged hippocampal slices: importance of oxygen supply[END_REF]. Whole-cell patch-clamp recordings were performed on mEC neurons with glass pipettes pulled from standard borosilicate glass tubing containing (in Electrophysiology analysis: All data were acquired using an AXON Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), low-pass filtered at 2 kHz, and digitized at 5 kHz using an Instrutech ITC-18 and Igor Pro (WaveMetrics) custom-made acquisition procedures.

Automatic off-line detection of Up and Down state transitions was performed using custommade procedures in Igor Pro as described previously [START_REF] Craig | Distinct roles of GABA B1a -and GABA B1b -containing GABA B receptors in spontaneous and evoked termination of persistent cortical activity[END_REF]. Briefly, during Up states, the membrane potential noise increases due to synaptic activity. We calculated the product of the ratio of the root mean square of the membrane potential in two successive analysis windows each containing 120 data points (24 ms at 5 kHz) and the ratio of the relative change in membrane potential between the current and the antepenultimate time window. Any change of more than 10% was considered as a Down-to-Up or Up-to-Down state transition.

All traces were visually inspected to confirm the detection of appropriate Up and Down state transitions. The spike frequency was measured over the duration of detected Up or Down state periods.

Calcium imaging: As for the electrophysiological experiments, slices were mounted on coverslips coated with 0.1% poly-L-lysine and transferred to a submerged-style recording chamber perfused with modified aCSF at 4-5 mL . min -1 at 30-32 ⁰C. Population imaging was done with a two-photon microscope (VIVO, 3i systems). Two-photon excitation was performed using a MaiTai Ti-Sapphire laser (Spectra-physics, Inc.) at 890 nm, coupled to a galvanometer scanning unit that was mounted on a Zeiss AxioExaminer Z1 upright microscope equipped with a 20x/1.0 NA objective. Fluorescence was acquired using non-descanned GaAsP detectors behind the objective. Recordings were performed with a 300 um x 300 um field of view (FOV), at an acquisition rate of 1-3 Hz. One, two, or three z-planes were recorded for 10-27 min per condition; for the analysis, only the z-plane with the highest number of cells was used.

Calcium data processing: To extract the calcium fluorescence signal, the raw fluorescence movies were analysed with Suite 2P [START_REF] Pachitariu | Suite2p: beyond 10,000 neurons with standard two-photon microscopy[END_REF]. This software automates analysis by performing the following steps: First, x-y drift is corrected by aligning the frames, ). We identified calcium events when the neuronal z-scored signal exceeded a threshold of 4 s.d. and had a prominent peak exceeding the neighboring local minimum by more than 4 s.d. A cell was identified as active if it was found to have a calcium event in any of the three recording conditions. The duration of the event was defined as the time between upward and downward crossing of the threshold value. Slices with at least 65% of their cells active were included in the analysis (5 out of 13 slices from Necab1-cre*GCaMP6f-floxed mice, all slices from Thy1-GCaMP6f mice). We cropped the recordings to equal length of 7 minutes.

Population events: Population events were detected by analysing the population mean of the z-scored and time-binned signal. First, for each cell the mean z-score was calculated during each one-second-long bin. Next, the values for each time bin were averaged across the cells for each slice. Population events were detected when the local maximum of the signal crossed a set threshold. The threshold was manually adjusted for each slice to capture the peaks. If multiple local maxima were detected within a single period during which the z-score exceeded 50% of the threshold value, only the peak with the highest value was included. A neuron was deemed to participate in the population event if it had a z-score value exceeding half of the population event threshold. The duration of a population event was defined as the time between upward and downward crossing of 50% of the peak value. For a cell to be considered to participate in population events, it had to be active in 70% of the detected population events.

The choice of the threshold did not affect the significance of the effects and was tested for the minimum level of participation set at 50, 60, 70 and 80%.

Statistical analysis:

The statistics for electrophysiology experiments were performed using Prism 5 (GraphPad Software Inc.) and R (https://www.r-project.org). Non-parametric tests were used throughout the study. Comparison between paired data sets was performed using Wilcoxon signed-rank test. One-way ANOVA on ranks was done using Kruskal-Wallis test, and two-way ANOVA on ranks (ART ANOVA) was done using standard ANOVA after data alignment and ranking using ARTool package in R. Data are reported as mean ± S.E.M. unless stated otherwise.

Statistical analysis of the calcium imaging data was performed using MATLAB and R. For statistical testing of the autocorrelations, we assumed a normal distribution of the signal values and applied Bartlett's formula for the estimate of the standard error. Bartlett's formula assumes that the random time series is generated by a moving average process where two sequential observations are correlated. Standard error was calculated as:

𝑆𝐸(𝑘) = 1 + 2𝛴 𝑘 -1 𝑖 = 1 𝑟 2 𝑖 𝑁
where k is the lag, r i is the autocorrelation at lag i and N is the number of observations in the recording. The non-zero-lag peak value r k of the autocorrelation was tested for the null hypothesis that the real autocorrelation value is not different from 0. The autocorrelation is said to be significant if the r k / SE(k) ratio was outside of the 0.95 quantile of the Norm(0, 1) distribution.

Code and data accessibility: Code used for the analysis of electrophysiological and calcium imaging data and to generate the figures can be accessed on the authors' GitHub site: https://github.com/przemyslawj/ento_ach_caimg. Electrophysiological and calcium imaging data will be shared on any reasonable request.

Drugs: Acetylcholine (Sigma-Aldrich) stock solution was made daily at an initial concentration of 5 mM and then diluted in aCSF to a final concentration of 100-500 nM. Atropine, pirenzepine AFDX-116 and mecamylamine were purchased from Tocris.

Results

Low physiological concentrations of ACh increase Up state duration and occurrence via muscarinic receptor activation

We monitored Up and Down state activity in spontaneously active slices of the mEC of juvenile mice (n = 40 animals aged P14-P19) using whole-cell current-clamp recordings from individual layer II-III principal neurons (n = 60). Previous reports indicate that cortical extracellular ACh concentration varies between 50 nM and 5 µM depending on the behavioral state [START_REF] Kametani | Alterations in acetylcholine release in the rat hippocampus during sleep-wakefulness detected by intracerebral dialysis[END_REF][START_REF] Marrosu | Microdialysis measurement of cortical and hippocampal acetylcholine release during sleep-wake cycle in freely moving cats[END_REF][START_REF] Hasselmo | High acetylcholine levels set circuit dynamics for attention and encoding and low acetylcholine levels set dynamics for consolidation[END_REF][START_REF] Parikh | Prefrontal acetylcholine release controls cue detection on multiple timescales[END_REF][START_REF] Zhang | Spatiotemporal coupling between hippocampal acetylcholine release and theta oscillations in vivo[END_REF]. We investigated how low physiological concentrations of bath applied ACh (100-500 nM), consistent with values measured during SWS [START_REF] Kametani | Alterations in acetylcholine release in the rat hippocampus during sleep-wakefulness detected by intracerebral dialysis[END_REF][START_REF] Marrosu | Microdialysis measurement of cortical and hippocampal acetylcholine release during sleep-wake cycle in freely moving cats[END_REF] similar to the intrinsic voltage-dependent oscillations previously observed in mEC stellate cells [START_REF] Dickson | Properties and role of I(h) in the pacing of subthreshold oscillations in entorhinal cortex layer II neurons[END_REF]. Washing out ACh (5 µM) restored Up and Down states, but the incidence and Up state morphology remained affected even after 30 minutes of wash-out (n = 2; Figure S1-1A).

We next asked which receptors mediate ACh modulation of Up states. Cholinergic effects are mediated through ionotropic nicotinic receptors and metabotropic muscarinic receptors. The ACh-induced (250-500 nM) increase in Up state duration and incidence was reversed by application of atropine (Atr; 1 µM), a muscarinic receptor antagonist (Duration: ACh, 3.89 ± 0.5 s vs Atr, 2.90 ± 0.26 s; Wilcoxon signed-rank test, W = 60; n = 11; p = 0.014; Incidence: ACh, 7.38 ± 0.82 min -1 vs Atr, 2.80 ± 0.40 min -1 ; Wilcoxon signed-rank test, W = 66, n = 11, p = 0.001; Figure 1A,B). Application of atropine alone did not affect the duration and incidence of Up states but prevented the ACh-induced modulation (Duration: Ctrl, 2.93 ± 0.62 s, Atr, 2.54 ± 0.22 s, Atr+ACh, 2.51 ± 0.21 s; Kruskal-Wallis test, n = 7, p = 0.96; Incidence: Ctrl, 4.01 ± 1.13 min -1 , Atr, 4.06 ± 1.28 min -1 vs Atr+ACh, 4.5 ± 1.07 min -1 ; Kruskal-Wallis test, n = 7, p = 0.90; Figure 1C). In contrast, bath application of the nicotinic receptor antagonist mecamylamine (Mec; 10 µM) did not change the duration of Up states when co-applied after ACh (ACh, 5.67 ± 0.8 s vs Mec+ACh, 6.52 ± 0.97 s; Wilcoxon signed-rank test, W = 11; n = 9; p = 0.20; Figure 1D,E), nor their incidence (ACh, 2.71 ± 0.29 min -1 vs Mec+ACh, 2.66 ± 0.27 min -1 ; Wilcoxon signed-rank test, W = 25.5; n = 9; p = 0.77; Figure 1D,E) and bathing of mecamylamine prior to ACh did not prevent ACh-induced modulation of Up states (Duration:

Ctrl, 6.41 ± 1.47 s, Mec, 5.73 ± 0.95 s, Mec+ACh, 15.83 ± 3.38 s; Kruskal-Wallis test, n = 7, p = 0.027; Incidence: Ctrl, 1.79 ± 0.31 min -1 , Mec, 1.54 ± 0.29 min -1 vs Mec+ACh, 2.4 ± 0.27 min -1 ; Kruskal-Wallis test, n = 7, p = 0.09; Figure 1F). Thus, Up and Down state modulation by a low concentration of ACh is mediated primarily by muscarinic receptor activation.

Subtypes of muscarinic receptors mediating ACh modulation of Up and Down states

We next asked which subtypes of muscarinic receptors are involved in the modulation of Up and Down states. The muscarinic receptor family consists of 5 subtypes of receptor (M1-M5)

differentially distributed in the cortex. M1/M3 subtypes are localised mainly on pyramidal neurons whereas M2/M4 are predominantly found presynaptically at inhibitory synapses [START_REF] Levey | Muscarinic acetylcholine receptor expression in memory circuits: implications for treatment of Alzheimer disease[END_REF][START_REF] Lucas-Meunier | Cholinergic modulation of the cortical neuronal network[END_REF][START_REF] Chaudhuri | Localization of m2 muscarinic receptor protein in parvalbumin and calretinin containing cells of the adult rat entorhinal cortex using two complementary methods[END_REF][START_REF] Bubser | Muscarinic receptor pharmacology and circuitry for the modulation of cognition[END_REF]. suggesting that M2/M4 muscarinic receptors also contribute to the cholinergic modulation of Up states. However, AFDX-116 application prior to ACh application did not prevent AChinduced modulation of Up state duration or incidence (Duration: Ctrl, 3.44 ± 0.47 s, AFDX, 3.33 ± 0.26 s, AFDX+ACh, 5.80 ± 1.37 s; Kruskal-Wallis test, n = 9, p = 0.02; Incidence: Ctrl, 2.26 ± 0.36 min -1 , AFDX, 2.66 ± 0.46 min -1 vs AFDX+ACh, 5.01 ± 0.69 min -1 ; Kruskal-Wallis test, n = 9, p = 0.18; Figure 2F). Thus, the cholinergic modulation of Up state activity in mEC principal neurons is mediated primarily by M1/M3 muscarinic receptors with a possible additional contribution from M2/M4 muscarinic receptors.

ACh reduces both excitatory and inhibitory charge transfer without affecting E/I balance

To better understand how ACh application modulates synaptic transmission during Up states,

ACh increases overall firing frequency and differentially modulates spiking activity between states and neurons

We next investigated the effect of a low concentration of ACh on the firing pattern of individual neurons by performing cell-attached recordings in loose patch configuration, a method that allowed us to observe spiking behaviour with minimal disturbance of the recorded cells [START_REF] Alcami | Measuring the firing rate of high-resistance neurons with cell-attached recording[END_REF]. Network activity was simultaneously monitored using voltage-clamp recording of a close-by cell to detect synaptic activity corresponding to the Up state (Figure 4A). 4H). Finally, we asked whether the location of the neurons influenced their response to ACh and observed that the proportion of neurons from layer III showing an overall increase in firing frequency during Up state was significantly higher than the proportion of neurons from layer II (Layer II, n = 2/11 neurons; Layer III, n = 9/13 neurons; two proportion z-test, p = 0.01; We defined population events in the time-binned signal as events in which the mean z-score crossed a manually adjusted threshold (Figure 6A). After adding ACh (250 nM) to the superfusate, the duration of population events significantly increased from 2.2 ± 0.3 s to 4.0 ± 0.6 s (Wilcoxon signed-rank test on median event durations, W = 45, n = 10, p = 0.008; Figure 6B) as well as their incidence from 1.4 ± 0.2 to 3.2 ± 0.3 min -1 (Wilcoxon signed rank test on event frequencies, W = 52, n = 10, p = 0.006; Figure 6C). This effect was reversed by coapplication of atropine with ACh: event duration significantly decreased from 4.0 ± 0.6 s to 2.0 ± 0.3 s (Wilcoxon signed-rank test on median event durations, W = 45, n = 10, p = 0.008;

Figure 6B), and the incidence decreased from 3.2 ± 0.3 to 1.3 ± 0.2 min -1 (Wilcoxon signedrank test on event frequencies, W = 55, n = 10, p = 0.002; Figure 6C). In addition, the ΔF/F 0 amplitude of the population event peaks significantly increased between the control and ACh condition from 0.14 ± 0.02 to 0.27 ± 0.04 (Wilcoxon signed-rank test on median peak ΔF/F values, W = 35, n = 10, p = 0.04; Figure 6D) but did not significantly decrease following the addition of atropine (Wilcoxon signed-rank on median peak ΔF/F 0 values, W = 24, n = 10, p = 0.24; Figure 6D). The increased frequency, duration and amplitude of population events were accompanied by an increase in the median frequency and duration of transient calcium events in individual cells (frequency: median increased from 0.4 ± 0.1 min-1 to 1.5 ± 0.3 min-1;

Wilcoxon signed-rank test on median event frequency in each slice, W = 54, n = 10, p = 0.008;

Figure 6E; duration: median increased from 2.1 ± 0.3 to 3.3 ± 0.5 s; Wilcoxon signed-rank test on median duration in each slice, W = 36, n = 10, p = 0.01; Figure 6F). Hence, the ACh effect on population event duration and incidence observed here is similar to the electrophysiological data.

Plots of population activity in ACh (Figure 6A) revealed a periodicity of the network activity, which we quantified by calculating the autocorrelation function (Figure 7A). We observed an increase in autocorrelation peaks in the presence of ACh (8 out of 11 slices showed significant non-zero-lag peaks in the autocorrelogram) compared to control condition (3 out of 11 slices showed significant autocorrelation peaks) and atropine condition (only 1 of the 11 slices showed significant autocorrelation peaks; Figure 7B). The average lag of the first non-zerolag peak in the autocorrelation in the ACh condition was 17 s (SD = 5.1 s, n = 8) with a peak value of 0.52 (SD = 0.26, n = 8; Figure 7C).

Next, we looked at how the ACh and ACh + atropine conditions affected the recruitment of cells during population events. In the analysis, we included 9 slices with more than one population event in each of the three conditions. The population events reliably recruited a large part of the network (Figure 6A): 61 ± 7% cells participated in ≥ 70% of population events during the control and 73 ± 8% of cells during the ACh condition (Wilcoxon signed-rank test on median count of cells participating in more than 70% of events, W = 31, n = 9, p = 0.07 two-tailed; Figure 8A). We assessed if the stability of cell assemblies was modulated by ACh by monitoring whether the cells that changed their participation from active to non-active and from non-active to active, returned to their previous state after application of atropine. We assessed the significance of this pattern by comparing it with a model of random reassignments. In the random model, each cell in the network had an equal probability of switching from active to non-active and vice-versa. We set the probability of the switch equal to the proportion of cells empirically observed to switch their participation. We simulated 1000 random reassignments governed by the experimentally calculated probabilities. The number of cells that followed a pattern of temporal switch controlled by ACh and reversed by atropine was significantly higher in 6 out of 9 slices compared to the simulated random reassignments (p < 0.05) and when tested on cells from all slices pulled together (p < 0.01; Figure 8B).

Hence, consistent with our intracellular recordings, these calcium imaging results show that ACh induces an increase in co-activity in the network through rhythmic population events of increased frequency and duration. Moreover, the neuronal pattern of activation appeared stable during population-wide bursts while ACh reversibly modulated individual cell recruitment during these events.

Discussion

We have examined how a low physiological concentration of ACh, similar to the estimated concentration during SWS, affects Up and Down state activity in acute slices of the mEC. We found that ACh increases the duration and incidence of Up states in layer II/III principal neurons via muscarinic receptor activation. Moreover, ACh reduces both excitatory and inhibitory recurrent synaptic transmission and causes ~50% of the neurons to increase their firing in Down states and ~50% to decrease their firing in Up states. Finally, at population level, we found that ACh promotes the synchronisation of mEC neurons and reorganises patterns of co-active neuronal populations. These observations suggest that a low tone of ACh controls the timing of neuronal co-activation during Up and Down states in the mEC, which might affect the hippocampal-neocortical information flow during SWS.

Low physiological concentrations of ACh prolong Up states and increase their occurrence via muscarinic receptor activation

We investigated how submicromolar concentrations of ACh affect the Up-Down state activity in the mEC. We found that ACh increases the duration of individual Up states as well as their incidence via muscarinic receptor activation (Figure 1) as previously reported in the neocortex [START_REF] Carracedo | A neocortical delta rhythm facilitates reciprocal interlaminar interactions via nested theta rhythms[END_REF]. We did not observe any effect of nicotinic receptor blockers on the ACh-induced modulation of Up and Down states. This is to be expected since most nicotinic receptors have an affinity for ACh in the micromolar range while α7 homomeric receptors, the only nicotinic receptors expressed in the cortex with submicromolar affinity for ACh, show fast desensitization [START_REF] Christophe | Two types of nicotinic receptors mediate an excitation of neocortical layer I interneurons[END_REF][START_REF] Hay | Orexin-dependent activation of layer VIb enhances cortical network activity and integration of non-specific thalamocortical inputs[END_REF][START_REF] Hay | Nicotinic transmission onto layer 6 cortical neurons relies on synaptic activation of non-α7 receptors[END_REF]. Of the 5 sub-families of muscarinic receptors (M1 to M5), which all have submicromolar affinities for ACh [START_REF] Levey | Muscarinic acetylcholine receptor expression in memory circuits: implications for treatment of Alzheimer disease[END_REF][START_REF] Lucas-Meunier | Cholinergic modulation of the cortical neuronal network[END_REF][START_REF] Brown | Muscarinic Acetylcholine Receptors (mAChRs) in the Nervous System: Some Functions and Mechanisms[END_REF], we found that both sub-families M1/M3 and M2/M4 receptors may contribute to the cholinergic modulation of Up state activity. An M1/M3 receptor blocker almost fully abolished ACh-induced Up state modulation while an M2/M4 receptor blocker affected Up state incidence but not their duration (Figure 2). M1/M3 receptors show apparently opposing effects on mEC neurons: they depolarise principal neurons and increase their excitability, but suppress excitatory synaptic transmission [START_REF] Klink | Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons[END_REF][START_REF] Richter | Muscarinic control of intracortical connections to layer II in rat entorhinal cortex slice[END_REF][START_REF] Yun | Cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus of the rat[END_REF][START_REF] Cheong | Cholinergic modulation of synaptic physiology in deep layer entorhinal cortex of the rat[END_REF][START_REF] Egorov | Graded persistent activity in entorhinal cortex neurons[END_REF][START_REF] Hamam | Cholinergic suppression of excitatory synaptic responses in layer II of the medial entorhinal cortex[END_REF][START_REF] Barrett | Contribution of muscarinic M1 receptors to the cholinergic suppression of synaptic responses in layer II of the entorhinal cortex[END_REF]. We observed a decrease in the excitatory synaptic charge transfer in the presence of ACh, consistent with a suppression of excitatory transmission by the activation of M1/M3 receptors (Figure 3). The concomitant decrease in inhibitory synaptic charge transfer could be due to the reduced recurrent excitatory input coupled with the activation of M2/M4 receptors, which are G i -coupled [START_REF] Levey | Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies[END_REF][START_REF] Levey | Muscarinic acetylcholine receptor expression in memory circuits: implications for treatment of Alzheimer disease[END_REF][START_REF] Chaudhuri | Localization of m2 muscarinic receptor protein in parvalbumin and calretinin containing cells of the adult rat entorhinal cortex using two complementary methods[END_REF][START_REF] Bubser | Muscarinic receptor pharmacology and circuitry for the modulation of cognition[END_REF]. The net effect was an increase in Up state duration and incidence (Figures 1 and2), which suggests that the network response to muscarinic receptor activation is mediated primarily through the depolarisation of excitatory neurons.

Whereas we report an increase in Up state duration coupled with a doubling of Up state occurrence in the presence of submicromolar concentrations of ACh, at a higher concentration

(5 µM), we observed a switch from Up and Down states to slow sinusoidal-like oscillations, similar to those observed in layer II stellate cells upon depolarisation [START_REF] Dickson | Properties and role of I(h) in the pacing of subthreshold oscillations in entorhinal cortex layer II neurons[END_REF].

The lengthening of Up states and shortening of Down states with low concentrations of ACh are consistent with another recent report in entorhinal cortex, which used carbachol in submicromolar concentrations [START_REF] Nghiem | Cholinergic switch between two types of slow waves in cerebral cortex[END_REF]. However, the effect of higher Our study revealed an increase in Up state duration in neurons recorded in current clamp mode (Figure 1), but no change in duration of Up state-associated synaptic activity in voltageclamp mode (Figure 3). Several mechanisms may contribute to this apparent discrepancy.

First, neurons with broadened spiking periods may not have been as strongly recurrently connected to the network involved in the Up states. This might explain how the increased spiking activity seen in the period immediately preceding synaptic Up states did not produce synaptic currents (Figure 4). However, it is unclear whether this is due to an anatomical lack of recurrent connectivity or a cholinergically induced change in functional connectivity through activation of presynaptic inhibitory muscarinic receptors. Additionally, some neurons may display frequency depression of synaptic transmission onto their postsynaptic targets, and it is also possible that the increase in Up state duration in current clamp in some neurons results from the activation of a cholinergically-induced intrinsic plateau potential [START_REF] Egorov | Graded persistent activity in entorhinal cortex neurons[END_REF][START_REF] Klink | Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons[END_REF], without the occurrence of action potentials in the later part of the Upstate-associated depolarisation, thus explaining the paucity of synaptic events towards the end of Up states (Figure 1).

Action potential discharge across Up and Down states

Our cell-attached recordings indicate that around 50% of the neurons increase their firing rate in response to a low concentration of ACh while 50% show a decrease (Figure 5). This result might appear surprising since we observed an increase in Up state duration and incidence (Figures 1 andS1-1). Nevertheless, the proportion of neurons showing an increase in spiking activity is consistent with the proportion of neurons (30-100%) that shows sustained activity in response to cholinergic activation in the presence of glutamate and GABA receptor blockers [START_REF] Tahvildari | Ionic basis of on and off persistent activity in layer III lateral entorhinal cortical principal neurons[END_REF][START_REF] Jochems | Cholinergic receptor activation supports persistent firing in layer III neurons in the medial entorhinal cortex[END_REF][START_REF] Batallán-Burrowes | Dopamine suppresses persistent firing in layer III lateral entorhinal cortex neurons[END_REF]. Thus, it is plausible that muscarinic receptor-induced depolarisation in neurons expressing M1/M3 receptors leads to the observed increase in firing. In contrast, in neurons that show less expression of M1/M3 receptors, the combination of synaptic suppression and reduced spiking activity in 50% of neurons might reduce synaptic excitation and lead to a decrease in firing rate.

Up states are network events characterised by an increase in neuronal and synaptic activity [START_REF] Steriade | Thalamocortical oscillations in the sleeping and aroused brain[END_REF][START_REF] Steriade | A novel slow (< 1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components[END_REF]. Our understanding of Up and Down states comes mainly from recordings made in anaesthetised animals and wholecell recordings in slices. In anaesthetised animals, the majority of action potential discharges occur during Up states [START_REF] Saleem | Methods for predicting cortical UP and DOWN states from the phase of deep layer local field potentials[END_REF] but most anaesthetics are associated with low activity of modulatory neurons, including basal forebrain cholinergic neurons [START_REF] Manns | Rhythmically discharging basal forebrain units comprise cholinergic, GABAergic, and putative glutamatergic cells[END_REF]. This might explain why action potential discharge during Down states is infrequently reported in anaesthetised preparations. Moreover, previously published studies often defined Up state boundaries using multi-unit activity as a marker of Up state, limiting the possibility to appreciate firing during Down states (see, for example, [START_REF] Jercog | UP-DOWN cortical dynamics reflect state transitions in a bistable network[END_REF]. Whilst GABAergic interneurons display firing activity during Down states [START_REF] Zucca | Slow wave sleep (SWS) is characterised by a low tone of acetylcholine (ACh), which modulates sharp wave ripple incidence in the hippocampus and neocortical slow wave activity. The medial entorhinal cortex (mEC) couples hippocampal and neocortical activity during SWS and is highly sensitive to ACh. Using electrophysiology and Ca2+ imaging, we show that a sleep-like tone of ACh modulates mEC Up states via muscarinic receptors and reorganises how neuronal circuits are recruited during Up states[END_REF], further investigation in naturally-sleeping animals would be required to investigate to what extent cortical principal neurons fire during Down states.

Correspondence between population events and Up states

We used calcium imaging to study neuronal activity during population events (Figures 5678), which we defined as peaks in the mean fluorescence changes across neurons. Because the change in GCaMP6f fluorescence is primarily caused by action potentials [START_REF] Chen | Ultrasensitive fluorescent proteins for imaging neuronal activity[END_REF], and neurons in our slice preparation predominantly fire during Up states (Figure 4), we surmised that population events correspond to electrophysiologically defined Up states. This correspondence was visible in the elevated ΔF/F 0 signal during simultaneously recorded calcium transients and electrophysiological Up states (Figure 5). However, the electrophysiologically recorded Up states were twice as frequent as the population events while their duration was comparable (Figures 1 and5). This difference in apparent incidence could be explained if some Up states are associated with firing of a too small number of neurons to give a population mean signal that is detected as a population event. This is particularly the case in control conditions where neurons showed low firing rates and thus likely small fluorescence changes. Moreover, we were technically constrained to low acquisition rates (<3 Hz), which might have limited our capability of capturing the calcium fluorescence peaks. Muscarinic receptor activation was reported to amplify the calcium rise from intracellular stores in response to a train of action potentials [START_REF] Power | Nuclear calcium signaling evoked by cholinergic stimulation in hippocampal CA1 pyramidal neurons[END_REF]. This effect might explain the observed increase in amplitude of fluorescence changes in the presence of ACh (Figure 6D); however, it should not affect the reported frequency or duration of the population events. Thus, even with some technical differences between the two sets of experiments, we feel confident that the Up states recorded at single cell level using the patchclamp technique and the population calcium events observed using two-photon microscopy reflect the same network events.

ACh increases synchronised periodic activity and reorganises network activity

Up and Down state transitions correspond to the alternation between synchronous population events and quiescent periods. The synchronous activity of regular periodicity gave rise to significant non-zero-lag peaks in the autocorrelation (Figure 7) with a period corresponding to the population event incidence. A similar increase in the periodicity of Up states was reported by muscarinic receptor activation in sensory cortices [START_REF] Carracedo | A neocortical delta rhythm facilitates reciprocal interlaminar interactions via nested theta rhythms[END_REF][START_REF] Lőrincz | A distinct class of slow (∼0.2-2 Hz) intrinsically bursting layer 5 pyramidal neurons determines UP/DOWN state dynamics in the neocortex[END_REF]. We observed that the population events reliably recruited the same assembly of cells throughout the duration of the recording (Figure 8A). ACh reorganised the active network with some cells reversibly dropping out and others reversibly joining the active assembly (Figure 8B). At the individual cell level, this change was reflected by an increase in activity of some cells and a decrease in others (Figure 4C,H). It is conceivable that the combination of intrinsic depolarisation and excitatory synaptic suppression induced by ACh would favour neurons that are part of assemblies with strong synaptic interconnectivity, allowing the induction of cholinergic persistent activity in these cells while reducing the recurrent excitation that weaker assemblies would receive. This mechanism could promote spiking contrast between sets of neuronal assemblies.

Conclusion

We found that ACh increases Up state duration and modulates spiking activity in principal neurons of the mEC. As the main gateway between the hippocampus and the neocortex, the mEC Up states may control the degree of coupling between the hippocampus and the neocortex [START_REF] Hahn | Spontaneous persistent activity in entorhinal cortex modulates corticohippocampal interaction in vivo[END_REF]. Therefore, because the level of ACh varies during SWS [START_REF] Jing | An optimized acetylcholine sensor for monitoring in vivo cholinergic activity[END_REF], the cholinergic modulation of Up state duration in the mEC could serve as a mechanism to modulate the degree of coupling between the hippocampus and neocortex during SWS, and thereby influence memory consolidation [START_REF] Tonegawa | Memory engram cells have come of age[END_REF][START_REF] Van De Ven | Hippocampal offline reactivation consolidates recently formed cell assembly patterns during sharp wave-ripples[END_REF]. More research would be needed to establish to what extent ACh has a role in the modulation of this coupling.

Abbreviations:

ACh Cumulative distribution function for the proportion of cells that participated in a given percentage of population events. The dashed vertical line marks the threshold for a population event-active cell -a cell participating in >70% of population events. A hundred of cells were sampled with repetition from each slice to make each slice contribute equally to the plotted distribution. B: Changes in the cell population participating in network events. The lines track how some cells changed from population event active to inactive and from inactive to active.

The line thickness represents the relative fraction of cells that share the same pattern of activation across the three recording conditions. The dark grey lines track cells that returned to their previous state after atropine whether their activity changed by ACh or not. The light grey lines track cells that did not return to the previous activation state after atropine. The network reorganisation followed the atropine-reversible pattern represented by dark grey lines, better than a model of random reassignments (p < 0.01, cells from all slices pooled together). The dashed vertical line marks the threshold for a population event-active cell -a cell participating in >70% of population events. 100 cells were sampled with repetition from each slice to make each slice contribute equally to the plotted distribution. B: Changes in the cell population participating in network events. The lines track how some cells changed from population event-active to inactive and from inactive to active. The line thickness represents the relative fraction of cells that share the same pattern of activation across the three recording conditions. The dark grey lines track cells that returned to their previous state after atropine whether their activity changed by ACh or not. The light grey lines track cells that did not return to the previous activation state after atropine. The network reorganisation followed the atropine-reversible pattern represented by dark grey lines, better than a model of random reassignments (p < 0.01, cells from all slices pooled together).

90x52mm (600 x 600 DPI)

  mM): K-gluconate (110), Hepes (40), ATP-Mg (2), GTP (0.3), and NaCl (4) with a resistance of 4-6 MΩ for current clamp recordings and Cs-methylsulfonate (120), Hepes (10), EGTA (0.2), ATP-Mg (4), GTP(0.3), QX-314 (10), and CsCl (20) with a resistance of 3-4 MΩ for voltage clamp recordings. Both pipette solutions were adjusted to obtain a pH of 7.2-7.3 at 25 ⁰C and an osmolarity between 275 and 290 mosmol . L -1 . In addition, we performed cellattached recordings in loose-seal configuration (≤40 MΩ) using pipettes of 3-5 MΩ filled with aCSF. For current clamp experiments, cells exhibiting membrane potential positive to -55 mV were discarded. In addition, series resistance was monitored throughout the experiment. Cells were rejected if the series resistance was above 20 MΩ and/or if the change in series resistance in control condition versus drug was higher than 20% for voltage clamp experiments. No series resistance compensation or liquid junction potential correction was applied.

  photobleaching. The neuronal fluorescence change was calculated as ΔF/F 0 (t) = (F(t)-F 0 (t))/F 0 (t), where F(t) was the fluorescence signal at time t. The statistical analysis was performed on z-scored ΔF/F 0 values (i.e. mean subtracted and divided by the standard deviation (s.d.)). We identified calcium events when the neuronal z-scored signal exceeded a

  Coapplication of ACh with the selective M1/M3 muscarinic antagonist pirenzepine (Pir; 10 µM) entirely reversed ACh modulation of duration and incidence of Up states (Duration: ACh, 4.13 ± 0.76 s vs Pir 2.60 ± 0.25 s; Wilcoxon signed-rank test, W = 28; n = 7; p = 0.02; Incidence: ACh, 8.1 ± 0.79 min -1 vs Pir, 2.61 ± 0.66 min -1 ; Wilcoxon signed-rank test, W = 28, n = 7, p = 0.02; Figure2A,B) and application of Pirenzepine prior to ACh prevented ACh-induced modulation of Up states (Duration: Ctrl, 3.35 ± 0.6 s, Pir, 3.51 ± 0.71 s, Pir+ACh, 3.25 ± 0.79 s; Kruskal-Wallis test, n = 6, p = 0.72; Incidence: Ctrl, 3.43 ± 0.86 min -1 , Pir, 3.33 ± 0.63 min - 1 vs Pir+ACh, 3.9 ± 0.42 min -1 ; Kruskal-Wallis test, n = 6, p = 0.84; Figure2C). Application of AFDX-116 (1 µM), an M2/M4 antagonist, did not significantly affect the duration of Up states (Duration: ACh, 4.41 ± 0.43 s vs AFDX 3.61 ± 0.46 s; Wilcoxon signed-rank test, W = 24; n = 7; p = 0.11), but the incidence was significantly reduced (Incidence: ACh, 6.8 ± 0.39 min-1 vs AFDX, 4.04 ± 0.89 min-1; Wilcoxon signed-rank test, W = 28, n = 7, p = 0.016; Figure2D,E),

  Figure 3B). We asked whether a reduction of the membrane resistance during ACh could

Figure

  Figure 4I). Altogether these results indicate that a low tone of ACh reorganises network activity

Figure 1 :

 1 Figures captions

Figure 2 :

 2 Figure 2: Submicromolar ACh concentrations prolong Up state activity via type 1/3 and

Figure 3 :

 3 Figure 3: Effect of acetylcholine on excitatory and inhibitory synaptic transmission

Figure 4 :

 4 Figure 4: Acetylcholine modulates spiking behaviour across Up and Down states. A:

Figure 5 :

 5 Figure 5: Calcium signal in individual cells correlates with electrophysiologically

Figure 6 :

 6 Figure 6: ACh increases synchrony in the network. A: The effect of ACh and atropine (Atr)

Figure 7 :

 7 Figure 7: ACh induces rhythmic activity in the network. A: Autocorrelation of the mean

Figure 8 :

 8 Figure 8: Population event recruitment and reversible modulation by ACh. A:

Figure S1- 1 :

 1 Figure S1-1: Submicromolar ACh concentrations increase Up state duration and

Figure S6- 1 :

 1 Figure S6-1: Activity of all the slices imaged across the three recording conditions. z-

Figure 1 (Figure 8 (

 18 Figure 1(Black and White): Submicromolar ACh concentrations prolong Up state activity in the mEC via muscarinic receptor activation. A: Example of whole-cell recording from layer III principal neuron in control condition and after 500 nM ACh and ACh + atropine (Atr, 1 μM) superfusion. B-C: Comparison of Up state duration and Up state incidence in (B) control, ACh and ACh + Atr conditions (n = 11) and (C) control, Atr and Atr + ACh conditions (n = 7). D: Example of whole-cell recording from layer III principal neuron in control condition and after 500 nM ACh and ACh + Mecamylamine (Mec, 10 μM) superfusion. E-F: Comparison of Up state duration and Up state incidence in (E) control, ACh and ACh + Mec conditions (n = 9) and in (F) control, Mec and Mec + ACh conditions (n = 7). * indicates p < 0.05. 183x165mm (600 x 600 DPI)

Figure S1- 1 (

 1 Figure S1-1 (Black and White): Submicromolar ACh concentrations increase Up state duration and incidence in the mEC. A-B: Examples of two whole-cell recordings from layer III principal neurons. A constant bias current was used to set the membrane potential at -70 mV at the start of the recording. A shows succession of control condition, bath application of 400 nM and 5 µM ACh and washout. Insets show Up states in control, during 400 nM ACh and after 15 min washout; Up state morphology is modified by ACh and does not wash out fully. B shows control and after application of 5 µM ACh. Insets show Up and Down states in control condition and the oscillating membrane potential during ACh application. C-D: Comparison of Up state duration (C) and incidence (D) at 100 nM (n = 5), 200 nM (n = 3), 300 nM (n = 3), 400 nM (n = 2) and 500 nM ACh (n = 8) indicates no significant difference in duration or incidence in this range of concentration.157x97mm (600 x 600 DPI)

Acknowledgements: We are grateful to Drs Adam Hantman and Joshua Dudman at the Janelia Research Campus for providing Necab1-cre mice. We would like to thank Dr Melodie Borel for her help on Igor script analysis and Dr Michael T. Craig for the automated Up-Down state analysis. V.M. was initially supported by a Swiss National Science Foundation Early Postdoc Mobility Fellowship. This research was supported by the Biotechnology and Biological Sciences Research Council, U.K.


Cédric Gendrot
email: cgendrot@univ-paris3.fr

Martine Adda-Decker

Impact of duration on F1/F2 formant values of oral vowels: an automatic analysis of large broadcast news corpora in French and German

Formant values of oral vowels are automatically measured in a total of 50000 segments from four hours of journalistic broadcast speech in French and German. After automatic segmentation using the LIMSI speech alignment system, formant values are automatically extracted using PRAAT. Vowels with unlikely formant values are discarded (4%). The measured values are exposed with respect to segment duration and language identity. The gap between the measured mean Fi values and reference Fi values is inversely proportional to vowel duration: a tendency to reduction for vowels of short duration clearly emerges for both languages. These results are briefly discussed in terms of centralisation and coarticulation.

Introduction

The availability of large audio corpora and powerful automatic tools for alignment motivates the present study, which aims at observing the automatically determined formant values in oral vowels. French and German vowel systems will be compared and the obtained measures will be discussed as a function of segment length. By this experimental study we try to answer, at least partially, to several questions: -Is it possible to automatically extract reliable formant values from an automatically aligned "spontaneous" speech corpus? To what extent? What proportion of vowels achieves formant values close to the awaited targets? -How do formant values vary with respect to segment durations? Which vowels are most prone to variation? -Which differences can be observed between the variations of the two languages?

This work aims at contributing to the establishment of values of formants and their variability, especially for French as they are hardly documented at the present time. Our interest goes towards the variations in terms of opening/closing (correlated to F1) and the frontness/backness (roughly correlated to F2) and we will discuss the possible reduction phenomena in terms of centralisation/coarticulation.

Corpora and methodology

The French and German corpora correspond to radio and TV journalistic shows: articulation, without being emphasized, remains quite distinct, so that speech can be understood by a broad audience. Such speech cannot be described as fully spontaneous, but rather as prepared speech: only few hesitations, repetitions, and word fragments are observed and syntactic structures often remain close to written language. Reduced vowel phenomena, which we are interested in throughout this study, are undoubtedly less present here than in more conversational-style spontaneous speech.

Corpora

The French corpus corresponds to approximately 2 hours of speech (15 men and 15 women) mainly extracted from broadcast news of France Inter, recorded and transcribed orthographically at the French CTA/DGA. The present study was carried out in the framework of the MIDL (Modelisations for Identification of Languages) project. MIDL partners were LIMSI-CNRS, LPP Paris3, CTA/DGA/GIP, Télécom Paris, EA1483 Paris3. The German corpus corresponds to 2 hours of journalistic shows of ARTE (20 men and 10 women). These are resources which are available at LIMSI at research ends via various European projects (in particular LE-OLIVE and LE-ALERT). The acoustic quality of the German corpus is slightly poorer than that of the French corpus. For this reason we initially used more data in German (5 hours) than in French. In order to keep comparable proportions between both languages, the German corpus was limited to two hours (the comparison of average formant values measured on two and five hours did not show significant differences).

Automatic alignment

The LIMSI speech transcription system [START_REF] Gauvain | The Limsi Broadcast News Transcription System[END_REF] was used for corpus alignment. Orthographical transcriptions being known a priori the alignment system is used to locate phone boundaries, to choose among potential pronunciation alternatives (in particular "liaisons" and schwa), and to discard silences, breath and other noise segments. Contextindependent phone models are used for alignment. Whereas context-dependent (e.g. triphone) acoustic models produce better transcription performances (i.e. a lower word error rates), context-independent acoustic models are more reliable for phone boundary location For technical reasons, the segmentation resolution is limited to 10ms and the minimum duration of a segment is 30ms. Labelling thus produced is not a phonetic, but rather a phonological or phonemic labelling (corresponding in most cases to standard word pronunciations). Formant measures then allow to evaluate the variations observed in the acoustic realisation of phonemes.

Automatic formant extraction

Formant extraction makes use of the Burg algorithm implemented in the PRAAT software [START_REF] Boersma | Praat, a system for doing phonetics by computer[END_REF]. The detection of amplitude peaks is determined in a band lower than 5kHz for male speakers and lower than 5,5kHz for females. Measurements were taken respectively at 1/3, ½, 2/3 of the vowel segment, and then averaged to provide a single value. The interpretation of the extracted amplitude peaks as formants can raise controversy on a considerable number of segments: noises, too high fundamental frequency (voice of women and children), nasality... Two methodological safeguards are applied to prevent from errors: (i) French nasal vowels were excluded from the study, German vowels being exclusively oral. (ii) amplitude peak values are filtered in order to reject erratic items, with respect to the acoustics of the vocal tract. For each vowel, upper and lower formant value limits are given for the first three formants (cf. Table 4 in appendix): if one of the formants lies outside the specified ranges, the corresponding vowel segment is rejected. Formant ranges were chosen in a broad way. A hundred visual checks for each vowel were carried out in order to reject as "errors" only severe formant detection problems and not the "deviating" values which might be due to contextual assimilation effects, to prosody or to speaker's characteristics for example. The formant values of a vowel depend not only on the speaker, but also on the articulation context (mainly left and right phonemes) and on the position of the vowel relatively to stressed and boundaries. The concept of "target undershoot", i.e. the non-realisation of the awaited target values is a well-known phenomenon, studied by many researchers [START_REF] Lindblom | Spectrogaphic study of vowel reduction[END_REF][START_REF] Perrier | Dynamic modelling and control of speech articulators: application to vowel reduction[END_REF]. However, to the best of our knowledge, large-scale corpus measures have not yet been carried out and compared across languages. After this filtering, approximately 1000 vowels out of the 24000 oral French vowels were rejected (4% of segments rejected). The major part of these rejections corresponds to segments of very short duration (600 of the rejected segments have a duration smaller than 50ms). Listening to many of them shows that, at least for the shorter segments, the segmentation is not questionable. Other reasons may explain these rejections, in particular a partial or total devoicing of vowels, thus making formant detection more difficult (or even impossible) and potentially producing non-sense formant values. Similarly, when two formants of a vowel are close, especially in low frequency ranges, (which is the case for posterior closed vowels), the algorithm may detect only one formant instead of two, thus entailing a shift towards the higher order values. The /u/ is particularly prone to rejection, as all mentioned reasons may apply. For the /u/ this raises problems for the automatic control of formants (cf 3.1).

Analysis and results

Results are mainly given for French. The German corpus is used to check whether our observations hold for other languages than French.

Rejection rates of vowels

According to vowel identity: Table 1 indicates rejection rates obtained by the filtering described in 2.3. The overall rejection rate is about 4%. One can notice that rejection rates are highest for closed vowels (/i/, /y/, /u/). We may suggest that not only height would be involved in the rejection rate, but also the proximity of 2 formants for these vowels (namely F2 -F3 for /y/; F3 -F4 for /i/). /oe/1 is also frequently rejected because of its duration (cf. below). /u/ is strongly rejected for reasons explained in section 2.3. According to vowel duration: Table 2 indicates the rate of rejected segments for three duration intervals. Results show that filtering eliminates more frequently shorter segments. We hypothesise, in addition to the reasons invoked above for vowel rejection, that this can also be due to locally unclear pronunciations, where hypothesized segments are actually shorter than 30ms (cf. section 2.2.) and thus including in its segmentation a part of the preceding and/or following phonemes (consonants).

Variations in formants values according to duration

Average values of the vowels retained after filtering are shown in Table 5 (see appendix). As observed in Table 2 shorter segments are more prone to rejection than longer segments: this suggests that short vowels drive away from their F1 and F2 reference values as mentioned in the literature ( [START_REF] Lindblom | Spectrogaphic study of vowel reduction[END_REF]). To check this point statistically, ANOVAs with two factors were carried out, the two factors being "phoneme" and "duration". This may be interpreted for both languages as non-reached targets for the shorter vowels while the formants of longer vowels are close to references values as described in the literature (cf. table 3 for French, and [START_REF] Rausch | Untersuchung zur Vokalartikulation im Deutschen[END_REF] for German). More details and exact mean values of F1 and F2 formants according to duration intervals D can be found in [START_REF] Gendrot | Analyses formantiques automatiques de voyelles orales : évidence de la réduction vocalique en langues française et allemande[END_REF].

Table 3: Formant reference values used for French as inspired from read isolated sentences [START_REF] Calliope | La parole et son traitement automatique[END_REF].

Variations in formant values according to context

Duration of a segment is partly determined by the suprasegmental context. The "target undershoot" observed when looking at the figures gives a first impression of centralisation, as peripheral vowels shift to a more neutral position when shorter. However segmental context is an important parameter in determining vowels' spectral characteristics (as stated by Vaissière [START_REF] Vaissière | Etude des variations allophoniques de la voyelle /a/ et ses conséquences pour la reconnaissance automatique de la parole[END_REF] for French). Now if we consider that shortest vowels are more prone to coarticulation ([3]), i.e. taking spectral characteristics of the immediately preceding and following consonants, then short vowels would be more coarticulated and thus have a F2 transition pointing towards a locus corresponding to the place of articulation of neighbouring consonant (for example at approximately 600Hz for labial consonants -more specifically plosives -and 1800Hz for dentals [START_REF] Delattre | Acoustic loci and transitional cues for consonants[END_REF]. For most vowels, this tendency is coherent with centralisation effects except for some specific cases such as / pap / : short [a] will not shift to a neutral position like other vowels as its second formant will lower compared to its target value (1444 Hz for male and 1677 Hz for female speakers).

In order to check this point for our data, we isolated in both corpora vowels with identical left-right segment considering place of articulation (for labials : p, b, m, f, v and for dentals : t, d, n, l). We can notice on figures 4 and 5 that short vowels in labial phonemic context are more closed but also more posterior (lowering of F2). This is interpreted as a higher coarticulation, rather than more centralisation, as more centralisation would lead F2 to move towards 1500 Hz instead of lowering. This interpretation is also valid for other contexts although formant movements cannot distinguish coarticulation from centralization. This tendency can be observed for French but not for German and this will be the next point of investigation of this study.

Discussion and conclusion

Although many points remain to be described and deserve a more in-depth investigation, the presented study allows to give first answers to the questions listed in the introduction. All the measured vowel formants of the corpus (minus 4% rejected by filtering) occupy the vocalic space in an organised manner : for segments which are longer than 90 ms, the automatically measured values describe a "triangle" which is very close to the reference values published for French in the literature. For shorter durations the vocalic triangle shrinks progressively resulting in "concentric" surfaces for both French and German (see Figures 2 and3): the vocalic surfaces undergo a centripetal movement towards some central vowel position. The previous observations go in favour of the validity of automatic extraction from automatically aligned data, at least for the longer segments.

Comparing French and German, important differences in formant values could be measured for /y/ vowels between both languages. As German is a language with lexical accent, a more important tendency to reduction than for French could be expected (as suggested in [START_REF] Delattre | Comparing the prosodic features in English, German, Spanish, and French[END_REF]). However reductions are observed with a comparable degree in both languages, even if French high front vowels /i/, /y/, /e/ seem to be less prone to variation then German /i/, /y/, /Y/, /e/. This suggests that reduction is not an exclusively linguistic phenomenon, but admits also explanations of a physical or physiological nature.

Figure 1 :

 1 Figure 1: Overview of speech alignment : the audio stream is segmented and each segment is given an appropriate phonemic (or silence/breath/noise) label.

  For vowel duration D, the specified intervals [ D <= 50ms ]; [ 60 <= D <= 80ms ] and [ 90 <= D ] allowed the use of these numerical values as nominals for statistics. Results show that segment duration has a significant effect on the values of F1 [ F(49.25) p<0.0001 ] and F2 [ F(9.85) p<0.0001 ]. For German, observed variations as a function of vowel duration are shown in Figure 3 (see appendix): tendencies are globally similar to those observed for French.

Table 5 :

 5 Measured mean values of the first three formants of French according to vowel identity and speaker sex.

Figures 2 - 3 :Figures 4 - 5 :

 2345 Figures 2-3: Measures mean average values of F1 and F2 for French (left) and German (right) vowels according to their duration. By ascending order (black [30 -50ms], red [60 -80], blue [90 -110]). Male and female results are merged.

Table 1 :

 1 Proportion of rejected segments as a function of segment identity (in %).

	i	y	e	ϯ	a	oe ø	ѐ	o	u
	5	15 1	0.3 0.6 4	0.4 1	4.9 25

Table 2 :

 2 Proportion of segments measured and rejected as a function of segment duration D (ms).

	D duration	[30 -50]	[60 -80]	[90 -110]
	% proportion	39 %	38.5 %	22.5 %
	% rejection	6.1 %	2.8 %	2.4 %

Table 4 :

 4 Fi formant threshold values (minimum and maximum) in Hertz used for filtering.

	French -male

September, 4-8, Lisbon, Portugal

The schwa (/ђ/) and /oe/ are merged, as they share the same representation in the speech recognition system with the same acoustic model for both.

Acknowledgment

The present study was funded by the French CNRS in the framework of an interdisciplinary research program (STIC-SHS Société de l'Information MIDL project).