Muscarinic modulation of M and h currents in gerbil spherical bushy cells.

Descending cholinergic fibers innervate the cochlear nucleus. Spherical bushy cells, principal neurons of the anterior part of the ventral cochlear nucleus, are depolarized by cholinergic agonists on two different time scales. A fast and transient response is mediated by alpha-7 homomeric nicotinic receptors while a slow and long-lasting response is mediated by muscarinic receptors. Spherical bushy cells were shown to express M3 receptors, but the receptor subtypes involved in the slow muscarinic response were not physiologically identified yet. Whole-cell patch clamp recordings combined with pharmacology and immunohistochemistry were performed to identify the muscarinic receptor subtypes and the effector currents involved. Spherical bushy cells also expressed both M1 and M2 receptors. The M1 signal was stronger and mainly somatic while the M2 signal was localized in the neuropil and on the soma of bushy cells. Physiologically, the M-current was observed for the gerbil spherical bushy cells and was inhibited by oxotremorine-M application. Surprisingly, long application of carbachol showed only a transient depolarization. Even though no muscarinic depolarization could be detected, the input resistance increased suggesting a decrease in the cell conductance that matched with the closure of M-channels. The hyperpolarization-activated currents were also affected by muscarinic activation and counteracted the effect of the inactivation of M-current on the membrane potential. We hypothesize that this double muscarinic action might allow adaptation of effects during long durations of cholinergic activation.

Introduction

Cholinergic top-down connections exist at all levels of the auditory pathway to modulate sound information processing [1]. A well-documented descending system is the olivo-cochlear projection to the inner ear. Here, cholinergic activation reduces the excitability of the inner ear and facilitates unmasking of sounds in a noisy background by inhibiting the constant response to the noise and thus reducing wide-spread adaptation [2]. The olivo-cochlear bundle also sends collaterals into the cochlear nucleus [2,3-8]. In addition, about one quarter of cholinergic connections to the ventral cochlear nucleus comes from the pontomesencephalic tegmentum [8]. The function of these descending connections into the ventral cochlear nucleus (VCN) are not well understood. In the VCN, acetylcholine acts on stellate [9] and spherical bushy cells (SBC) [10] through both nicotinic and muscarinic receptors. The SBC resting membrane potential depolarized on two different time scales following a brief application of carbachol, a broad cholinergic agonist. Nicotinic α7 receptors mediate a fast, transient depolarization while muscarinic receptors cause a slow, long-lasting depolarization. Although immunohistochemical data show the presence of M3 muscarinic receptors on SBC bodies [5], the presence of other muscarinic subtypes and the currents involved in muscarinic depolarization remain unresolved. We hypothesize, that muscarinic modulation could act through slow voltage-activated currents, like the M- and h-currents, in spherical bushy cells.

The M-current (IM) is a slowly activating, non-inactivating voltage-gated potassium outward current which is sensitive to muscarine [11]. Muscarinic agonists induce a slow depolarization [11-13] by inhibiting IM and this effect is usually accompanied by a decrease in membrane conductance. The voltage-gated potassium channel Kv7 underlies IM [14] but there is evidence that other channels, like two-pore potassium channels, can also generate IM [15]. To our knowledge, the presence of IM in gerbil SBCs has not yet been demonstrated.

The hyperpolarization-activated cation current or Ih, is present in brainstem auditory neurons including SBCs [16]. It can affect the resting membrane potential and influence excitability [17-19]. Ih is mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels [20]. In vitro data have shown that Ih is sensitive to muscarinic activation in striatal cholinergic interneurons [21].

In this study, we identified the muscarinic receptor subtypes responsible for the SBC response to the application of cholinergic agents, and their associated currents. For that, whole-cell patch clamp recordings combined with pharmacology and immunohistochemistry were performed. We demonstrated that SBCs expressed IM and that both IM and Ih were affected by the application of oxotremorine-M (oxoM). Additionally, our immunohistochemical data showed that SBCs expressed two further subtypes of muscarinic receptor, M1 and M2.

Materials and methods

Animals

All experiments were performed in the Institute for Biology 2, RWTH Aachen University, Aachen, Germany in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and authorized by local state authorities (North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection, Recklinghausen, Germany). A total of 46 gerbils (Meriones unguiculatus) of either sex aged from post-natal day (P)14 to 30 were used.

Slice preparation

The animals were deeply anesthetized with isoflurane and quickly decapitated. The brain was removed from the skull and the brainstem was prepared in ice-cold cutting buffer for slicing. The cutting buffer contained (in mM): 215 sucrose, 10 glucose, 2.5 KCl, 4 MgCl2*6H2O, 0.1 CaCl2*2H2O, 1.25 NaH2PO4*2H2O, 25 NaHCO3, 3 myo-inositol (C6H12O6), 2 sodium pyruvate (C3H3NaO3), 0.5 L-ascorbic acid (C6H8O6), and was bubbled with 95% O2 and 5% CO2 to a pH value of 7.4 (308 mOsmol). The brainstem was cut either in frontal or parasagittal 180–250 μm slices by a vibrating microtome (VT1200S, Leica Biosystems, Nussloch, Germany). Then the slices were incubated for at least one hour in artificial cerebrospinal fluid (ACSF) at room temperature (RT, 25°C) containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 glucose, 3 myo-inositol (C6H12O6), 2 sodium pyruvate (C3H3NaO3), 0.5 L-ascorbic acid (C6H8O6), bubbled with 95% O2 and 5% CO2 to a pH value of 7.4 (314 mOsmol).

Electrophysiology

The slices containing the rostral part of the AVCN were placed in the recording chamber and perfused with oxygenated ACSF (100 ml/h) at RT under a fixed-stage microscope with IR-DIC and fluorescent imaging (Nikon Eclipse FN-1 microscope equipped with DS-Qi1MC camera and DC-U3 camera controller, Nikon Instruments, Japan). The patch electrodes were made from borosilicate glass filament capillaries (Science Products GmbH) with a horizontal DMZ Universal Puller (Zeitz-Instruments). The pipet resistance was between 2 to 5 MΩ (with overpressure) when the pipets were filled with a gluconate-based internal solution containing (in mM): 100 K-gluconate, 40 KCl, 0.1 CaCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 0.4 GTP, 0.1 Alexa-488 hydrazide (Thermo Fischer Scientific), 3 mg/ml biocytin (Thermo Fischer Scientific), adjusted to a pH of 7.2 with 1 M KOH (280 mOsm). The SBCs were recorded in whole-cell configuration with a HEKA EPC10 USB double patch clamp amplifier. The data were acquired by HEKA patchmaster software (HEKA Elektronik Dr. Schulze GmbH, Lambrecht/Pfalz, Germany). The recordings were low-pass filtered at 2.7 kHz and sampled at 50 kHz (100 kHz for the spontaneous miniature activity). The junction potential was corrected online for IM and Ih measurements (calculated at -11 mV). The series resistance (Rs) was uncompensated. Only cells with a Rs < 35 MΩ and with less than 30% change during the recordings were used. All electrophysiological recordings were made in slices from gerbils aged between P14-P25. This range was defined to get cell recordings as stable as possible after hearing onset, estimated at P12 [22].

IM current measurement

In voltage-clamp mode, the cells were initially held at -60 mV. The cells were then clamped to -20 mV for 1.8 s and hyperpolarizing steps from -70 to -30 mV with 10 mV increment were applied for 2 s before returning to -20 mV. For each voltage step, IM amplitude was measured as the difference between the instantaneous current (Iinst; maximum current in the first 100 ms of the 2 s voltage step) at the beginning of the step, caused by the leak conductance (or very rapidly voltage activated conductances), and the steady state current (Iss; average current of the last 150 ms of the 2 s voltage step) at the end. ZD7288 (20 μM; Bio Techne/Tocris UK) was added to ACSF to block Ih. In Fig 1, α-Dendrotoxin (αDTX, 50nM, Biozol, Germany) was also added in ACSF to block the Kv1.1 and Kv1.2 channels. Oxotremorine-M (oxoM, 1 μM; Bio Techne/Tocris UK) was locally applied with a perfusion pencil in absence or after washing in XE991 dihydrochloride (XE991, 10 μM; Bio Techne/Tocris UK).

Fig 1

Presence of IM in SBCs.

(A) Activation protocol (upper part) and its current response (lower part); scale: 500 pA. (B) Deactivation protocol (upper part) and its current response (lower part); scale: 200 pA. (C) Higher magnification of the current response shown in B at voltage step -50 mV; scale: 100 ms/100 pA.

Ih current

In voltage-clamp mode, the cells were held at -60 mV and stepped to voltages from -110 mV to -30 mV with 10 mV increment for 2 s. The current Ih was calculated as the difference between the instantaneous current and the steady-state current. IM was blocked by adding XE991 (10 μM) to ACSF. OxoM (1 μM) was locally applied in absence or after washing in ZD7288 (20 μM).

Membrane potential and excitability monitoring

In current-clamp mode, a set of 10 current steps was injected onto the SBCs every 30 s for 20 minutes. From every set of step currents a current-voltage curve was constructed from which the input resistance (IR) was derived as a linear fit to the subthreshold part of the current-voltage curve. Also, the resting membrane potential (RMP) was taken from the mean of the current clamp recordings prior to stimulus current injection. From a strongly hyperpolarizing current step the amount of “voltage sag” was measured as the difference between the maximum and the steady-state voltage deflection. From a suprathreshold current step the AP threshold potential was determined by thresholding the relation between the change of membrane potential (in mV/ms) and the membrane potential (i.e. a “phaseplane” plot). A threshold rate of change of 50mV/ms was taken as indicative of the onset of the rising phase of the AP. This procedure yields the membrane potential at which the steep incline of the AP begins, which we took as an estimate of the AP threshold. Changes in RMP, IR, voltage sag and AP threshold potential were observed upon wash-in of carbachol (carbamylcholine chloride, 500 μM; Sigma Aldrich Germany), oxotremorine-M (1 μM) or 4DAMP (1 μM; Bio Techne/Tocris UK). The first 2.5 min of these experiments were discarded (duration that the cell needed to stabilize). The values for RMP, IR, voltage sag and AP threshold potential were normalized to the mean of the values obtained during the 2.5 min control condition. For statistical analysis, the mean of the values obtained during 2.5 min per cell for each condition were averaged and compared (i.e., all values for control condition, 2.5 min where the effect was maximal with the agent tested and the last 2.5 min for washout, unless stated otherwise) by one way ANOVA with repeated measures.

Evoked and spontaneous miniature EPSCs

An Iso-Flex stimulus isolator (A.M.P.I., Jerusalem, Israel) was connected to a 250 μm bipolar tungsten electrode (Micro Probe Inc., Gaithersburg, USA) of 1.5 MΩ impedance placed in the AVCN near the patched cell to electrically stimulate AN fibers as described in [10]. The stimulus intensity was set to 1.2 times the threshold intensity of the activation of one single input (range: 14–28 V). The stimulation protocol consisted of a stimulus train of 30 stimuli at 50 Hz, 100 Hz or 200 Hz, repeated 10 times with 30 s interval and was performed before and after washing in carbachol (500 μM) for 10 minutes. The EPSC 10%-90% rise time was determined and the EPSC decay time constant was calculated by fitting a single exponential function to the decaying slopes of EPSC. The eEPSC amplitude was determined by the difference between the crossing point of the rising slope and the baseline linear fits, and the maximal deflection point. To quantify short-term depression, the amplitude of eEPSCs for each stimulus was averaged over 10 repetitions and normalized to the first eEPSC amplitude. The size of the readily-releasable pool was estimated with the back-extrapolation method [23] from 100Hz pulse-trains. Using the estimated pool-size in nA and the amplitude of the first EPSC, the initial release probability was calculated. We furthermore estimated the amount of asynchronous release as in [24].

Spontaneous miniature EPSCs (mEPSCs) were recorded for several minutes before, during and after application of carbachol (500 μM). The mEPSCs were detected by a custom-made automated detection algorithm briefly described below. This algorithm is based on an algorithm developed by [25]. First, ten to twenty mEPSCs were detected manually for each cell and averaged to create a template. Second, low-frequency noise was removed by subtracting a polynomial function (9th-order, unconstrained fitting procedure) fitted to the whole 1000ms current trace. Third, the template was slid along the current trace. To fit the data, the template was optimally scaled and offset at each position of the trace. Finally, a detection criterion was determined by the template scaling and the goodness of the fit at each point of the data. The event was detected when this criterion crossed a threshold set manually for each cell.

All EPSC recordings were made in presence of glycine and GABA receptor blockers: strychnine (1 μM; Sigma Aldrich Germany), gabazine (10 μM; Abcam UK) and CGP 55845 hydrochloride (2 μM; Bio Techne/Tocris UK).

Biocytin-streptavidin staining

During recordings, the cells were filled with biocytin contained in the internal solution (see above) to morphologically confirm the cell type afterwards. At the end of the experiment, the slices were immersed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight for fixation and transferred in PBS. After rinsing several times with PBS, the slices were washed with 0.1% Triton X-100 in PBS. Then, the slices were incubated for 2.5h at RT in a streptavidin solution containing: 0.1%Triton X-100, 1% bovine serum-albumine (BSA) in PBS, Alexa Fluor® dye streptavidin conjugates (1/800, Cat. No. S11223, Thermo Fisher Scientific, Germany). The slices were washed several times, first with PBS, second with 0.3% Triton X-100 in Tris buffered saline (TBS) and finally with TBS only. At the end, a 4`-6-diamidin-2-phenylindol-dye (DAPI) staining was performed (0.1μg/ml in PB). Each slice was placed on a coverslip inside of a 240 μm thick adhesive frame (Grace Bio-Labs, USA). Fluoprep (bioMérieux, UK) was applied and the slices sealed in with another coverslip placed firmly on the adhesive frame. This technique allowed direct imaging of slices up to 240μm without distortions due to pressure exerted by the cover slip. Neurons in the slices were imaged with a laser-scanning confocal microscope (TCS SP2, Leica microsystems, Germany). The z-resolution in stacks of confocal images was set to 0.5 μm. The contrast and the brightness of all images were adjusted using ImageJ (RRID: SCR_003070).

Immunohistochemistry

For immunohistochemistry, P30 gerbils (n = 3) were very deeply anesthetized with an intraperitoneal injection of ketamine/xylazine (150 and 10 mg/kg, respectively). Then, the sternum was cut and removed to expose the heart. The right atrium was immediately opened to sacrifice the animal. A 21G cannula was carefully inserted and clamped into the left ventricle through which 15 ml ice-cold PBS was perfused first. Then 15 ml ice-cold fixative (4% PFA in PBS) was perfused. The animal was decapitated, the brain was removed from the skull and kept in 4% PFA in PBS at 4°C overnight. Then the brain was successively immersed in 10 and 30% sucrose solutions at 4°C for cryoprotection until sunk.

The part of the brainstem containing the CN was fully covered by Tissue-Tek (Sakura Finetek, AJ Alphen aan den Rijn, The Netherlands). Parasagittal and frontal sections of 30 μm were cut using a cryotome (CM3050S, Leica Biosystems, Wetzlar, Germany). The CN sections were collected on coated slides (Scientific Menzel-Gläser Superfrost Plus) and double stained with the following steps. First, the sections were washed with PBS and incubated at RT for 3h with a blocking solution containing: 4% normal horse serum (NHS), 0.4% Triton X-100, 1% BSA in PBS. Second, the sections were incubated for 24h at 4°C with a primary antibody solution containing: 1% NHS, 0.3% Triton X-100, 1% BSA in PBS and the primary antibodies. Third, after washing with PBS, the sections were incubated for 3h at RT with the secondary antibody solution containing: 0.02% Triton X-100, 1% BSA in PBS and the secondary antibodies. Finally, the sections were rinsed in PBS and stained with DAPI (1μg/ml in PB). Once coverslipped (in Fluoprep, bioMérieux, UK) and sealed, the sections were stored in dark at 4°C until imaging with a laser-scanning confocal microscope (TCS SP2, Leica microsystems, Germany). The contrast and the brightness of all images were adjusted using ImageJ (RRID: SCR_003070).

The primary antibodies used were: goat anti-calretinin antibody (1/500, Merck Millipore, Germany), rabbit anti-M1 muscarinic receptor (443–458) antibody (1/200, #AMR-010, RRID: AB_2340994, Alomone labs, Israel) and rabbit anti-M2 muscarinic receptor antibody (1/200, #AMR-002, RRID: AB_2039995, Alomone labs, Israel). The secondary antibodies used were: donkey anti-goat alexa488 (1/500, Life Technologies, USA) and donkey anti-rabbit alexa546 (1/500, Life Technologies, USA). The M1 staining was performed on a total of 7 slices and M2 staining on 13 slices.

Statistics

We performed one- and two-way analysis of variance (ANOVA) with repeated measures using custom MATLAB (RRID: SCR_001622) code. For this we constructed repeated measure models from our data using the function fitrm.m and performed two-way ANOVA with repeated measures for the factors time/pulse number, pharmacological condition and the interaction of these factors. In the result section we report the F-statistics as F(a,b), with a and b being the degrees of freedom of factor and error, respectively. We never assumed sphericity and thus always use the conservative Greenhouse-Geiser correction of probability values. One-way ANOVA with repeated measures was performed accordingly for only one factor (pharmacological treatment). In case of significant outcomes of the repeated measure ANOVA, pairwise comparisons were performed with the MATLAB function multcompare.m for each factor. Here, probabilities were Tukey-Cramer corrected. Significance was determined for an α value of <0.05. In several occasions we also performed regular analysis of variance (ANOVA) and pairwise t-tests. Unless noted otherwise all data are expressed as mean ± standard error of the mean (SEM).

Results

Inhibition of M-current by oxotremorine-M in SBCs

In voltage clamp, both activation- and deactivation-, (or relaxation) protocols (upper parts in Fig 1A and 1B, respectively) permit measurement of IM. For the former, the cell was held at -60 mV, and stepped to more depolarized values. In presence of Kv1.1/2 and HCN channel blockers, the slowly developing outward current (Fig 1A, lower part) reflected the slow opening of M-channels. In Fig 1A, IM was estimated at 690 pA at +10 mV step. For the deactivation protocol, the voltage was first clamped to -20 mV, which activated a substantial amount of M-channels and keep a constant outward IM (Fig 1B). Applying hyperpolarizing steps from this holding potential induced an instantaneous inward current followed by a slow inward current relaxation corresponding to the gradual closure of M-channels (Fig 1B and 1C). Please note that this protocol was thus designed to demonstrate non-inactivating, slow voltage activated conductances: other outward currents like A-type potassium currents would be inactivated by the high holding potential. Rapidly gated, non-inactivating outward currents, like Kv3-mediated potassium currents, are mostly deactivated during the “instantaneous” phase and thus do not pollute the slow relaxation current. A true identification of the non-inactivating, slow voltage activated outward current as M-current however must involve pharmacological experiments (see below). At -50 mV, the inward relaxation (difference between Iinst and Iss) was estimated at 96 pA. Stepping back to -20 mV resulted in a smaller instantaneous current (I’inst) coincident with a reduction of the conductance caused by the closure of M-channels (Fig 1C). The slow opening of M-channels could be visualized by the slow outward tail IM current equal to 193 pA at -50 mV (Fig 1C). For the most hyperpolarizing steps, a rapidly activating and rapidly inactivating outward current could be observed matching the kinetics of the potassium A-current (indicated by the arrow in Fig 1C).

In the activation protocol, measurements of IM are potentially contaminated by slow activation of other ion channels, unless a combination of blockers are added to the ACSF. The inward relaxation observed in the deactivation protocol however is considered to be mainly mixed with the activity of HCN channels. For this reason, the following quantification of IM, before and after application of pharmacological agents, were only made with the deactivation protocol (Fig 2A) in the presence of ZD7288, a HCN channel blocker, in 16 cells (from gerbils aged from P14 to P25,cell capacitance = 23 ± 2 pF, IR = 156 ± 18 MΩ, Rs = 16.3 ± 1.5 MΩ).

Fig 2

IM suppression by oxoM in an example cell.

(A) Deactivation protocol with voltage steps (2 s) from -70 mV to -30 mV. (B) Example traces of current recordings in control (ctrl) condition (left part), after XE991 wash-in (middle part) and application of oxoM in presence of XE991 (right part); scale: 200 pA. The insets on the top right are magnifications of the -50 mV step; scale: 100 ms/50 pA. (C) Higher magnification of the -50 mV step in B (left part) showing how the inward relaxation, IM, was measured, i.e. by the difference between instantaneous Iinst and steady state Iss currents; scale 100 ms/100 pA. A clear reduction of the inward relaxation was observed between the ctrl and XE991 conditions at -50 mV, see insets in B left and middle parts, while no further substantial change was noticed when oxoM is applied (B, right part). (D) Values of IM at each voltage for the cell shown in B. (E) Example traces of current recordings in control condition (left part) and after application of oxoM (right part); scale: 200 pA. The insets are the magnification of the -50 mV step; scale: 100 ms/50 pA. The inward relaxation IM was clearly reduced by oxoM. (F) Values of IM at each voltage for the cell shown in E.

The inward relaxation current was measured before and after wash-in of XE991, a blocker of Kv7 channels known to substantially contribute to IM. The results for one example cell are presented in Fig 2. The maximum values of IM for this example cell in control conditions were measured at -50 and -40 mV steps for this cell, 108 and 111 pA, respectively (Fig 2C and 2D). The relaxation current was almost non-existent at -70 mV (Fig 2B and 2D) and reversed between -80 and -70 mV (not shown). The application of XE991 strongly reduced IM at all voltages (Fig 2B, middle part and 2D). Next, we applied oxoM which also induced a strong reduction (Fig 2E and 2F) from 74 to 19 pA at -50 mV. However, a substantial part of IM (20–30%) was still present at -50 and -40 mV after perfusion of both XE991 and oxoM. In this example, oxoM did not induce further substantial reduction in presence of XE991 (Fig 2B) since IM was almost entirely inhibited by XE991. Nonetheless, at -50 and -40 mV, a small additional reduction was discernable (Fig 2B, insets in middle and right parts and 2D).

In Fig 3A we show group data for the same experiments as described in Fig 2. On average, both step-voltage (F(4,16) = 6.35, p<0.05; n = 5) and XE991 treatment (F(1,4) = 14.69, p<0.05; n = 5) had a significant effect on IM amplitudes when tested with two-way ANOVA with repeated measures. There was also a significant interaction between the two factors (F(4,16) = 7.39, p<0.05; n = 5). Post-hoc pair-wise testing revealed significant differences between the control and the XE991 condition for the step voltages -40mV (p<0.05), -50mV (p<0.01) and -70mV (p<0.05). At -30mV (p = 0.09) and -60mV (p = 0.06) the differences were not statistically significant. The average of IM amplitude at -50 mV (n = 5) was reduced from 57 ± 15 pA to 5 ± 3 pA after washing in XE991. Noticeably, IM was almost completely abolished at more hyperpolarized voltage steps (-70 and -60 mV) whereas a persistent inward relaxation was recorded at -40 and -30 mV (17 ± 1 pA and 19 ± 4 pA, respectively, Fig 3A).

Fig 3

Quantification of IM suppression by oxoM in SBCs.

(A) Voltage-current relationship of the mean of the inward relaxation current IM (n = 5) in control and after application of XE991. (B) Voltage-current relationship of the mean of the inward relaxation current IM (n = 8) in control and after application of oxoM. (C) Voltage-current relationship of the mean of the inward relaxation current IM (n = 5) in presence of XE991 and after application of oxoM. (D) Percentage of IM at -50 mV inhibited by the application of XE991 (black), oxoM (grey) and oxoM in presence of XE991 (white). Application of XE991 or oxoM alone strongly reduced the inward relaxation current IM at -50 mV. Application of oxoM only inhibited a small amount of the residual IM when the cell was pre-treated with XE991. * p<0.05; ** p<0.01.

In Fig 3B we show group data for the muscarinic modulation of IM (cf. Fig 2F). On average, both step-voltage (F(4,28) = 19.6, p<0.001; n = 8) and oxoM treatment (F(1,7) = 32.8, p<0.001; n = 8) had a highly significant and very strong effect on IM amplitudes (two-way ANOVA with repeated measures). There was, again, a significant interaction between the two factors (F(4,28) = 8.42, p<0.001; n = 8). Post-hoc pair-wise testing revealed significant differences between the control and the oxoM condition for all step voltages (-30mV: p<0.01; -40mV: p<0.01; -50mV: p<0.001; -60mV: p<0.01) except -70mV (p = 0.16).

The small XE991-resistant current was not seen when oxoM was applied (Fig 3B). In presence of XE991 (n = 5), further change of inward relaxation caused by oxoM was minimal or none (Fig 3C). This is reflected in the statistical analysis comparing the XE991 condition with the XE991+oxoM condition: neither voltage (F(4,16) = 0.86, p = 0.41) nor pharmacological condition (F(1,4) = 7.41, p = 0.053) was a significant factor, nor was any interaction between the factors left (F(4,16) = 2.36, p = 0.17).

In Fig 3D, for each agent tested, the percentage of IM inhibition was calculated (% inhibition = (1-(Itest/Ictrl)) x 100) at -50 mV to further illustrate our findings. In control condition, XE991 and oxoM inhibited IM by 94 ± 6% and 82 ± 7%, respectively (ANOVA p<0.001, df = 2, F = 13.76; post-hoc test p>0.9999 XE991 (black) vs. oxoM (grey)). On the other hand, oxoM application in presence of XE991 had a weak effect on residual IM (13 ± 22%) compared to the other conditions (post hoc p<0.01, oxoM vs. XE991+oxoM; p<0.001, XE991 vs. XE991+oxoM), indicating a broad overlap of affected conductance between XE991 and oxoM.

The strong reduction of the inward relaxation by XE991 application showed the presence of IM most likely caused by Kv7 (KCNQ-type) channels in SBCs. This current was strongly modulated by the application of a muscarinic agonist suggesting that IM plays a role in the cholinergic influence on SBCs.

Inhibition of Ih by oxotremorine-M in SBCs

Ih current was recorded (Fig 4A) in presence of XE991 to avoid contamination by IM. Ih is mediated by the family of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) and can be blocked by ZD7288 (Fig 4B, left and middle parts). The amplitude of Ih was measured in 14 cells (from gerbils aged from P19 to P25, cell capacitance = 18 ± 1pF; IR = 102 ± 8 MΩ, Rs = 19.9 ± 1.4 MΩ). The slow inward current, mostly present at voltages more negative than -60 mV (Fig 4B), reflected the slow opening of HCN channels. Ih was almost completely abolished (from 402 to 46 pA at -110 mV) after applying ZD7288 for at least 10 minutes (Fig 4B, middle part). No further noteworthy change was observed after application of oxoM (Fig 4B, right part). In contrast to this, in absence of ZD7288 oxoM prominently decreased Ih amplitude from 382 to 59 pA at -110 mV (Fig 4E). Fig 4E and 4F summarize the effect of ZD7288 and oxoM on Ih amplitude for each voltage step for this example cell. The profile of the two IV curves was similar in response to the two agents tested. At -30 and -40 mV in control condition, Ih was negligible (17 and 22 pA in Fig 4D and 15 and 12 pA in Fig 4F, respectively). In the presence of either ZD7288 or oxoM, Ih was strongly reduced at each voltage but the most robust decrease was observed at the highest hyperpolarized value tested (Ih reduced by 8 times with ZD7288 and 7 times with oxoM, see Fig 4D and 4F). Fig 4D shows that there was no further effect of oxoM on membrane currents upon hyperpolarizing voltage steps when ZD7288 was already present in the ACSF.

Fig 4

Ih suppression by oxoM in an example cell.

(A) Deactivation protocol with voltage steps (2 s) from -110 mV to -30 mV. (B) Example traces of current recordings in control (ctrl) condition (left part), after ZD7288 wash-in (middle part) and application of oxoM in presence of ZD7288 (right part); scale: 500 pA. (C) Step at -110 mV from B (left part) showing how the inward current Ih was measured. Ih was clearly visible at the more hyperpolarized voltage steps in ctrl but was almost completely gone in presence of ZD7288. (D) Values of Ih at each voltage for the cell shown in B. (E) Example traces of current recordings in control condition (upper part) and after application of oxoM; scale: 500 pA. (F) Values of Ih at each voltage for the cell shown in E. Ih was also considerably reduced by the application of oxoM alone.

Group results for the same experiments as shown in Fig 4 are presented in Fig 5. The average amplitudes of Ih (Fig 5A) were significantly influenced by voltage (F(8,32) = 50.23, p<0.001; n = 5) and ZD7288 (F(1,4) = 80.9, p<0.001; n = 5). Post-hoc pair-wise comparisons revealed, that the reduction of Ih by ZD7288 was statistically significant and strong for step voltages of -70 mV (reduction of -129 pA ± 14.7 pA, p<0.001) and all more hyperpolarized values (-80 mV: -178 ± 19.5 pA, p<0.001; -90 mV: -261 ± 31 pA, p<0.01; -100 mV: -319 ± 29.3 pA, p<0.001; -110 mV: -375 ± 46.5 pA, p<0.01).

Fig 5

Quantification of Ih suppression by oxoM in SBCs.

(A) Voltage-current relationship of the mean of the current Ih (n = 5) in control and after application of ZD7288. (B) Voltage-current relationship of the mean of the current Ih (n = 5) in control and after application of oxoM. (C) Voltage-current relationship of the mean of the current Ih (n = 6) in presence of ZD7288 and after application of oxoM. (D) Percentage inhibition of Ih at -110 mV induced by the application of ZD7288 (black) or oxoM (grey) in control condition or by the application of oxoM in presence of ZD7288 (white). Application of ZD7288 or oxoM alone significantly reduced Ih at -110 mV. Application of oxoM did not reduce Ih when the cell was pre-treated with ZD7288. * p<0.05; ** p<0.01.

OxoM application (Fig 5B) on average also strongly reduced H-currents in n = 5 spherical bushy cells. Again, both voltage (F(8,32) = 34.9, p<0.01; n = 5) and oxoM (F(1,4) = 26, p<0.01; n = 5) significantly influenced Ih. Again, there was a significant interaction between the factors (F(8,32) = 43.6, p<0.001). Similar to the results for the ZD7288 experiment, oxoM significantly reduced Ih for step voltages of -70 mV (reduction of -73 ± 8.7 pA, p<0.01) and lower (-80 mV: -105 ± 15 pA, p<0.01; -90 mV: -141 ± 24 pA, p<0.01; -100 mV: -182 ± 32 pA, p<0.01; -110 mV: -218 ± 36 pA, p<0.01).

The presence of ZD7288 before oxoM application prevented large effects of oxoM on Ih (Fig 5C). When we tested the ZD7288 vs. the ZD7288+oxoM conditions in two-way ANOVA with repeated measures, voltage was still a weak but significant factor for Ih (F(8,40) = 13.6, p<0.01; n = 6) but not pharmacological condition (F(1,5) = 1.18, p = 0.32; n = 6). A significant interaction between the factors (F(8,40) = 9.6, p<0.01; n = 6) was apparent. This finding was supported by the post-hoc pair-wise testing: For voltages of -90 mV (reduction of -19 ± 8.1 pA, p<0.01) and -100 mV (reduction of -50 ± 16 pA, p<0.05) oxoM further reduced the average currents, suggesting incomplete block of Ih by ZD7288 or an oxoM effect on leak channels that are ZD7288 insensitive. We will further discuss this finding in a later section of this paper.

At -110 mV, ZD7288 and oxoM similarly inhibited Ih by 80 ± 7% and 81 ± 4%, respectively (Fig 5D, ANOVA p<0.0001, df = 2, F = 31.3; post-hoc test p>0.9999 ZD7288 vs. oxoM) while the effect of oxoM in presence of ZD7288 was considerably reduced, 22 ± 8% (oxoM (grey) vs. ZD7288+oxoM, p<0.0001; ZD7288 vs. ZD7288+oxoM, p<0.0001).

Overall these data showed that Ih was strongly decreased by the application of a muscarinic agonist in SBCs. This suggests that both IM and Ih could potentially play a role as effectors in the cholinergic modulation of spherical bushy cells.

Expression of M1 and M2 muscarinic receptors in AVCN

The expression of M3 receptor, known to modulate the M-current, was already shown in the neuropil of gerbil SBCs [5]. However, several studies have shown that M1 also played a role in IM [26-30]. In addition, Ih can be modulated by the intracellular signaling pathway triggered by the Gi/oα protein, which is activated by M2 and M4 muscarinic receptors. For this reason, the presence of other muscarinic receptors was evaluated in parasagittal slices of the CN. Here we present the results obtained for M1 and M2. The calretinin-positive auditory nerve terminals helped to differentiate the SBCs (filled arrows) in the AVCN from other cell types (Fig 6A). A strong M1 signal was uniformly present in the entire AVCN and localized on cell bodies (Fig 6B). All AVCN neuronal cell bodies seemed to present the M1 staining suggesting that SBCs are not the only cell type expressing M1 in the AVCN (Fig 6C and 6D, empty arrows).

Fig 6

Expression of M1 and M2 muscarinic receptors in gerbil AVCN.

(A) Calretinin (CR) staining in a parasagittal slice of gerbil cochlear nucleus. The filled arrows point to strong calretinin staining corresponding to big auditory nerve fiber terminals, the endbulb of Held. Scale: 100 μm. (B) M1 muscarinic staining was found on all AVCN cell bodies. (C) Merged image of A and B. Filled arrows show M1-positive SBCs. Empty arrows show M1-positive cells which are not SBCs. (D) Digital magnification of the white square in C. Scale: 50 μm. (E) Calretinin staining in a parasagittal slice of gerbil cochlear nucleus in which endbulb of Held could be identified. Scale: 100 μm. (F) M2 muscarinic staining was mostly present in the anterior part of AVCN, around SBCs. Filled arrowheads indicate sparse but strong M2 staining close to cell bodies. (G) Merged image of E and F. (H) Digital magnification of the white square in G. Empty arrowheads point to M2-positive puncta on SBC cell bodies. Scale: 50 μm (magnification: 63x). D = dorsal; P = posterior; NR = nerve root.

Fig 6E and 6F show calretinin and M2 staining, respectively. Contrary to M1, M2 appeared weaker, more intense near the lateral edges of AVCN and mainly localized in the neuropil of AVCN cells. Few intense M2 signals could be noticed around cell bodies (Fig 6F, 6G and 6H, filled arrowheads) but no co-localization with calretinin-positive endbulbs was observed. In addition, sparse M2-positive puncta were observed on SBC cell bodies (Fig 6H, empty arrowheads) implying that M2 acts on the postsynaptic site. This suggests that M2 in the gerbil AVCN might not function as heteroceptor on the presynaptic site, as was previously reported [31] for AN terminals.

These results confirmed the presence of both M1 and M2 muscarinic receptors in AVCN. Staining against M1 was stronger and mainly found on the postsynaptic site of both SBCs and non-SBCs while M2 was predominantly localized in the neuropil surrounding SBCs. In addition to the M3 muscarinic receptor expression we have shown before [5], SBCs express a variety of muscarinic receptors of partially overlapping function. The non-uniform distribution of M2 signals throughout the AVCN suggests the possibility of a diversity of muscarinic responses in SBC.

No effect of carbachol on synaptic transmission at the endbulb of Held

Since immunohistochemical results from our previous work pointed out evidence of presynaptic nicotinic receptors [5,10] and we showed the expression of M2 in AVCN (which can act as presynaptic heteroceptor), the effect of a cholinergic agonist on the synaptic transmission between auditory nerve and SBC was evaluated. For that, synaptic transmission was investigated by recording evoked EPSCs (n = 6, from gerbils aged from P15 to P17, cell capacitance = 33 ± 4 pF, IR = 234 ± 16 MΩ, Rs = 10.9 ± 1.2 MΩ) and spontaneous miniature EPSCs (n = 8, from gerbils aged from P16 to P20, cell capacitance = 30 ± 3 pF, IR = 276 ± 50 MΩ, Rs = 12.3 ± 1.1 MΩ) before and after 10 minutes of carbachol application.

A frequency-dependent depression of evoked EPSC amplitudes (Fig 7B1-3, n = 6) was evident in our recordings. Even though in the 200 Hz conditions many missing values (which represent insufficient EPSC at that given pulse number) made statistical analysis difficult, the pulse number generally had a highly significant influence on EPSC amplitude (two-way ANOVA with repeated measures, 50 Hz: F(29,145) = 45.2, p<0.001; 100 Hz: F(29,145) = 52.7, p<0.001; 200 Hz: F(29,29) = 37.9, p = 0.1). However, depression of EPSC amplitudes was not statistically different between the control and the carbachol groups (50 Hz: F(1,5) = 0.89, p = 0.38; 100 Hz: F(1,5) = 1.56, P = 0.26; 200 Hz: F(1,1) = 1.5, p = 0.43) and very weak or no significant interaction between pulse-number and pharmacological treatment was observed (50 Hz: F(29,145) = 3.25, p = 0.04; 10 0Hz: F(29,145) = 2.51,p = 0.15; 200 Hz: F(29,29) = 1.34, p = 0.45). The 10%-90% rise time of eEPSC1-15 and the decay time constant of eEPSC1-15 were determined for all frequencies and in both conditions (Fig 7B4-6 for rise time, 7B7-9 for decay time constant). For the 10%-90% rise time we found a robust effect of pulse number (two-way ANOVA with repeated measures, 50 Hz: F(13,65) = 15.9, p<0.001; 100 Hz: F(13,65) = 11.7, p<0.01; 200 Hz: F(13,39) = 12.1, p<0.01), however we did not find a statistically significant influence of pharmacological condition (50 Hz: F(1,5) = 0.06, p = 0.8; 100 Hz: F(1,5) = 0.002, p = 0.96; 200 Hz: F(1,3) = 0.68, p = 0.46) or any interactions (50 Hz: F(13,65) = 0.38, p = 0.79; 100 Hz: F(13,65) = 0.41, P = 0.65; 200 Hz: F(13,39) = 2.1, p = 0.22). For the decay time constant of EPSC1-15, neither pulse number, pharmacological condition nor the interaction of the factors had a statistically significant effect when tested with a two-way ANOVA with repeated measures.

Fig 7

No effect of carbachol on evoked EPSCs and short term depression.

(A) Example traces of recorded eEPSCs at 100Hz during the 30 stimulus train in control (ctrl) condition (upper part) and in presence of carbachol (carb, lower part); scale: 50 ms/3 nA. In both cases, a strong short-term depression was observed. (B1-3) Normalized average amplitude of eEPSCs for 50 Hz (B1), 100 Hz (B2) and 200 Hz (B3) compared between control (black) and carbachol (grey) conditions. No significant effects of carbachol were found. n = 6. (B4-6) Rise time of eEPSCs for 50 Hz (B1), 100 Hz (B2) and 200 Hz (B3) compared between control (black) and carbachol (grey) conditions. No significant effects of carbachol were found. n = 6. (B7-9) Decay time constant of eEPSCs for 50 Hz (B1), 100 Hz (B2) and 200 Hz (B3) compared between control (black) and carbachol (grey) conditions. No significant effects of carbachol were found. n = 6. (C) Estimated amount of asynchronous release (given as the amount of area under the curve of the second half of the stimulus duty cycle, divided by the area under the curve of the first half of the stimulus duty cycle) steeply increased with stimulus frequency but is not affected by carbachol. Please note: individual values (open circles) for the 200Hz condition masked by the marker for the group mean (closed circles). (D) The estimated release probability of the endbulb of Held (given as the amount of current released by the first EPSC divided by the estimated size of the readily-releasable pool) does not significantly depend on stimulus frequency or carbachol.

In addition to EPSC dynamics we estimated the amount of asynchronous release of the synapse during steady-state stimulation at three different stimulation frequencies (Fig 7C). While a highly significant increase of estimated asynchronous release with frequency was evident (two-way ANOVA with repeated measures, F(2,10) = 218, p<0.001), pharmacological condition had no influence (F(1,5) = 0.54, p = 0.49), neither did we find any interaction of factors (F(2,10) = 0.25, p = 0.7).

Finally we wanted to roughly estimate the release probability of the synapse at the different conditions. For this we estimated the readily-releasable pool from our pulse-train data with a back-extrapolation method and expressed the release probability as the fraction of the pool (in nA) expended by EPSC1 (Fig 7D). Neither stimulation frequency, nor pharmacological condition or the interaction of the factors had a statistically significant effect on the release probability when tested with a two-way ANOVA with repeated measures.

We therefore concluded, that we could not find any significant influence of cholinergic signalling on endbulb of Held synchronous and asynchronous evoked synaptic transmission in the gerbil.

Next we wanted to analyze the effect of carbachol on spontaneous release of vesicles at the endbulb of Held and thus recorded spontaneous miniature synaptic currents (mEPSC). The mEPSCs were recorded during at least 5 minutes. Example traces are shown in Fig 8A. The average amplitude, the frequency and the kinetics of mEPSCs were measured before, during and after applying carbachol. The mean mEPSC amplitude was 39 ± 17 pA (n = 8) in control conditions. No significant change was observed in presence of carbachol (35 ± 15 pA, n = 8) and after wash-out (32 ± 14 pA; Fig 8B, n = 8; one-way ANOVA with repeated measures: F(2,14) = 2.29, p = 0.16). The average frequency was 0.55 ± 0.16 Hz (n = 8) in control condition and remained at, 0.55 ± 0.15 Hz (n = 8) in the presence of carbachol and 0.56 ± 0.14 Hz after wash-out (Fig 8C, F(2,14) = 0.07, p = 0.82). The mean 10%-90% rise time was 405 ± 88 μs (control, n = 8), 415 ± 89 μs (carbachol, n = 8) and 460 ± 110 μs (wash-out, n = 8, Fig 8D). There was a significant difference between the wash-out and the carbachol condition (p<0.05 after one-way ANOVA with repeated measures, F(2,14) = 5.86, p<0.05, post-hoc t-tests are Bonferroni corrected). However, the mean effect size was very small and there was no difference between the control and the wash-out condition (p = 0.07). The mean time constants of mEPSC decay were 1.02 ± 0.25 ms (control, n = 8), 1.16 ± 0.41 ms (carbachol, n = 8) and 1.24 ± 0.47 ms (wash-out, n = 8, Fig 8E), differences were not significant (F(2,14) = 4.1, p = 0.06). Overall we conclude that there was no influence of carbachol on spontaneous vesicle release at the endbulb of Held of the gerbil.

Fig 8

No influence of carbachol on spontaneous mEPSCs.

(A) Example traces of recorded mEPSCs in control condition (ctrl, left part), in presence of carbachol (carb, middle part) and after washout carbachol (washout, right part); scale: 2 s/50 pA. The red dots represent the automatically detected mEPSCs. (B) Average amplitude, (C) average frequency, (D) average rise time and (E) average decay time of mEPSCs before, during and after washing out carbachol. No difference was observed between the three conditions for amplitude and rise- or decay kinetics. Although there was a significant difference between carbachol and wash-out for the mEPSC rise-time (asterisk in D) the effect was only very small.

Taken together, our measurements of evoked and spontaneous synaptic events at the endbulb of Held suggested that cholinergic transmission in the AVCN predominantly affected the excitability of the spherical bushy cell and did not significantly affect synaptic release or short term plasticity.

Slow muscarinic effects on SBC membrane potential and excitability

The application of oxoM decreased IM and Ih currents but the physiological meaning of this was unclear. In a set of current clamp experiments (Fig 9 & Fig 10), functional physiological parameters were monitored over several minutes every 30 s before, during and after bath application of carbachol (broad cholinergic agonist), oxoM or 4DAMP (M3 antagonist) in a total of 34 cells (from gerbils aged from P14 to P22; cell capacitance = 22 ± 2 pF; IR = 78 ± 3 MΩ, Rs = 15.8 ± 1.5). The average RMP in control condition was -61.95 ± 0.36 mV.

Fig 9

RMP and IR monitoring during application of cholinergic compounds.

(A) Voltage response to 10 current step injections in control (left), after 3 min (middle) and 10 min (right) of carbachol perfusion; scale: 200 ms/30 mV. (B) Current-voltage curves of the recordings shown in (A). (C) Mean of RMP change over time (upper part). The thicker black line on the X axis represents the duration of carbachol perfusion. The 3 arrows indicate the time of the recordings shown in A. The average of 2.5 min was plotted for each condition (lower part). (D) Mean of IR change over time (upper part) and average over 2.5 min in each condition (lower part). (E) Mean of RMP change over time (upper part). The thicker green line on the X axis represents the duration of the perfusion of ACSF (control experiment). (F) Mean of IR change over time (upper part) and average over 2.5 min in each condition (lower part). (G) Mean of RMP change over time (upper part). The thicker blue line on the X axis represents the duration of the oxoM perfusion. (H) Mean of IR change over time (upper part) and average over 2.5 min in each condition (lower part). (K) Mean of RMP change over time (upper part). The thicker red line on the X axis represents the duration of the 4DAMP perfusion. (L) Mean of IR change over time (upper part) and average over 2.5 min in each condition (lower part). Shaded areas represent the standard error.

Fig 10

AP threshold and voltage sag monitoring during application of cholinergic compounds.

(A) Voltage response to de- and hyperpolarizing current injections in control (left), during (middle; wash-in) oxotremorine-M perfusion and after (right; recovery) oxotremorine-M perfusion; scale: 50 ms/25 mV. Thin grey lines illustrate the difference between maximum- and steady-state depolarization, which we analyze here as voltage sag caused by activation of Ih. (B) Phaseplane plots illustrating the relation between the rate of change of the membrane potential and the membrane potential during the occurrence of the action potentials shown above. A value of >50 mV/ms was taken as AP threshold (marked with an x; note that due to the low sample number during the rapid rise the exact value of the threshold can be different in the three plots). It becomes clear that the AP threshold shifts towards hyperpolarized values during oxoM application (middle panel: thick grey line = oxoM, thin black line = control) and recovers after wash-out (right panel: thin grey line = oxoM, thick black line recovery). (C-F) Mean of AP threshold change over time for carbachol (C), ACSF (D), oxotremorine-M (E) and 4DAMP (F). The thicker line on the X axis represents the duration of perfusion. The arrow in C highlights the transient reduction of AP threshold caused by carbachol. (G-K) Mean of voltage sag change over time for carbachol (G), ACSF (H), oxotremorine-M (J) and 4DAMP (K). Shaded areas represent the standard error.

Fig 9A shows examples of voltage traces under injection of 10 current steps comprised between -0.3 to 0.3 nA (adjusted according the cell recording) before the application of carbachol (left), after 3 min (middle) and after 10 min (right) of carbachol perfusion. At suprathreshold current injection, a single action potential was observed at the beginning of the step in each condition. The initial resting potential for this cell was measured at –62 mV (dashed grey line). After 3 min in presence of carbachol, the resting potential was shifted to –59 mV and came back to the –62 mV even before washing out carbachol. The IV curve for the 3 recordings shown in Fig 9A was plotted in Fig 9B and used to estimate the RMP and the IR. The average change in RMP was plotted against time in Fig 9C (upper part, n = 5, shaded areas = SE). The thicker black line on the X axis represents the duration of carbachol application. Carbachol induced a depolarization reaching a maximum of +3.0 ± 1.1 mV (n = 5, Fig 9C, lower part) at the beginning of the perfusion and the RMP came back to the initial value before the end of carbachol application. Nevertheless, this change failed to be significant when comparing the maximum change (one-way ANOVA with repeated measures F(2,8) = 3.50, p = 0.06, n = 5, Fig 9C, lower part). This depolarization was accompanied by a significant change of –7.7 ± 4.7 MΩ (n = 5) in IR (F(2,8) = 4.55, p<0.05, n = 5; post-hoc test: ctrl vs. carb, p<0.05, Fig 9D). This transient decrease in IR, reflecting an increase in conductance, could be explained by the opening of nicotinic receptors. Noteworthy, after getting back to the initial value, the IR continued to increase until the end of the recording. This was associated with an instable and weak depolarization (Fig 9C, upper part) that could be mediated by muscarinic receptors. As a control, the same protocol was applied but the cholinergic agent was omitted (Fig 9E and 9F). No significant change was observed in both RMP (F(2,6) = 0.48, p = 0.57, n = 4) and IR (F(2,6) = 0.06, p = 0.85, n = 4).

In Fig 9G (upper part), the resting potential distinctly hyperpolarized upon oxoM perfusion reaching a max change of -2.4 ± 1.0 mV and went back to the initial value when oxoM was washed out. This was marginally significant in one-way ANOVA with repeated measures (F(2,28) = 4.9, p<0.05; n = 15, Fig 9G, lower part), however variability was high due to opposite (n = 2) or no (n = 3) response from 5 cells. No significant variation of the IR (F(2,28) = 0.68, p = 0.44, n = 15) could be measured for oxoM. The same scheme, though stronger (maximum change equal to -4.2 ± 1.5 mV), was visible with 4DAMP perfusion in Fig 9K (upper part). At the end of 4DAMP perfusion, the RMP increased slowly toward its initial value. Again, three cells showed opposite effects (depolarization) preventing statistical significance (F(2,18) = 2.25, p = 0.16, n = 10; lower part in Fig 9K). The effect of 4DAMP perfusion on IR was unclear (Fig 9L, upper part). The high variation between the cells and the lack of any recovery afterwards (Fig 9L, lower part) suggested an insignificant change (F(2,18) = 0.67, p = 0.46, n = 10).

We then analyzed two further functional parameters, namely the action potential threshold and the amplitude of the voltage sag during hyperpolarizing current injections in the same dataset (cf. Fig 9). This data is presented in Fig 10 in a similar format as Fig 9. Fig 10A shows current clamp recordings from a SBC before (Fig 10A, left), during 1 μM oxoM perfusion (Fig 10A, middle) and after recovery (Fig 10A, right). This SBC responded with strong hyperpolarization to the oxoM perfusion (RMP went from -60.1 ± 0.7 mV to -81.9 ± 1.0 mV and recovered to -63.9 ± 0.6 mV). The voltage sag, i.e. the difference between the maximum and the steady-state potential upon hyperpolarizing current injection, is changed during oxoM application. In this example cell the difference between peak- and steady-state hyperpolarization was 8.1 ± 0.2 mV during control, was reduced to 5.5 ± 0.2 mV during oxoM perfusion and recovered to 6.8 ± 0.5 mV after oxoM washout. This finding agrees with a reduction of Ih during oxoM application (cf. Fig 4F). In Fig 10B we show an example of the AP threshold analysis. We identified the AP threshold by a rate threshold, i.e. the membrane potential where the rate of membrane potential change first exceeded 50 mV/ms. This is visualized for the control in Fig 10B. During the control the AP threshold was -37.7 ± 0.3 mV for this cell, was then reduced to -61.3 ± 0.7 mV during oxoM perfusion and recovered back to -41.8 ± 0.9 mV after oxoM washout. Similar to the data shown in Fig 9 we performed the measurements shown in Fig 10A and 10B every 30s for several minutes before, during and after perfusion of cholinergic agents. In Fig 10C & 10G we show the effect of carbachol perfusion on AP threshold and voltage sag. While AP threshold is only transiently reduced by carbachol immediately after wash-in (arrow in Fig 10C; reduction of -8.1 ± 7.3 mV, n = 5), carbachol causes a lasting reduction of the voltage sag (Fig 10G, on average reduction of -1.8 ± 1.9 mV). When tested with one-way ANOVA with repeated measures, these changes are statistically not significant though (AP threshold: F(2,8) = 0.07, p = 0.83, n = 5; voltage sag: F(2,8) = 2.9, p = 0.15, n = 5). Omitting the cholinergic agent (Fig 10D & 10H) neither caused a reduction in AP threshold (F(2,6) = 0.54, p = 0.53, n = 4) nor voltage sag (F(2,6) = 1.1, p = 0.37, n = 4), but showed how variable these metrics can unfortunately be.

Both washing in of oxotremorine-M (Fig 10E & 10J) and 4DAMP (Fig 10F & 10K) caused a slower and longer lasting reduction in AP threshold (oxoM: reduction of -3.4 ± 5.5 mV; 4DAMP: reduction of -4.3 ± 8.8 mV), but again due to the high variability of the data the results were not statistically significant (oxoM: F(2,28) = 1.1, p = 0.35, N = 15; 4DAMP: F(2,18) = 2.02, p = 0.17, n = 10). Surprisingly, washing in of oxotremorine-M did, averaged over all cells, only cause a small (non-significant) change in voltage sag (Fig 10J; F(2,28) = 0.99, p = 0.37, n = 15). 4DAMP caused a transient reduction of the voltage sag (reduction of -1.7 ± 3.6 mV, Fig 10K), which was statistically not significant (F(2,18) = 0.8, p = 0.41, n = 10) due to high variability.

Although these data are hard to interpret due to high variability we can nevertheless conclude that a cholinergic, and specifically muscarinic, influence on both AP threshold and voltage sag appears likely for SBCs, which would corroborate our results from the voltage clamp experiments.

In conclusion, long-lasting perfusion of carbachol during current-clamp recordings from SBCs indeed allowed the visualization of depolarizing changes caused by the combined action of both nicotinic and muscarinic effects. Perfusion of M3 antagonists had a hyperpolarizing effect on the unclamped RMP, likely due to blocking of tonically activated muscarinic signaling (cf. [10]) contributing to the RMP. Surprisingly, the muscarinic agonist oxoM on average also hyperpolarized the RMP of SBCs. However, a considerable diversity regarding both direction of effects and temporal profiles of effects of muscarinic transmission in SBCs was evident.

Discussion

We demonstrated that IM was present in SBCs. Both IM and Ih were reduced by the perfusion of a muscarinic agonist. In addition, the presence of two muscarinic receptors M1 and M2 in the AVCN was confirmed by immunohistochemistry. M1 was somatic while M2 was mainly located in the neuropil and sparsely on the soma of SBCs. Although M2 is known to be localized on the presynaptic site, no effect was visible on the endbulb of Held to SBC synaptic transmission. Finally, we showed that muscarinic activation induced differential effects on the resting membrane potential of SBCs. The activation of all muscarinic receptors by oxoM on average hyperpolarized the RMP (-2.4 ± 1.0 mV, Fig 9G). The inhibition of M3 shifted the RMP to even more negative values (-4.2 ± 1.5 mV, Fig 9K).

IM is a voltage-dependent outward potassium current. When the membrane potential shifts to more positive values, M channels slowly open and thereby hyperpolarize the membrane potential and bring the cell closer to its resting state. The M-channels consist of a heteromeric assembly of Kv7.2 and Kv7.3 potassium channel subunits [32,33]. The presence of low-threshold potassium conductance in SBCs, which could correspond to M conductance, has been already reported by [34]. In this study we confirmed that IM is present in SBCs. Indeed, the perfusion of the Kv7 blocker XE991 almost completely abolished the inward relaxation current. The activation of all muscarinic receptors via local application of oxoM also abolished the inward relaxation (Fig 3). In addition, we showed that long application of M3 muscarinic receptor antagonist (4DAMP) induced a negative shift of SBC resting membrane potential (Fig 9K) similar to the change caused by tolterodine (broad muscarinic antagonist) bath application and confirming a tonic activation of muscarinic receptors [10]. Immunohistochemical data confirmed the presence of M3 on the soma of SBCs [5]. These results indicate that M3 receptor activation likely causes the long-lasting depolarization through the closure of M-channels. Several studies have demonstrated that another muscarinic receptor, M1, was also involved in IM inhibition [28,35,36]. Our immunohistochemical staining exhibited strong M1 expression on the soma of AVCN cells including SBCs (Fig 6A–6D). M1 and M3 are both Gq/11 protein-coupled metabotropic receptors triggering the same intracellular signaling (Fig 11). Acetylcholine (or any other cholinergic agonist) binding to M1/M3 leads to the cleavage of the phosphatidylinositol-4,5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3) through activation of phospholipase-Cβ (PLCβ). However, the exact secondary messenger which directly induces the closure of M-channels is still debated.

Fig 11

Hypothetical signaling cascade in response to acetylcholine release.

On one hand, acetylcholine (Ach) binds the M1/M3 muscarinic receptor which induces the activation of the phospholipase C (PLC) via the Gαq/11 protein. The activated PLC depletes the phosphatidylinositol biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 concentration increase releases calcium from the internal cell storage. Directly or indirectly, one or several of these 2nd messengers trigger the closure of Kv7 channels thereby depolarizing the cell membrane. On the other hand, acetylcholine may bind the M2 muscarinic receptor inhibiting the adenylate cyclase (AC) via the Gαi protein which induces a reduction of cyclic AMP. Thus, for the same membrane potential, more HCN channels are closed inducing the hyperpolarization of the cell. Cell membrane and ion channels were designed by “Servier Medical Art” (https://smart.servier.com) provided by Les Laboratoires Servier (https://servier.com/en), licensed under Creative Commons Attribution 3.0 Unported License.

A noteworthy result is that XE991 failed to completely abolish the inward relaxation current at the most depolarized values tested (Fig 3A) while oxoM inhibits the current at all voltages. This could be explained by the difference in the application system of the drugs. OxoM was locally applied via a perfusion pencil just above the area where the cell was located while XE991 was perfused in the bath chamber. The former could allow a faster activation of muscarinic receptors whereas the replacement of normal ACSF by ACSF containing XE991 took several minutes. However, this was compensated by waiting at least 10 min of XE991 bath application before recordings, leaving enough time to totally replace the ACSF in the bath chamber and affect all cells. An alternative explanation is that Kv7 channels are not the only ion channel type involved in IM. Indeed, only an HCN blocker was added to the ACSF in these experiments. The remaining current had an activation threshold more positive than IM (starting around -40 mV) which matches with the delayed-rectifier potassium current (IKDR). Although IKDR has faster kinetics than those of IM, our measurements of the inward relaxation did not consider the kinetics and consequently a role of IKDR in this cannot be excluded. Other channels could also be involved in IM. For example, two-pore-domain potassium channels were closed by the activation of M1/M3 muscarinic receptors causing the thalamo-cortical neurons to depolarize [15].

Ih is a depolarizing cationic current, mainly due to an influx of sodium. The molecular correlate is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel. The name Ih comes from its unusual voltage-dependency. Indeed most of the channels are open when the membrane potential is hyperpolarized but a proportion of HCN channels stay tonically open at resting potential and maintain a resting state close to action potential threshold [20,37]. Four different isoforms of HCN channels exist, HCN1 to HCN4. Mouse BCs strongly expressed the isoforms HCN1, moderately HCN2 and HCN4 [16]. The local application of oxoM strongly reduced Ih at every voltage tested (Fig 5) meaning that muscarinic activation here inhibits a depolarizing current, thereby hyperpolarizing the cell membrane. Part of this effect can result from the decrease of adenosine-3’,5’- cyclic monophosphate (cAMP) and/or guanosine-3’,5’- cyclic monophosphate (cGMP). These cyclic nucleotides regulate HCN channels by shifting the voltage of half-maximal activation (V1/2) to more positive values [20,37]. Thus, reducing the cyclic nucleotide availability shifts the activation curve to a more negative value. In that case, the probability that HCN channels are open is reduced for the same voltage. Two muscarinic receptors, M2 and M4, induce a decrease of cAMP [31] by inhibiting the adenylate cyclase (AC). This enzyme is inhibited by the α-subunit of Gi/o protein, itself activated by M2/M4 receptors (Fig 11).

Our immunohistochemical data revealed the presence of M2 muscarinic receptors in the AVCN (Fig 6E–6H). The M2-positive signal was mostly detected in the neuropil of BCs, implying either a dendritic localization on SBCs or an expression on the presynaptic site. However, no overlap was observed between calretinin-positive auditory nerve terminals and M2 staining. In addition, presynaptic parameters, estimated by recording evoked and spontaneous EPSC, were unchanged upon wash-in of carbachol (Figs 7 and 8). However, if a tonic activation of muscarinic receptors occurs, as previously suggested by [10], a small effect might be more easily revealed by the application of an antagonist. Nonetheless, the absence of cholinergic effect on the synaptic transmission are in accordance with our immunohistochemical results. On the other hand, M2 localization on calretinin-negative terminals (possibly of cholinergic axon coming from the olivary complex or from the ponto-mesencephalic tegmentum) could not be refuted based on our results. Indeed, M2 is known to be an autoreceptor that can regulate acetylcholine release [31]. Furthermore we cannot exclude presynaptic localization on inhibitory terminals, which strongly influence responses of SBC. M2 staining was also localized on the soma of SBCs (Fig 6H, empty arrowheads) confirming that M2 was present on the postsynaptic side in SBCs. The existence of M2 on the soma and dendrites of postsynaptic cells has previously also been demonstrated in hippocampal interneurons [38].

Recently, [21] have demonstrated that cholinergic interneurons expressing M2 exhibited a decrease of around 20% of Ih under application of oxoM. However, the strong inhibition of Ih observed here is unlikely to be explained only by the M2-mediated decrease in cAMP alone. This suggests that other phenomena influence HCN channels. There was evidence that another second messenger, PIP2, affects the activation curve of HCN channels [39]. In that study, adding PIP2 shifted the activation of HCN2 channels to positive voltages similarly to cAMP. Even more compelling, adding both cAMP and PIP2 shifted V1/2 to even more positive voltages than cAMP or PIP2 alone. M1/M3 receptors expressed by SBCs then could indirectly be responsible for a significant part of Ih inhibition (Fig 11, grey dashed arrow) through the cleavage of PIP2. Furthermore, HCN channels are also regulated by their phosphorylation status. The protein kinase A (PKA), activated by cAMP, positively shifted the activation curve of Ih (HCN4) isoform in mouse sinoatrial node [40] while the protein kinase C, activated by PLC, phosphorylated HCN1 and reduced Ih in hippocampal neurons [41]. Both HCN channel isoforms are present in BCs [16]. In summary, a combination of possible intracellular mechanisms through which muscarinic activation can modulate Ih seems likely and could explain the strong effects seen in our recordings.

Muscarinic activation in SBCs induced two opposite effects. M1/M3 activation blocked the M-current which should depolarize the RMP while M2 activation reduced Ih and then supposedly leads to a hyperpolarization of the cell membrane. This result raises one important question: why does the same signal induce two opposite effects? The changes of the resting membrane potential, the input resistance and the action potential threshold were monitored under application of carbachol, oxoM or 4DAMP to clarify the resulting effect of the global muscarinic activation on the excitability of the neuron. Long application of carbachol activated both nicotinic and all types of muscarinic receptors. The RMP depolarized in the first three minutes after application of carbachol and went back to its initial value before washing out carbachol. Action potential threshold briefly decreased as well which, together with the depolarization, should strongly but transiently increase the spike probability of the SBC. This spontaneous return to the initial value before the end of carbachol application was previously also noticed in vivo in SBC spontaneous firing rate under iontophoretic application of carbachol [10]. Additionally, the transient depolarization was accompanied by a transient decrease in the input resistance consistent with the opening of nicotinic receptors generating a sodium inward current. The desensitization of nicotinic receptors can explain the spontaneous return of IR and RMP to their normal values.

Noteworthy, the input resistance continued to slowly increase even after the end of carbachol application. This high increase in the input resistance (almost 20 MΩ at 15 min, Fig 9D, upper part) could reflect the closure of both Kv7 and HCN channels though no concomitant significant change was observed in the RMP. This contradicts the long-lasting depolarization observed after carbachol puff application shown in [10]. Nevertheless, the RMP was unstable during the IR increase. One hypothesis is that the hyperpolarizing effect of Ih reduction counteracts the depolarizing effect of IM blockade to stabilize the cell potential. The differential results between the two studies could be explained by the different system of carbachol application. The puff applications of carbachol used in [10] were phasic and very localized on the dendrites and part of the soma close to the primary dendrite while the perfusion pencil application of carbachol used in this study was sustained and reached every cell in a local volume of the slice and all the cellular compartments (whole dendritic tree, soma and initial axon segment). The long-lasting application can lead to the activation of a greater absolute numbers and a different ensemble of muscarinic receptors and might trigger their desensitization/internalization inducing a different overall change.

Surprisingly, long application of oxoM showed a distinct response. Naturally, the transient nicotinic effect was absent since only muscarinic receptors were activated. Nonetheless, the average RMP clearly hyperpolarized in presence of oxoM (Fig 9G, upper part) without a clear effect on the IR. The lack of effect on the IR might be due to big inter-individual variation. We also cannot rule out potential effects of high or fluctuating series resistance during the long recordings (cf. “Electrophysiology”in the Material & Method section). The hyperpolarization upon oxoM application could result from a disequilibrium between Ih and IM in favor of Ih inhibition. This can be caused by more activated M2 receptors than both M1/M3 receptors. Yet, our immunohistochemical data revealed a stronger M1 than M2 expression (Fig 6D and 6H respectively) which might be in conflict with this hypothesis. However, it seems that oxoM binds more selectively to M2 than M1 in comparison to carbachol [42] favoring Ih inhibition causing the hyperpolarization. The 4DAMP perfusion caused a strong hyperpolarization. The blockade of M3 activation against the background of a tonic cholinergic signaling in SBC [10] leads to 1) IM inhibition only caused by M1 activation and 2) Ih inhibition via M2 activation alone, resulting in a net negative shift of RMP.

Noticeably, few cells in both oxoM (n = 2) and 4DAMP (n = 3) perfusion experiments showed an opposite effect to the average. This can be explained by an imbalance in Ih and IM inhibition depending on the proportion of M1/M3 vs. M2 expression. Indeed M1 seemed to be strongly expressed on all AVCN cells while M2 expression was sparse and heterogeneous (Fig 6). These results suggest an underlying diversity of cholinergic signaling in SBC based on factors not yet identified.

The variability observed in response to muscarinic activation might be explained by the range of gerbil age we chose. Indeed, all electrophysiological recordings were made in hearing gerbils aged between P14 to P25. However, the expression levels of both KCNQ and HCN are age-dependent in central neurons [43]. In our results, while the currents Ih were larger in older animals, no age-related difference could be observed for M currents. In addition, no correlation could be made between the age and the diversity of RMP responses consecutive to the application of muscarinic modulators suggesting that the diversity observed in SBCs is unlikely age-dependent.

In general, the muscarinic effect observed on the currents and on the RMP in the different whole-cell recordings could have been underestimated. Indeed, we proposed a model in which secondary messengers (like cAMP, IP3) of muscarinic activation act on the channels but they might have been diluted/washed out over time. This might also have contributed to the variability in the results. Furthermore the artificial extracellular calcium concentration and the reduced recording temperature might exert differential effects on different second messenger pathways. We would thus suggest to perform perforated-patch recordings at physiological temperature and ion concentrations as a control in future experiment to rule these possible problems out.

In this study, muscarinic receptors were activated by the application of oxoM, but this agonist has two downsides that could affect the interpretation of our results. First, we argued that oxoM inhibited M currents by blocking Kv7 (KCNQ) channels via the activation of muscarinic receptors (see Figs 2 and 3). However, it has been demonstrated that oxoM can also directly block KCNQ2/3 channels [27]. If this direct mechanism on Kv7 channels occurred here, we could not distinguish whether the inhibition of M currents resulted from muscarinic activation or from a direct blockade of KCNQ2/3 channels by oxoM. However, Kv7 blockade should logically lead to cell depolarization; but, on average, the cell RMP hyperpolarized (see Fig 9) upon oxoM application. We suggested that this hyperpolarization resulted from Ih inhibition consecutive to M2 activation. This effect cannot be explained by the direct effect of oxoM on KCNQ2/3. In addition, a muscarinic antagonist tolterodine shifted the cell RMP to more negative values [10] which supports the hypothesis that muscarinic activation depolarizes the cell, supposedly by blockade of Kv7 channels. Secondly, it was shown that oxoM directly and indirectly potentiates NMDA currents [27]. However in mature AVCN the transmission is mostly mediated by AMPA receptors [44]. Thus this effect likely does not play a large role here.

One hypothesis to explain the purpose of having two opposite effects (depolarization vs. hyperpolarization) triggered by the same molecule is the following. In the case of a brief application (like puff application), carbachol binds with a higher probability M1/M3 receptors (due to higher expression) resulting in a depolarization of the resting membrane potential. During long lasting bath application, carbachol reached every cell part leading to more M2 activation which can counteract the effect of M1/M3 depolarization and bring the RMP back to its initial value. This phenomenon could represent a form of adaptation in case of a strong and long-lasting cholinergic activation and could also explain the spontaneous return of the normal spontaneous firing rate of SBCs in the in vivo experiments under high concentration of carbachol [10]. As described in [10], the transient cholinergic excitation (RMP depolarization) likely enhances sound localization by providing more well-timed action potentials and seems to improve the detection of tones in noisy background by expanding the dynamic range of responses. Depending on the exact kinetics of the partially opposing effects of cholinergic transmission on SBC we demonstrated, one could speculate about the listening situations each would have the greatest impact in. In listening situations rich in transients, with rapidly changing stimulus levels, the depolarizing effects of cholinergic transmission could dominate. This would render SBC more excitable and counteract peripheral cholinergic gain control as discussed in [10]. However, during long durations of intense stimulation (e.g. constantly loud surroundings) the hyperpolarizing muscarinic effects we saw in our wash-in experiments could dominate. This might act as an additional central gain control that reduces tonic activation (and thus masking effects) of SBC by the background noise. Direct physiological evidence for these ideas is not yet available unfortunately.

In conclusion, both IM and Ih are present in SBCs and are both modulated by the activation of muscarinic receptors. IM inhibition is most likely mediated through the activation of both M1 and M3 receptors whereas Ih is most likely decreased by M2 activation. The resting membrane potential might be tuned by the balance of these currents and then depends on the relative proportion of M1/M3 and M2 receptor activation. Further experiments are needed to understand the intracellular signaling caused by M1/M3 and M2 activation and the physiological meaning of two opposite effects induced by the same modulator in the same cell.

24 Jul 2019

PONE-D-19-18119

Muscarinic modulation of M and h currents in gerbil spherical bushy cells

PLOS ONE

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Reviewer #1: The manuscript by Gillet, Kurth and Kuenzel investigates the presence and effect of muscarinic receptors on spherical bushy cells in the anteroventral cochlear nucleus. The authors use a combination of pharmacology and immunohistochemistry to identify the presence of M1 and M2 receptors on those cells which are differentially expressed on the cell soma and neuropil, respectively. Using agonists for muscarinic receptors (Oxotremorine M) and antagonist for Kv7 (XE991) and HCN channels (ZD7288), the authors show that the effect of mACh activation largely overlaps with block of Kv7 and HCN channels and conclude that muscarinic receptors in SBCs interact with Kv7 and HCN channels resulting in long lasting changes of resting membrane potential.

Finally, the authors propose a model in which the endogenous release of acetylcholine acts on both M1/M3 and M2 receptors. Activation of M1/M3 receptors will depolarize the cell by closure of Kv7 channels while activation of M2 receptors hyperpolarizes the cell by closure of HCN channels.

Overall, the manuscript provides a detailed investigation of the role of muscarinic acetylcholine receptors on SBC and is in line with previous reports by the same authors, suggesting an intriguing role of cholinergic modulation on SBCs. The data is solid and the interpretation and conclusions are mostly appropriate and close to the data.

However, the use of pharmacological agents to dissect metabolic pathways has its inherent caveats and I feel that some points need further clarification to strengthen the author’s interpretation.

Major comments:

1. The expression of M1/M2 receptors on SBCs has been investigated by immunohistochemistry and important conclusions are based on those data. However, the staining against M1 suggests a predominantly cytoplasmic localization, rather than membrane bound, which might be caused by unspecific binding of the antibody. Did the authors confirm the specificity of the antibody or performed the control experiment using an antibody that bounds another epitope (e.g. Alomone Labs Cat# AMR-001, RRID:AB_2039993 binds the third intracellular loop, rather than the C-terminus of M1 receptors)?

2. The series resistance during ephys recordings was not compensated, but the authors do not report Rs values during the recordings or if there were any differences between groups or changes over time. While the influence on the slow and rather small IM currents might be negligible, a high Rs will likely affect the fast, larger IInst currents which in turn could affect the analysis of IM. Likewise, the amplitude and kinetics of AP-evoked EPSCs (Figure 7) will critically depend on the recording’s Rs due to the non-linearity of Rs compensation. Unless the uncompensated Rs values during recordings were either small (<5 MOhm) or very similar between groups this could introduce considerable errors when estimating amplitude and kinetics. The authors should therefore also report the Rs values for the individual recordings or declare if recordings with high Rs (e.g >>10 MOhm) were discarded.

3. The authors recorded from P14-P30 gerbils of either sex. In their previous study (Gillet, 2018, J. Comp. Neurol.), the authors reported an increase in cholinergic innervation between P15 and P28 and another recent study (Mueller et al, 2019, Front. Cell. Neurosci., DOI: 10.3389/fncel.2019.00119) suggest that some properties of mouse CN neurons develop after hearing onset. It is not clear why the age range was chosen so close to the animal’s hearing onset and no information is provided on the age distribution for the different experiments (except for immunohistochemistry at P30). Considering the contrasting effects of oxoM and 4DAMP in some of the recordings (Figure 9, some cells show increase in RMP, other decrease), did the authors split the data set by age or sex to determine if those factors might account for the observed variability, e.g. activation of mACh might be depolarizing in young, but hyperpolarizing in older animals or vice versa. Since the authors have those data readily acquired, it might be worth adding a statement, if any difference in sex/age was observed. If a certain age range was used for some of the recordings for experimental reasons (e.g. recordings more stable), this should be mentioned.

4. Based on fiber stimulation and mEPSC recordings with application of carbachol, the authors conclude that cholinergic transmission does not affect synaptic transmission at SBCs. However, the authors also show that application of 4DAMP (an M3 antagonist) hyperpolarizes SBCs, suggesting a tonic activation of M3 receptors in the slice. If ACh receptors were tonically activated at the endbulb of Held, the effect on synaptic transmission would likely be negligible when using ACh agonists but would require the use of ACh antagonists. This possibility should be tested or at least discussed.

5. The authors performed fiber stimulation at 100 Hz and analyzed the EPSC kinetics for the first EPSC. Did the authors also test higher (e.g. 300 Hz) stimulation rates, which would more closely mimic in vivo activity? A potential ACh modulation of synaptic vesicle release might be more pronounced during ongoing activity. Similarly, the authors analyzed EPSC kinetics for the first EPSC only. Since the first EPSC after a period of quiescence is somewhat artificial (considering the high spontaneous rates of most auditory brainstem neurons), it might be worthwhile to also analyze the EPSCs later in the train (if the signal-to-noise ratio permits such analysis).

6. Application of the mACh receptor agonist oxoM hyperpolarized the SBC and the authors argue that an indirect block of Kv7 channels causes this effect. While this argument is conceivable and has been shown in other systems, the authors should be aware that oxoM can directly block Kv7 channels without the activation of mACh receptors (see Zwart et al., 2016, Eur. J. Pharm. DOI: 10.1016/j.ejphar.2016.08.037). Did the authors perform control experiments with oxoM + atropine to block mACh receptors or used a more specific mACh agonist (e.g. oxotremorine or xanomeline) which has been shown to not interact with KCNQ channels? If not, the authors should at least discuss this possibility and how it might affect their conclusions.

7. The model in Figure 10 proposes two opposing effects of endogenous ACh, one leading to depolarization by Kv7 closure, the other to hyperpolarization by HCN closure. The authors speculate that brief ACh application will predominantly bind to M1/M3 receptors (highly expressed), thereby depolarizing the cell, while during prolonged application the hyperpolarization by M2 (weakly expressed and located at the neuropil) activation dominates. While this explanation is reasonable in the current experimental setting, it might not apply in vivo (since the origin and activation dynamics of those inputs are barely known). The authors should discuss if either of those conditions might be favorable during varying acoustic stimulation (e.g. hearing in noise, natural sounds).

8. The proposed model suggests the involvement of secondary messengers (cAMP, PLC etc) to act on Kv7 and HCN channels. While this is reasonable and has been shown in other systems, I wonder if some of the reagents have been washed out/diluted over time in the whole-cell experiments, thus underestimating the observed effect. This could be tested by e.g. gramicidin-perforated patch-clamp experiments. Do the authors have any data on this? If not, new experiments are not required, but the authors should acknowledge and discuss this possibility.

9. Line 502: “oxoM induces closure of M channels […] and depolarizes the resting membrane potential”. This conclusion is not immediately evident from the data, since application of oxoM (Figure 9G) resulted in an, on average, small hyperpolarization. The authors might argue that block of M3 channels hyperpolarized the cell, but unless M1/M3 channels were specifically activated and result in depolarization, this statement is too strong given the underlying data.

10. Previous studies by the corresponding author and others (e.g. PMIDs: 21411667, 25164657, 28945194, 27855778, 23345233) point to an important role of inhibition in shaping SBC responses, particularly during complex or naturalistic acoustic stimulation. Did the authors find any evidence for ACh receptors on inhibitory inputs contacting the SBCs?

Minor comments:

11. Slices were incubated at room temperature and all recordings were performed at room temperature and at supra-physiological calcium (2 mM vs. 1.2-1.5 mM). Since the kinetics of G-protein mediated signal transduction pathways are temperature and calcium dependent, the observed effect in the experiment might be different to in vivo conditions, especially considering the proposed “balance” between M1/M3 (depolarizing) and M2 (hyperpolarizing) mediated pathways. While I acknowledge that the authors performed similar experiments at 37C in a previous publication (Figure 4 in Goyer et al, 2016, eNeuro), it might be worth discussing the effect of temperature and calcium concentration in the light of the two proposed pathways.

12. The manuscript would benefit from a statistics section within the Methods. Although the statistics for this data set is mostly straight forward, the authors should describe how sample size and statistical tests were selected (e.g. testing for normal distribution, etc.).

13. Line 98: Please state how the junction potential was estimated, by calculation or experimentally. Was the junction potential also corrected for the voltages shown in Figure 9B?

14. Line 156: Please state the concentration of Triton X-100 in PBS. Was it 0.1 % as in the subsequent steps?

15. Line 79: Please indicate which temperature was considered room temperature (e.g. 25C).

16. For antibodies and software used, please include RRIDs where available (e.g. line 192 “rabbit anti-M1 muscarinic receptor (Alomone Labs Cat# AMR-010, RRID:AB_2340994)”)

17. In the Methods, the authors describe that cells were filled with biocytin during recordings, but its purpose appears unclear, since the immunohistochemistry was performed on animals not used during slice recordings. If the biocytin staining was used to morphologically verify the cell type after the end of recordings (e.g. to identify SBC vs. stellate cells), it should be stated in the Methods and Results section.

18. Line 143: Please indicate how many orders were used for the polynomial fit and if any constraints were used.

19. The manuscript frequently reports p-values as p=1. While I understand that p-values may be >1 after applying the Bonferroni correction on the actual p-values rather than the alpha value of the test, it is better to report such p-values as p>0.9999, since p=1 would only be possible if the means of two groups are identical.

20. Figure 1: In the bottom part of Panel A, the response to multiple depolarization steps is shown, but the indication of IM, ISS etc. is for one of the traces only, which might be confusing. Consider indicating for which trace the labels apply, e.g. using different colors or line styles.

21. Figure 1: Consider replacing the title of Panel C “IM at -50mV” with “Determination/Identification of IM at -50mV” or alike, since the panel shows all currents, not only IM

22. Figure 2: The panel organization is hard to follow. Consider moving the current-voltage graphs next to the corresponding traces. Also, it appears that the same data is labeled “XE991” in panel D and “ctrl” in panel G. While I understand the rationale of doing so, it might be clearer to combine those two panels into a single graph (labeled as ctr, XE991, XE991+oxoM which is then consistent with the raw traces). It might also be easier to follow, if the y-labels of panels D/F/G were labeled “Inward relaxation current IM”, as to link it to IM in panel C.

23. Figure 2: The y-labels on panels D/F/G is not centered on the y-axis

24. Figures 3-5: Please be consistent regarding the nomenclature of the currents. For instance, in Figure 3A-C the current is labeled “inward relaxation current”, but labeled “IM” in panel D. In my understanding those are the same currents but using different names is potentially confusing. Similar in Figures 4/5 with most panels labeled “inward current”, but “inhibition of Ih” in Fig. 5D. In my understanding, the term “inward current” is incorrect in this context, since it only refers to Ih, not the total inward current (as indicated in Figure 4C)

25. Figure 3 & 5: The currents at -50 mV (Figure 3) and -110 mV (Figure 5) were analyzed using paired t-tests. Since multiple measures have been taken from the same cells (ctr vs. drug and at different voltages) I feel that a two-way repeated measures ANOVA would be more appropriate and informative. In this case, the data should be analyzed using the within-subject factors treatment (ctrl vs. drug) and voltage.

26. Figure 6: The DAPI labeling looks unusual and different from previous papers from the same authors (e.g. Gillet et al, 2018, J. Comp. Neurol.), i.e. nuclei are barely labeled (see the “holes” in the cells in panel D, which should contain the cell nuclei) and most of the labeling is constrained to the auditory nerve root. Since the DAPI labeling is not critical for the conclusion (and not referred to in the manuscript), consider removing this channel from the images.

27. Figure 6: Please include which magnification (e.g. 60x) was used for imaging.

28. Figure 8: Since the detection of mEPSCs is highly variable and critically dependent on threshold selection, it would be helpful to indicate detected mEPSCs in the representative traces shown in panel A, so as the reader has a better understanding of which events were considered mEPSCs

29. Figure 8 & lines 395-404: The data are analyzed using ANOVA, but not details are provided as to which type of ANOVA was used. Given the experimental design, it seems a one-way repeated measures ANOVA would be appropriate. Please indicate this in the text.

30. Figure 9: Please rescale the y-axis in panel H bottom to avoid cutting off data points.

31. Figure 10: Please label the small red circles as “Acetylcholine”.

32. Line 223: “Array of blockers” awkward wording, perhaps replace with “combination/cocktail of blockers”

33. Line 360: consider replacing “bigger M2 signals” with “intense M2 signals”

34. The authors should consider replacing the bar charts in Fig. 7/8 with box plots but keeping the individual data points to better display the data distribution.

35. Lines 465-475: The authors explained that when excluding parts of the data set (cells with no or depolarizing effect) the statistical tests became significant. While this is not surprising (even a normal distribution with enough N will show “statistical significance” when limiting the data set to only positive or negative values), it implies that those excluded cells “prevented” statistical significance. Unless those cells were excluded based on criteria (e.g. age, sex, recording quality etc.) other than their response to drug application, this exclusion seems unjustified and potentially misleading. The authors did a good job in pointing out the differences in responses but should probably remove the statistics of the partial data set (after exclusion of those cells).

36. Line 498: “For the first time […] IM was shown in SBCs”. This statement is too strong considering that Marx & Manis, 1991 (J Neurosci, DOI: 10.1523/jneurosci.11-09-02865.1991) reported a very similar low-threshold conductance and originally discussed the presence of an M current in bushy cells.

37. Line 515-416: unclear what is meant by “other synaptic functions”? The authors did not test the effect of cholinergic transmission on e.g. LTP or homeostatic plasticity, so this statement is vague and should be rephrased.

38. Lines 26 & 564: Consider removing/replacing the subjective term “interestingly”.

39. Line 143: typo in “substracting” (remove second “s”)

40. Line 149: replace “glycinergic and GABAergic blockers” with “glycine and GABA receptor blockers”.

41. Line 374: replace “immunohistocheminal” with “immunohistochemical”

42. Line 424: “cell conductance” should probably read “cell capacitance”.

43. Line 430: replace “showed in (A)” with “shown in (A)”

44. Line 569: missing “t” in “spontaneous”

45. Line 539: please explain what “nACSF” stands for, if the additional “n” is not a typo.

46. Line 540: replace “a HCN blocker” with “an HCN blocker”.

Reviewer #2: The muscarinic modulation of M and H currents in gerbil spherical bushy cells by Gillet and colleagues has potential to be of interest to the wide audience of PLOSONE, but it requires a thorough revision to improve its clarity.

Major issues

The manuscript needs some grammar and language polishing. Some sentences seem incomplete or awkward. For instance: pg 12, ln 273 “in control conditions of by the …”.

In some cases (Fig. 2 legend), the description of the data is too succinct.

Although the authors do explain that they use whole cell recordings to identify the muscarinic receptor subtypes and the currents involved in spherical bushy cells, and this is indeed very interesting an sound, the manuscript would benefit from a clearly stated hypothesis.

The age range of the animals is 14-30 days. While 14 days is juvenile, 30 days is considered as young adult, which may lead to increased variability of the results. Also, the expression levels of KCNQ and HCN channels is age dependent (see (Buskila et al., 2019) and should be discussed.

It is unclear which protocol was used to measure Ih? How can it be the same as for Im (P6)? Please explain

Please explain why you chose the concentration of Carbachol, as 500uM seems a bit high.

Fig 1. I’m not aware about all the plethora of K+ channels expressed in BSC, but the fact that one can see an outward current not necessarily imply that this current is Im. The authors should add a selective blocker to confirm the identity of this current, as done in fig 2.

Also, I’m not sure why fig. 1 and fig. 2 are not joined. I don’t see any added value for both figures

Fig. 3d – is not clear. Please re-label the graph to clearly identify the difference between XE991ctrl and XE991, OxoM-xe991 and OxoM ctrl.

Fig. 9E legend– perfusion of what? nACSF? What is it, not explained in the text.

Fig. 9G – the current data show no significant difference if all data is included and becoming significant if some data is excluded. If there is a good reason for excluding data, please do so. If the authors convinced that there is a trend, they should increase the n-number.

Statistical tests – It is not clear which ANOVA was used? One way, two way? What was the significance level and df? These should be clearly stated.

Oxotremorine-M potentiate NMDA currents; however, the authors seems to neglect this. How can it affect the results? Ca2+ and Na+ entry?

Immunohistochemistry data– how many slices were examined? No stat is reported.

Although the authors looked into the impact of Im and Ih on the subthreshold activity in BSC (RMP, IR), the suprathreshold physiological activity (firing rate, resonance, SFC’s) was not tested. If the authors have the data, they should present it as it would strengthen this study findings.

Minor issues

Pg 3, Ln 39 – please identify VCN as the ventral cochlear nucleus

Pg 3, Ln 40 – please identify SBC

Pg 6, Ln 115 – a 10 current steps protocol should state the range of injected currents.

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Reviewer #2: No

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14 Oct 2019

Detailed responses to comments: see coverletter of revision

Submitted filename: Responses.docx

Click here for additional data file.

29 Oct 2019

PONE-D-19-18119R1

Muscarinic modulation of M and h currents in gerbil spherical bushy cells

PLOS ONE

Dear Dr Kuenzel

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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Reviewer #1: The authors have addressed all my concerns raised during the previous review. The new analyses and extended discussion further strengthen the authors’ conclusions and the current manuscript is much improved.

Consequently, I have only a few minor points I would like to point out:

- Line 334: “OxoM” should probably be lowercase.

- Line 364: “washing” should perhaps read “wash-in”

- Figure 7C: The 200Hz condition seems to show the mean values only with individual cells missing.

- Figure 10A: The Figure legend (Line 600f) describes the right panel as “after 10 min of oxoM perfusion” but the figure panel is labeled “recovery” (i.e. some time after drug application has been stopped)

- Figure 10A: Please label the middle panel “oxoM wash-in”, since multiple drugs (carbachol, oxoM, 4DAMP) have been used in the figure.

Reviewer #2: Although the authors corrected many grammatical errors, I suggest a further editing and proof reading to aid clarity and make the text more readable. Moreover the affiliation of the manuscript was a bit awkward as the figure legends were mixed into the text, without the figures, which to my opinion just adds to the confusion. Additional concerns:

In the methods section, the authors state that they chose to include cells with quite high Rs (up to 35 MOhm) and allowed fluctuations of 30%. All these parameters can significantly affect their recordings and therefore conclusions. As the authors state that no more experiments can be made, I suggest commenting on this in the discussion.

Ln 138. Please rephrase to “One way ANOVA with repeated measures”, as well as other places in the text.

Results section, ln 286 onwards: this section is written like a figure legend, which is quite repetitive, why?

Results in Fig 3A are a bit odd. The plot shows that there is almost no difference between the data points at -70mV, yet its deemed significant as there is a small Im current at this voltage?

Fig. 3D – maybe I’m missing something, but in the legend the authors state that the comparison was between the treatment to control conditions (no treatment), yet in the plot it seems like the authors compared between the different treatments. Please clarify. Also, does the results with OXOM+XE991 means that there was no inhibition of Im, or that compared to XE991 alone, there was no additional inhibition? This needs clarification!

The description of the measured currents (Im, Ih) repeat itself too many times ( methods, results and legend).

Ln. 385 – the conclusion that “ The presence of ZD7288 before oxoM application prevented large effects of oxoM on Ih” does not make any sense, as in the presence of ZD7288, there is no Ih, so where this expectation came from…

Ln 409: the overall conclusion is wrong. Essentially, all you show is that cholinergic agonist decrease the Ih, there is no evidence to support the conclusion that Ih play a role in the cholinergic modulation of SBC’s.

Fig 10 legend: the plots in A are not 10 current steps (as only two traces are shown). Moreover the labels in the plot suggest “wash-in” and “recovery”, while the legend suggest it is 3 min and 10 min post carbachol application. I think there is a mix-up with fig 9A, Please clarify.

I found result 10J a bit puzzling, and essentially contradict Fig 4f. So did OXOM decrease Ih or not?

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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5 Dec 2019

Detailed responses to comments: see coverletter of revision.

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

11 Dec 2019

Muscarinic modulation of M and h currents in gerbil spherical bushy cells

PONE-D-19-18119R2

Dear Dr. Kuenzel,

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Additional Editor Comments (optional):

Reviewers' comments:

19 Dec 2019

PONE-D-19-18119R2

Muscarinic modulation of M and h currents in gerbil spherical bushy cells

Dear Dr. Kuenzel:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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