extfieldsofstudy: Biology Medicine

text:
Synaptic plasticity of inhibitory synapses onto medial olivocochlear efferent neurons

Abstract The descending auditory system modulates the ascending system at every level. The final descending, or efferent, stage comprises lateral olivocochlear and medial olivocochlear (MOC) neurons. MOC somata in the ventral brainstem project axons to the cochlea to synapse onto outer hair cells (OHC), inhibiting OHC‐mediated cochlear amplification. MOC suppression of OHC function is implicated in cochlear gain control with changing sound intensity, detection of salient stimuli, attention and protection against acoustic trauma. Thus, sound excites MOC neurons to provide negative feedback of the cochlea. Sound also inhibits MOC neurons via medial nucleus of the trapezoid body (MNTB) neurons. However, MNTB–MOC synapses exhibit short‐term depression, suggesting reduced MNTB–MOC inhibition during sustained stimuli. Further, due to high rates of both baseline and sound‐evoked activity in MNTB neurons in vivo, MNTB–MOC synapses may be tonically depressed. To probe this, we characterized short‐term plasticity of MNTB–MOC synapses in mouse brain slices. We mimicked in vivo‐like temperature and extracellular calcium conditions, and in vivo‐like activity patterns of fast synaptic activation rates, sustained activation and prior tonic activity. Synaptic depression was sensitive to extracellular calcium concentration and temperature. During rapid MNTB axon stimulation, postsynaptic currents in MOC neurons summated but with concurrent depression, resulting in smaller, sustained currents, suggesting tonic inhibition of MOC neurons during rapid circuit activity. Low levels of baseline MNTB activity did not significantly reduce responses to subsequent rapid activity that mimics sound stimulation, indicating that, in vivo, MNTB inhibition of MOC neurons persists despite tonic synaptic depression. Key points Inhibitory synapses from the medial nucleus of the trapezoid body (MNTB) onto medial olivocochlear (MOC) neurons exhibit short‐term plasticity that is sensitive to calcium and temperature, with enhanced synaptic depression occurring at higher calcium concentrations and at room temperature. High rates of background synaptic activity that mimic the upper limits of spontaneous MNTB activity cause tonic synaptic depression of MNTB–MOC synapses that limits further synaptic inhibition. High rates of activity at MNTB–MOC synapses cause synaptic summation with concurrent depression to yield a response with an initial large amplitude that decays to a tonic inhibition.

Introduction
Mammalian auditory systems encode acoustic stimuli that occur over many orders of magnitude of sound intensity. Animals require mechanisms to shift the gain of the auditory system between softer and louder sounds both to enable soft sound detection and to prevent system saturation during loud sounds. The cholinergic responses in the OHC to SK or BK potassium channels, OHCs are inhibited by MOC activity (reviewed in Fuchs & Lauer, 2019). This inhibition reduces OHC activity, suppressing active processes in the organ of Corti and in turn, reducing auditory nerve activity with broadened tuning curves (Art et al., 1985;Fex, 1962a;Galambos, 1956;Mountain, 1980;Siegel & Kim, 1982;Wiederhold & Kiang, 1970). Thus, the MOC system is implicated in cochlear gain control, as well as detection of salient sounds, protection from noise trauma and auditory attention (Boero et al., 2018;Delano et al., 2007;Glenn & Oatman, 1977;Kawase et al., 1993;Maison et al., 2013;Oatman, 1976;Rajan, 1988Rajan, , 1995Reiter & Liberman, 1995;Taranda et al., 2009;Terreros et al., 2016;Tong et al., 2013;Winslow & Sachs, 1987) via their actions on OHCs.
The patterns of activity of MOC synapses onto OHCs in the cochlea are governed by MOC somatic activity, which is in turn determined by interplay between intrinsic electrical neuronal properties and synaptic inputs that provide information about sound stimuli. In vivo recordings from MOC axons demonstrate low sound thresholds, narrow, V-shaped tuning curves qualitatively similar to auditory nerve tuning curves, and short-latency responses to loud sounds (Brown, 1989;Fex, 1962b;Maison et al., 2003;Robertson, 1984;Robertson & Gummer, 1985). Many MOC neurons have zero to low rates of action potentials in vivo in the absence of sound (Brown, 1989;Maison et al., 2003;Robertson & Gummer, 1985). The presynaptic circuitry driving MOC neuron activity is incompletely known, but anatomical (Brown et al., 2003Darrow et al., 2012;De Venecia et al., 2005) and recent functional (Romero & Trussell, 2021) work indicates that T-stellate cells of the ventral cochlear nucleus (VCN) are the primary sound-driven inputs to MOC neurons, with contributions from cells of the cochlear nucleus (CN) small shell cap (Hockley et al., 2022). MOC neurons are also strongly excited by descending projections from the inferior colliculus (IC) (Romero & Trussell, 2021). In contrast to these excitatory synaptic inputs, we previously demonstrated that MOC neurons receive inhibitory synaptic inputs from neurons of the medial nucleus of the trapezoid body (MNTB), which can suppress spontaneous action potentials in MOC neurons in brain slice preparations in vitro (Torres Cadenas et al., 2020). MOC neurons may also receive additional synaptic inputs from brain regions involved in sound perception, as well as those involved in other roles such as attention Caicedo & Herbert, 1993;Christian Brown et al., 2013;Faye-Lund, 1986;Gómez-Nieto, Horta-Junior et al., 2008;Groff & Liberman, 2003;Horvath et al., 2003;Ota et al., 2004;Suthakar & Ryugo, 2017;Thompson & Thompson, 1993;Vetter et al., 1993). How these various synaptic inputs integrate and impact MOC neuron activity remains an important unanswered question in auditory processing and cochlear gain control.
To increase our understanding of how MOC neurons adjust their activity over the course of auditory stimuli, we characterized the short-term plasticity of the MNTB-MOC synapse. We show that short-term plasticity is affected by temperature, specifically that synaptic depression is reduced at physiological temperature. We also demonstrate that extracellular calcium concentrations that are known to modulate presynaptic neurotransmitter release affect plasticity at this synapse. MNTB-MOC synapses exhibit depression at both physiological and high extracellular calcium concentrations, and exhibit facilitation at low extracellular calcium concentrations. By applying low rates of stimulation of MNTB axons to mimic baseline spontaneous in vivo activity we examine the subsequent MOC synaptic responses to higher rates of activity, such as might occur at sound onset. Stimulation of MNTB-MOC synapses at typical baseline spontaneous rates does not appreciably alter subsequent synaptic activity. Importantly, we show that stimulation of the MNTB-MOC synapse at higher rates of activity significantly reduces the subsequent effect of MNTB-MOC synapses due to synaptic depression. J Physiol 600.11 Finally, we test the response of MNTB-MOC synapses to long, fast stimulation to maximally depress the synapses. From this we demonstrate that MNTB neurons can maintain sustained inhibition of MOC neurons even during highly active periods. This sustained inhibition ultimately summates to reach a steady state to generate a tonic inhibition of MOC neurons.

Ethical approval
Animal procedures followed National Institutes of Health guidelines, as approved by the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communication Disorders Animal Care and Use Committee. Male (n = 46) and female (n = 39) experimental mice were generated from a cross between ChAT-IRES-Cre transgenic mice on a C57BL/6J background strain (The Jackson Laboratory, Bar Harbor, ME, USA, cat. no. 028861) and tdTomato reporter mice (Ai14, The Jackson Laboratory, cat. no. 007914). Mice were housed on a 12/12 h light-dark cycle, with continuous availability of food and water. Data include recordings from 99 neurons in 92 brain slices from 85 animals. Mice were euthanized by carbon dioxide inhalation at a rate of 20% of chamber volume per minute, then decapitated.

Animals and slice preparation
Brain slices were prepared from post-natal day (P) 14−23 mice of either sex resulting from a cross between ChAT-IRES-Cre transgenic mice on a C57BL/6J background strain (The Jackson Laboratory, cat. no. 028861), with tdTomato reporter mice (Ai14, The Jackson Laboratory, cat. no. 007914). Hemizygotes were used to prevent deleterious effects noted in ChAT-IRES-Cre homozygotes (Chen et al., 2018). As noted in the mouse line descriptions, this ChAT-IRES-Cre strain is occasionally prone to ectopic expression of Cre in vasculature, glia or non-cholinergic neurons due to the presence of the neo cassette, causing tdTomato labelling in non-cholinergic cells. If any ectopic expression patterns were observed, the tissue from the animal was not used. Following euthanasia, the brain was removed in cold artificial cerebrospinal fluid (aCSF) containing (in mm): 124 NaCl, 1.2 CaCl 2 , 1.3 MgSO 4 , 5 KCl, 26 NaHCO 3 , 1.25 KH 2 PO 4 , 10 dextrose. One millimolar kynurenic acid was included during slice preparation. The pH was equal to 7.4 when bubbled with 95% O 2 -5% CO 2 . Three hundred micrometre coronal brain slices containing nuclei of the SOC including the MNTB and VNTB were cut with a vibrating-blade microtome (Leica Biosystems, Wetzlar, Germany, VT1200S) in cold aCSF. The slices were stored in a custom interface chamber at 32°C for 1 h and then allowed to cool to room temperature for electrophysiological recordings. The slices were used within 4 h of preparation.

Patch-clamp electrophysiological recordings
Brain slices were transferred to a recording chamber continuously perfused at a rate of ∼2-3 ml/min with aCSF bubbled with 95% O 2 -5% CO 2 . The majority of voltage-clamp recordings were performed at physiological temperature (35 ± 1°C), although some were performed at room temperature (24 ± 1°C), where indicated. In experiments to test the effect of temperature on synaptic properties, the different temperatures were applied in random order. The slices were viewed using a Nikon (Tokyo, Japan) FN-1 microscope with DIC optics and a Nikon NIR Apo ×40/0.80 N/A water-immersion objective. The images were collected with a QICLICK, MONO 12BIT camera (Nikon) using NIS-Elements software (Nikon). MOC neurons were identified for whole-cell voltage-clamp recordings by their position in the VNTB (MOC neurons in the dorsal peri-olivary region were not recorded from in this study) and visibility using red epifluorescence (546 nm emission filter, Lumencor Sola lamp). The recordings were performed using a MultiClamp 700B and DigiData 1440A controlled by Clampex 10.6 software (Molecular Devices). The recordings were sampled at 50 kHz and filtered on-line at 10 kHz. The internal solution for MOC recordings contained (in mm): 56 CsCl, 44 CsOH, 49 d-gluconic acid, 1 MgCl 2 , 0.1 CaCl 2 , 10 Hepes, 1 EGTA, 0.3-Na-GTP, 2 Mg-ATP, 3 Na 2 -phosphocreatine, 5 QX-314, 0.25% biocytin, 0.01 AlexaFluor-488 hydrazide. The Clequilibrium potential was ∼−20 mV.
Recording pipettes were pulled from 1.5 mm outer diameter borosilicate glass (Sutter Instrument Co., Novato, CA, USA) to tip resistances of 3−6 M . Series resistances were corrected 50−85%. The cells were voltage-clamped at −60 mV unless stated otherwise. Membrane voltages were not adjusted for a measured liquid junction potential of −2 mV (caesium gluconate solution). MNTB axons were electrically stimulated to evoke neurotransmitter release and generate postsynaptic currents (PSC) in MOC neurons by a large diameter glass pipette of ∼10 or ∼30 μm diameter filled with aCSF and 0.01 AlexaFluor-488 hydrazide connected to an Iso-Flex Stimulus Isolation Unit (A.M.P.I., Jerusalem, Israel), placed in the MNTB axon bundle within or at the lateral edge of the MNTB. The stimulus intensity was initially set to zero, then was gradually increased until PSCs were evoked. The stimulus was then adjusted until PSCs were evoked during less than 50% of stimulations, defined as 'minimal stimulation' . The stimulus intensity was then increased until a maximal evoked PSC (ePSC) amplitude was reached, and then reduced to an intermediate intensity that reliably evoked PSCs at a smaller amplitude than for maximum stimulation, 320 ± 180 μA. In previous work using the same methodology, this intermediate stimulus intensity evoked PSCs that were approximately 70% of the maximal ePSC amplitude (Torres Cadenas et al., 2020). 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (5 μm) was included in all experiments to block excitatory transmission through AMPA-type glutamate receptors by addition to the recirculating aCSF solution. Drugs and chemicals were obtained from Thermo Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St Louis, MO, USA).

Experimental design and statistical analysis
Evoked PSC amplitudes were measured in Clampfit 10.6 (Molecular Devices, San Jose, CA, USA) by manually placing cursors at the immediate onset of the PSC (for trains of stimuli, during the decay of the preceding PSC immediately prior to the stimulus artifact), and at the peak of the PSC. The latency to the PSC response was measured from the beginning of the stimulus artifact to the onset of the response. The PSC time constant of decay was measured from the peak of the evoked PSC to the baseline plateau and was fit with a single exponential. In some experiments to measure the effect of spontaneous background activity on synaptic plasticity of inhibitory synaptic inputs, MNTB axons were first stimulated for 20 pulses at 10 or 100 Hz, followed by stimulation at higher rates of 50-500 Hz. The amplitude of ePSCs, probability of recording an evoked PSC, and facilitation index (S x −S 1 )/S 1 was computed during synaptic responses to stimulus trains as in our previous work (Torres Cadenas et al., 2020).
All statistical analyses were performed using Origin v2020 (OriginLab Corp., Northampton, MA, USA) or R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria) using packages 'lme4' and 'lmerTest' . Linear mixed models were fit by REML (restricted (residual) maximum likelihood). t-Tests used Satterthwaite's method ('lmerModLmerTest'). Data were examined for normal distribution using the Shapiro-Wilk test. Non-parametric tests were applied given the non-normal distribution of many variables studied. The differences between two independent groups were assessed using a Mann-Whitney U-test.
Differences between multiple groups were tested with a Wilcoxon rank sum test. Differences from a defined value of 0 or 1 were tested with a One sample Wilcoxon signed-rank test. To evaluate the effects of stimulation rates on short-term plasticity at MNTB-MOC synapses, linear mixed-effects models were run with facilitation index as the dependent variable, stimulation rates and trains of electrical stimulation as fixed-effect independent variables. The random effects include random intercept for neurons and random slope for stimulation rate. We used the Akaike information criterion (AIC) to assist with selecting the appropriate statistical model. A lower AIC value indicates better quality of fit. The differences were considered significant if P < 0.05 and indicated by asterisks in figures. For figures, single traces were lowpass filtered at 2 kHz and in cases where more than one voltage-clamp trace was averaged, the data were not filtered. Summary box plots indicate the median and quartiles, with 10 and 90 percentiles indicated by error bars. A square within the box plot indicates the mean. Individual data points are overlaid. All data are presented as median ± median absolute deviation (MAD) unless otherwise stated. The figures were prepared in Origin v2020 and Adobe Illustrator CC 2021 (Adobe Inc., San Jose, CA, USA).

Results
Whole-cell patch-clamp recordings were performed from identified MOC neurons in brain slices from ChAT-IRES-Cre × tdTomato mice by targeting red fluorescent neurons in the VNTB. Using this approach, PSCs that were evoked (ePSCs) by electrical stimulation of presynaptic MNTB axons ( Fig. 1A) occurred in MOC neurons that were consistent with previous recordings (Torres Cadenas et al., 2020). With the high chloride concentration used here (60 mm), both excitatory and inhibitory PSCs were inward at a holding potential of −60 mV. AMPA receptors were blocked in all experiments with CNQX (5 μm) to isolate inhibitory PSCs. After establishing this baseline, we tested whether there was a difference in strength of inhibitory synaptic inputs in male vs. female mice. We compared the amplitude of PSCs evoked by minimal stimulation (interpreted as the stimulation of a single axon) and found no difference (ePSC amplitude: female: 24.4 ± 7.25 pA, male: 28.1 ± 8.2, Mann-Whitney U-test, U = 302, Z = −0.76, P = 0.44; ePSC time constant of decay: female: 11.6 ± 5.1 ms, male: 12.1 ± 4.5 ms, Mann-Whitney U-test, U = 348, Z = 0.04487, P = 0.96; n = 23 female, n = 30 male). Due to a lack of difference in evoked PSCs of male vs. female mice, mice of both sexes were pooled for further analyses.

Calcium dependence of plasticity at MNTB-MOC synapses
Many auditory neurons in the CN and SOC fire rapid action potentials through the duration of a sound stimulus, so short-term synaptic plasticity due to high rates of synaptic activity has important implications for auditory neuron circuit function. Short-term synaptic plasticity is governed by mechanisms that affect both pre-and postsynaptic elements, including extracellular calcium concentrations (Neher & Sakaba, 2008;Regehr, 2012). To characterize calcium dependence of short-term synaptic plasticity at the MNTB-MOC synapses, we applied trains of electrical stimulation to MNTB axons at rates of 10−100 ms in extracellular calcium concentrations from 0.5 to 2.0 mm calcium. The effect of stimulation rates was analysed with a linear mixed-effect model. By including random intercept for neurons and random slope for stimulations rate, we assume that the facilitation index and the effect of stimulation rates vary for each neuron. This was the best fitting model (AIC = −456.95) compared to a model without random effects (AIC = −87.43) and a model with only a random intercept (AIC = −279.62). Similar to our previous work (Torres Cadenas et al., 2020), electrical stimulation-evoked PSCs exhibited synaptic depression in 2.0 mm extracellular calcium at stimulation rates of 10, 50 and 100 Hz, with 100 Hz stimulation evoking a significantly greater degree of depression relative to 10 Hz stimulation (β = −0.35, SE: 0.086, t = −4.05, P = 0.0098, Fig. 2Aa). Recent work suggests that an extracellular calcium concentration of 1.2-1.5 mm is thought to approximate in vivo levels (Borst, 2010). In 1.2 mm extracellular calcium, trains of stimulation of MNTB axons did not evoke synaptic depression at the second pulse in the train, consistent with previous work using paired pulse stimulation and an ISI of 10 ms (Torres Cadenas et al., 2020). However, with sustained synaptic activity through the train, synaptic depression was observed at stimulation rates of 10−100 Hz (one sample Wilcoxon signed-rank test indicated facilitation index was significantly less than zero, index = −0.398, V = 43,775, P < 2.2 × 10 −16 , Fig. 2Ab). Synaptic depression was significantly greater in 2.0 mm compared to 1.2 mm extracellular calcium at train rates of 100 and 50, but not 10 Hz, as measured by facilitation index (100 Hz: 2.0 mm −0.299 ± 0.38, 1.2 mm −0.105 ± 0.18, Wilcoxon rank sum test W = 29,680, P = 2.2 × 10 −16 ; 50 Hz: 2.0 mm −0.084 ± 0.15, 1.2 mm −0.090 ± 0.15, Wilcoxon rank sum test W = 18,780, P = 3.4 × 10 −7 ; 10 Hz: 2.0 mm −0.029 ± 0.11, 1.2 mm −0.114 ± 0.21, Wilcoxon rank sum test W = 19,690, P = 0.27, n = 6 neurons, Fig. 2B-D).
These results indicate that short-term synaptic depression of MNTB-MOC synapses is not as strong in physiological conditions (1.2 mm calcium) compared to higher calcium (2.0 mm) conditions, but synaptic depression in 1.2 mm calcium is significant with sustained synaptic activity.

Calcium dependence of recovery from short-term plasticity
Following a period of sustained activity that elicits short-term synaptic plasticity, synaptic responses typically recover to baseline levels with the recovery time indicating when synaptic responses will return to their maximal effect. We investigated recovery of MNTB-MOC synapses after synaptic depression following trains of electrical stimulation of MNTB axons of 20 pulses at 100 Hz, as above, and also probed the calcium dependence of recovery in extracellular calcium concentrations of 1.2 and 2.0 mm. The amplitudes of PSCs evoked by MNTB stimulation were normalized to the first PSC in the train. The facilitation index of the final PSC in the train showed significant depression, as above, but was not different between 1.2 and 2.0 mm calcium (1.2 mm calcium: index = −0.56 ± 0.32, Wilcoxon rank sum test W = 34, Z = 2.17, P = 0.030, n = 8 neurons; 2.0 mm: index = −0.68 ± 0.29, Wilcoxon rank sum test W = 50, Z = 2.24, P = 0.025, n = 10 neurons, 1.2 vs. 2.0 mm calcium: Mann-Whitney U-test, U = 39.5, Z = 0, P = 1.00; Fig. 3A and B). This lack of difference in the facilitation index at the last pulse in the train  Data are presented as median ± MAD. Fast rates of MNTB axon stimulation were applied at the rates indicated ('sound-like' stimulation rate), either in control conditions or following two seconds of mild (10 Hz) or strong (100 Hz) background stimulation.
indicates that the synapses recovered from a similar degree of synaptic depression in both 1.2 and 2.0 mm calcium. Following the last pulse in the train, MNTB axons were again stimulated after a recovery interval ranging from 20 ms to 15 s ( Fig. 3A and B).  Fig. 3C and inset), demonstrating a calcium dependence of recovery from synaptic depression.

MNTB-MOC synaptic function following tonic background MNTB activity
MNTB neurons have a high background firing rate up to 189 Hz in the absence of sound, with average rates around 10−20 Hz documented in multiple species (Hermann et al., 2007;Kadner & Berrebi, 2008;Kopp-Scheinpflug et al., 2003Smith et al., 1998;Sommer et al., 1993). This high rate of background firing suggests that the MNTB-MOC synapses may be tonically depressed in vivo, even in quiet. Therefore, we mimicked the tonic activity level present in vivo at the MNTB-MOC synapse to determine if this baseline activity could alter ePSCs in MOC neurons at faster activity rates, such as might occur during the transition from quiet to sound-driven activity. Experiments were performed in 1.2 mm extracellular calcium and at physiological temperature. To mimic tonic firing, we applied 'background' stimulation to MNTB axons at 10 Hz, a typical spontaneous rate for MNTB neurons. This firing rate evokes mild synaptic depression at MNTB-MOC synapses, while a high rate of baseline stimulation at 100 Hz evokes strong synaptic depression at the MNTB-MOC synapse, as shown above. After 2 s of mild (10 Hz) or strong (100 Hz) baseline stimulation we then applied a 400 ms stimulation, at 50, 100, 200 or 500 Hz, to mimic enhanced MNTB synaptic activity associated with sound ( Fig. 4A and B). We compared the peak current amplitude reached in the control condition to the peak current amplitude reached following background stimulation. Mild background stimulation at 10 Hz did not reduce the subsequent responses to faster stimulation, but faster (100 Hz) background stimulation inhibited the response to subsequent stimulation at 200 Hz ( Fig. 4C-G and Table 1). Thus, if a given MNTB neuron has a typical, mild rate of background activity, the MNTB synapse will have a significant effect on the MOC neuron at sound onset. However, for the MNTB neurons with a high background activity, further synaptic activity evoked by sound may have a limited effect on the postsynaptic MOC neuron.

Rapid, sustained activity at MNTB-MOC synapses
Sound-driven responses of MNTB neurons in vivo can be up to 500−700 Hz (Kopp-Scheinpflug et al., 2008). Because MNTB-MOC synapses depress during repetitive stimulation, we asked whether these synapses can maintain activity even when rapid stimulation rates are applied for sustained durations. For this work we applied trains of electrical stimulation to MNTB axons at rates of 200 and 500 Hz for a duration of 1 s. During this sustained stimulation we found that evoked responses summated and became indistinct over the duration of the trains to reach a peak amplitude that incorporated both summation and short-term plasticity ( Fig. 5A and B). peak amplitude, there was a trend towards a faster latency from stimulation onset to peak in MNTB-MOC synapses stimulated at 500 compared to 200 Hz (latency to peak: 200 Hz: 45.36 ± 11.09 ms, n = 5 neurons; 500 Hz: 26.44 ± 8.46 ms, n = 6 neurons, Mann-Whitney U-test: U = 26, Z = 1.92, P = 0.055, Fig. 5D). Following this peak in activity, the total current had an exponential decay with a time constant that was not different between stimulation rates (200 Hz exponential decay: 715.92 ± 106.31 ms, n = 5 neurons, 500 Hz decay: 447.23 ± 277.47 ms, n = 6 neurons, Mann-Whitney U-test: U = 15, Z = 0.42, P = 0.68, Fig. 5E) and also decayed to a plateau current that was not different between stimulation rates (plateau current: 200 Hz: 92.4 ± 35.2 pA, n = 5 neurons, 500 Hz plateau: 144.6 ± 93.8 pA, n = 6 neurons, Mann-Whitney U-test: U = 10, Z = −0.82, P = 0.41). Thus, during sustained MNTB-MOC synapse activity such as may occur during a sustained sound, the evoked responses build to a peak followed by decay but are able to maintain a sustained inhibitory current.

Discussion
Functional evidence describing the complexity of synaptic inputs to MOC neurons corroborates existing histological evidence that multiple classes of neurons form synapses onto olivocochlear efferent neurons, indirectly influencing cochlear function. Recent work demonstrates that MOC neurons are excited by both ascending sound-driven inputs from CN T-stellate and small cell cap cells and descending excitation from the IC (Hockley et al., 2022;Romero & Trussell, 2021). MOC neurons are also inhibited by ascending sound-driven pathways via MNTB neurons (Torres Cadenas et al., 2020). There are likely additional sources of excitation, inhibition, or modulation from auditory and other brain regions as indicated by histological studies Caicedo & Herbert, 1993;Faye-Lund, 1986;Groff & Liberman, 2003;Horvath et al., 2003;Mulders & Robertson, 2000Ota et al., 2004;Suthakar & Ryugo, 2017;Thompson & Thompson, 1993;Vetter et al., 1993). The integration of these synaptic inputs to govern MOC activity depend on the relative strength and postsynaptic effect of inputs, and their plasticity during changing hearing conditions. Here we further characterize the short-term plasticity of inhibitory inputs to MOC neurons from neurons of the MNTB, in mouse. MNTB-MOC synaptic inputs depress over a range of physiological calcium and stimulation rate conditions, yet maintain sustained synaptic function. This holds even during high rates of activity and following background spontaneous rates of activity.

Temperature and calcium effects on MNTB-MOC plasticity
Increasing the temperature of tissue speeds the kinetics of biological processes, including those regulating vesicular neurotransmitter release (Sabatini & Regehr, 1996). Consistent with this idea, raising the temperature of a brain slice from RT to PhT during patch-clamp recordings of MNTB-MOC synaptic responses both decreased the latency and sped up the kinetics of evoked PSCs. Using PhT during recording has also been shown to decrease the latency and speed of evoked PSCs in neurons from other auditory and non-auditory brain areas (Kushmerick et al., 2006;Taschenberger & von Gersdorff, 2000;Zhang & Trussell, 1994). However, we saw no change in the amplitude of single evoked PSCs in PhT compared to RT. In other cell types an increase in evoked PSC amplitudes with increased temperature has been observed (Hardingham & Larkman, 1998;Kushmerick et al., 2006;Taschenberger & von Gersdorff, 2000;Zhang & Trussell, 1994), sometimes also associated with an increase in the mini-PSC (mPSC) amplitude (Kushmerick et al., 2006;Van Hook, 2020). In other systems that also have an increase in evoked PSC amplitude with increased temperature, this effect was not associated with an increase in mPSC amplitude (Hardingham & Larkman, 1998). It is unclear why we do not see an increased PSC amplitude with temperature. This may simply reflect a lack of change in quantal size with temperature combined with fewer release sites from MNTB onto MOC neurons, especially compared to other studies discussed here that examined synapses with many release sites. Changes in short-term synaptic plasticity with temperature have been observed at other central synapses. While many mechanisms affect both pre-and postsynaptic plasticity, net plasticity can be in part attributed to a balance between release probability and vesicle recruitment. Release probability has been observed to increase with temperature at many (Hardingham & Larkman, 1998;Pyott & Rosenmund, 2002;Volgushev et al., 2004) but not all (Allen & Stevens, 1994) synapses and is typically associated with increased synaptic depression. Increased vesicle recruitment occurs with increased temperature (Kushmerick et al., 2006;Pyott & Rosenmund, 2002) and is associated with decreased synaptic depression. However, at many synapses the net effect is a reduction in short-term synaptic depression at PhT relative to RT (Brenowitz et al., 1998;Hardingham & Larkman, 1998;Kushmerick et al., 2006;Pyott & Rosenmund, 2002;Taschenberger & von Gersdorff, 2000;Volgushev et al., 2004). Consistent with these studies, we find that there is less synaptic depression of MNTB-MOC synapses at PhT compared to RT, extending this principle to inhibitory synapses.
In addition to temperature effects, short-term synaptic plasticity involves interplay between many mechanisms, including presynaptic calcium dynamics, vesicle and release site properties, neurotransmitter diffusion and clearance from the synaptic cleft, and postsynaptic receptor dynamics, among other mechanisms. Various mechanisms for the strong influence of extracellular J Physiol 600.11 calcium on presynaptic mechanisms of plasticity have been proposed, including vesicle pool replenishment or depletion, changes in vesicle release probability, and effects on presynaptic calcium channels (Catterall & Few, 2008;Fioravante & Regehr, 2011;Mochida, 2011;Neher & Sakaba, 2008;Sakaba et al., 2002;Zucker & Regehr, 2002;reviewed in Regehr, 2012). In our previous work there was no difference between short term depression at MNTB-MOC synapses in 2.0 vs. 1.2 mm calcium when measured with paired pulse stimulation (Torres Cadenas et al., 2020), suggesting that synaptic plasticity is independent of extracellular calcium at this synapse. While unusual, this has been observed at other auditory synapses in the CN that were tested with high vs. physiological (lower) divalent cations in aCSF (Xie & Manis, 2017). However, extending the duration of synaptic activity to trains of stimulation of MNTB-MOC synapses demonstrated less synaptic depression in 1.2 compared to 2.0 mm calcium. With a further decrease in the calcium concentration to 0.8 mm, MNTB-MOC synapses were facilitated, confirming the calcium dependence of plasticity at the MNTB-MOC synapse via an unknown mechanism at the MNTB axon terminal.
The recovery of a synapse from depression is also calcium dependent, including dependence on mechanisms such as vesicle pool refilling (Dittman & Regehr, 1998;Neher & Sakaba, 2008). Indeed, MNTB-MOC synapses recovered to baseline levels faster in higher calcium conditions. Interestingly, the time constant of recovery from depression of inhibitory synapses to MOC neurons was faster than that of excitatory synapses to MOC neurons from either T-stellate or inferior colliculus neurons (Romero & Trussell, 2021). This suggests that if excitatory and inhibitory inputs to MOC neurons experience similar activity rates, inhibitory synapses will recover from depression faster than excitatory synapses.

Effect of spontaneous background activity on MNTB-MOC synaptic inhibition
The GBC-MNTB pathway that inhibits MOC neurons is remarkably fast and powerful, with high-fidelity synaptic transfer in response to stimulation up to ∼800 Hz at the calyx of Held synapse in vitro (Futai et al., 2001;Joshi et al., 2004;Kopp-Scheinpflug et al., 2003;Spirou et al., 1990;Taschenberger & von Gersdorff, 2000;. In vivo, the background spontaneous rate of MNTB activity in the absence of sound averages about 10−20 Hz and can range up to 190 Hz (Hermann et al., 2007;Kadner et al., 2006;Kopp-Scheinpflug et al., 2003Smith et al., 1998;Sommer et al., 1993). In response to sound stimulation, MNTB neurons have a primary-like with notch firing pattern (Guinan & Li, 1990;Kopp-Scheinpflug et al., 2008;Paolini et al., 2001;Smith et al., 1998;Tolnai et al., 2009) and spike at rates of up to hundreds of hertz in vivo (Kopp-Scheinpflug et al., 2008). Work from other labs has demonstrated that the calyx of Held synapses onto MNTB neurons have smaller evoked responses and more action potential failures following spontaneous background activity, but exhibit less synaptic depression during fast trains (Hermann et al., 2007). In addition, there is a transient synaptic facilitation following background stimulation (Müller et al., 2010). The in vivo activity patterns of MNTB-MOC synapses are unknown, but we assumed that, similar to the MNTB soma, they may have some baseline spontaneous activity that evokes tonic depression, followed by faster rates of activity in response to sound. MNTB inhibition of MOC neurons was not reduced by a low, 10 Hz background synaptic stimulation rate, but was reduced by a prior high, 100 Hz synaptic stimulation rate. Therefore, most MNTB neurons with low background spontaneous rates will likely inhibit MOC neurons upon sound onset. In contrast, MNTB neurons with high background activity levels may instead exert a tonic inhibition of MOC neurons that prevents MOC activity except during a strong sound stimulus.

Effect of inhibition on MOC activity
MOC neurons in guinea pig and cat rarely fire action potentials in quiet in vivo, and have low threshold responses to acoustic stimuli (Liberman, 1988;Liberman & Brown, 1986;Robertson, 1984;Robertson & Gummer, 1985). In contrast, in young mice (P14-P23), post-hearing onset MOC neurons sometimes have spontaneous action potentials in vitro (Torres Cadenas et al., 2020). MOC neurons recorded from brain slices of mice older (P30-P48) than those used in the present study lack spontaneous action potentials (Romero & Trussell, 2021). Activity from inhibitory MNTB inputs is likely silent in these experiments on more mature mice because spontaneous activity from MNTB neurons is largely absent in in vitro brain slices (Hermann et al., 2007). This suggests that changes in intrinsic electrical properties that occur with maturation, not inhibition, are likely responsible for the low spontaneous spike rate of mature MOC neurons in vivo, although evoked inhibition from the MNTB persists in the mature MOC neurons at the same levels as in younger animals (Torres Cadenas et al., 2020).
Instead of controlling the MOC neuron resting membrane potential, inhibition from MNTB neurons likely has a primary role in modulating MOC neuron responses to sound-driven excitatory synaptic inputs. We propose a model in which low rates of spontaneous MNTB-MOC synaptic activity in quiet provides mild inhibition and electrical shunting of MOC neurons that may raise the action potential threshold in response to excitation. Higher rates of MNTB-MOC activity at sound onset suppress and delay MOC responses to concurrent excitatory synaptic inputs, consistent with the long and variable latencies (from ∼5 to ∼65 ms) of MOC neurons in response to sound (Liberman & Brown, 1986;Robertson, 1984;Robertson & Gummer, 1985). Indeed, 100 Hz, but not slower, stimulation of MNTB axons using the same methodology as in the current work delays spontaneous MOC action potentials (Torres Cadenas et al., 2020). Through a long-duration sound, the MNTB-MOC synapses would undergo short-term depression that reduces the influence of inhibition. The time course of decay of sustained MNTB-MOC inhibition is in the range of hundreds of milliseconds, which is too long to account for the spike latencies of MOC neuron sound responses. Therefore, activation of MOC neurons by both ascending excitatory drive from the CN (Hockley et al., 2022;Romero & Trussell, 2021) and descending, facilitating excitation from the IC (Romero & Trussell, 2021) likely overcomes inhibition to evoke action potentials in MOC neurons. This hypothesized interaction of excitation and inhibition in MOC neurons, combined with the low synaptic release probability and subsequent facilitation of MOC effects on cochlear OHCs (Ballestero et al., 2011), is consistent with the build-up of 'fast' MOC effects as measured by distortion product otoacoustic emissions (∼100 ms; Guinan, 2011). However, this model does not inform 'slow' MOC effects lasting seconds (Guinan, 2011), which is likely due to properties of excitatory and modulatory synaptic inputs to MOC neurons. In contrast to the majority of MOC neurons that are likely receiving low rates of spontaneous background MNTB synaptic activity, MOC neurons receiving high rates of spontaneous background MNTB synaptic activity are likely insensitive to moderate sound. However, MOC neurons with rapid background inhibition may have a short latency response to onset of a loud sound due to the tonic synaptic depression of their inhibitory synaptic inputs. This is consistent with in vivo work showing a decrease in MOC response latency with increased sound intensity (Liberman & Brown, 1986). Therefore, reduced inhibitory drive to a subpopulation of MOC neurons at sound onset due to high rates of tonic inhibitory synaptic activity and subsequent depression may allow for faster negative feedback to the cochlea in response to loud sounds. Overall, inhibitory synapses from the MNTB to the MOC are likely characterized by tonic activity that under most conditions of low background activity would continuously inhibit MOC function in quiet, and robustly inhibit MOC neuron function at sound onset. These functions combine to delay MOC suppression of cochlear activity during rapidly modulating sound.


A cathelicidin antimicrobial peptide from Hydrophis cyanocinctus inhibits Zika virus infection by downregulating expression of a viral entry factor

Zika virus (ZIKV) is a re-emerging flavivirus that causes conditions such as microcephaly and testis damage. The spread of ZIKV has become a major public health concern. Recent studies indicated that antimicrobial peptides are an ideal source for screening antiviral candidates with broad-spectrum antiviral activities, including against ZIKV. We herein found that Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus in our previous work, conferred protection against ZIKV infection in host cells and showed preventative efficacy and therapeutic efficacy in C57BL/6J mice, Ifnar1−/− mice, and pregnant mice. Intriguingly, we revealed that Hc-CATH decreased the susceptibility of host cells to ZIKV by downregulating expression of AXL, a TAM (TYRO3, AXL and MERTK) family kinase receptor that mediates ZIKV infection, and subsequently reversed the negative regulation of AXL on host’s type I interferon response. Furthermore, we showed that the cyclo-oxygenase-2/prostaglandin E2/adenylyl cyclase/protein kinase A pathway was involved in Hc-CATH-mediated AXL downregulation, and Hc-CATH in addition directly inactivated ZIKV particles by disrupting viral membrane. Finally, while we found Hc-CATH did not act on the late stage of ZIKV infection, structure–function relationship studies revealed that α-helix and phenylalanine residues are key structural requirements for its protective efficacy against initial ZIKV infection. In summary, we demonstrate that Hc-CATH provides prophylactic and therapeutic efficacy against ZIKV infection via downregulation of AXL, as well as inactivating the virion. Our findings reveal a novel mechanism of cathelicidin against viral infection and highlight the potential of Hc-CATH to prevent and treat ZIKV infection.

3
The Second Affiliated Hospital of Soochow University, Suzhou, China

Edited by Craig Cameron
Zika virus (ZIKV) is a re-emerging flavivirus that causes conditions such as microcephaly and testis damage. The spread of ZIKV has become a major public health concern. Recent studies indicated that antimicrobial peptides are an ideal source for screening antiviral candidates with broad-spectrum antiviral activities, including against ZIKV. We herein found that Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus in our previous work, conferred protection against ZIKV infection in host cells and showed preventative efficacy and therapeutic efficacy in C57BL/6J mice, Ifnar1 −/− mice, and pregnant mice. Intriguingly, we revealed that Hc-CATH decreased the susceptibility of host cells to ZIKV by downregulating expression of AXL, a TAM (TYRO3, AXL and MERTK) family kinase receptor that mediates ZIKV infection, and subsequently reversed the negative regulation of AXL on host's type I interferon response. Furthermore, we showed that the cyclo-oxygenase-2/ prostaglandin E2/adenylyl cyclase/protein kinase A pathway was involved in Hc-CATH-mediated AXL downregulation, and Hc-CATH in addition directly inactivated ZIKV particles by disrupting viral membrane. Finally, while we found Hc-CATH did not act on the late stage of ZIKV infection, structurefunction relationship studies revealed that α-helix and phenylalanine residues are key structural requirements for its protective efficacy against initial ZIKV infection. In summary, we demonstrate that Hc-CATH provides prophylactic and therapeutic efficacy against ZIKV infection via downregulation of AXL, as well as inactivating the virion. Our findings reveal a novel mechanism of cathelicidin against viral infection and highlight the potential of Hc-CATH to prevent and treat ZIKV infection.
Zika virus (ZIKV) is a single-stranded, enveloped, and positive-sense RNA virus belonging to the Flaviviridae family (1). This group of approximately 70 viruses include members such as dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus (2,3). In all flaviviruses, the RNA genome encodes a polyprotein that is processed into three structural proteins (core C, membrane precursor prM, and envelope E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1,4). The structural proteins form the virus particle and the nonstructural proteins assist in replication and packaging of the genome. Aedes mosquitoes are the main vectors for the transmission of ZIKV (5). Besides, ZIKV can also be transmitted through sexual, vertical, and blood transmission (6,7). Initially, ZIKV did not arouse widespread concern in society, and there were few related cases of ZIKV (8). Recent epidemiological studies have shown that ZIKV infection can cause a variety of serious diseases, which can lead to microcephaly, congenital malformations, brain calcification, fetal death, multiple organ failure, thrombocytopenia, thrombocytopenic purpura, testis damage, and male infertility (9,10). In addition, ZIKV infection can also cause blindness and a variety of eye abnormalities, including retinal spots, lens subluxation, and optic neuritis (2,11). Therefore, it is urgent to develop safe and effective drugs for the prevention and therapy of ZIKV infection.
Many molecules expressed on the cell surface are involved in viral infection, including the DC cell surface c-type lectin receptor superfamily (DC-SING), DC-SING-related proteins (L-SING) (12,13), and phosphatidylserine (PS) receptors (11,14). PS receptors play a critical role in flavivirus infection, including members of the TIM (T-cell immunoglobulin and mucin domain) family and TAM (TYRO3, AXL and MERTK) family (15,16). TAM receptors belong to the tyrosine receptor family, a transmembrane protein that transmits signals from the extracellular environment to the cytoplasm and nucleus. TAM receptors regulate various cellular functions, including clearance of apoptotic cells by macrophages, platelet aggregation, and differentiation of natural killer cells (17). AXL belongs to the TAM family and can be expressed in brain and neural progenitor cells. It has been reported that AXL plays an important role in ZIKV infection (18)(19)(20)(21). On the one hand, AXL can act as a cofactor for the entry of ZIKV to host cells (15,22,23). On the other hand, AXL can regulate the innate ‡ These authors contributed equally to this work. * For correspondence: Lin Wei, weilin1005@126.com; Wei Xu, xuwei828@ suda.edu.cn.
immune response of host cells by negatively regulating interferon (IFN) signaling and the expression of inflammatory factors, thus promoting ZIKV infection (22,24,25). These molecules involved in viral infection are potential targets for the prevention and therapy of ZIKV infection. Antimicrobial peptides (AMPs), also called host defense peptides, are a group of relatively small cationic peptides. They are an important part of the innate immune system and play an important role in fighting against bacterial infection (26). Recently, more and more studies have shown that AMPs can also exert antiviral effects by inhibiting virus adsorption, disrupting virus envelope, and regulating host antiviral immune response (27)(28)(29). AMPs have become an important source for screening antiviral peptides with broad antiviral spectrum against herpes simplex virus (30,31), human immunodeficiency virus (32), hepatitis C virus (33), and influenza A virus (34). Therefore, AMPs can be used as new candidates for the development of antiviral drugs. In mammals, the two major AMP families are cathelicidins and defensins, among which cathelicidins have direct antimicrobial activity against a variety of microorganisms and actively participate in host's immune regulation (35). Cathelicidins are also found in the venom of various snakes. Previous studies have shown that cathelicidins derived from snakes have strong antibacterial activity with broad spectrum, including clinically isolated drug-resistant bacterial strains (36,37). Besides, snake-derived cathelicidins have extremely low cytotoxicity and hemolytic activity, showing a high clinical application prospect. In our previous study, we identified a novel cathelicidin peptide from the venom of the sea snake, Hydrophis cyanocinctus, which is designated as Hc-CATH (26). It showed a broad antibacterial spectrum as well as potent anti-inflammatory activity with high stability and low cytotoxicity (26). However, few attention was paid to its antiviral activity. In this study, we tried to evaluate the effects of Hc-CATH on ZIKV infection, including its effect on ZIKV-caused cytopathic effect, ZIKV replication in vitro, and ZIKV replication in vivo. The mechanism of action against ZIKV infection was also investigated.

Hc-CATH inhibits ZIKV infection in vitro
In order to explore whether Hc-CATH has antiviral activity against ZIKV, Vero cells were infected with ZIKV in the presence of Hc-CATH or PBS (solvent of peptide) as indicated in Fig. 1A, and the effect of Hc-CATH on ZIKV-caused cytopathic effect and ZIKV replication were detected. As shown in Fig. 1, B and C, ZIKV infection obviously caused cytopathic effect in Vero cells, and Hc-CATH markedly reversed the cytopathic effect caused by ZIKV infection in a dose-dependent manner. Hc-CATH also reduced the intracellular ZIKV RNA (Fig. 1D), NS3 protein (Fig. 1E), E protein (Fig. 1F), and extracellular ZIKV particles (Fig. 1, G and H) in a dose-dependent manner. At concentration of 5 μM, Hc-CATH reduced about 70.3% cytopathic effect, 52.7% ZIKV RNA, 67.4% NS3 protein, and 71.6% ZIKV particles. Besides, Hc-CATH did not have cytotoxicity against mammalian cells at concentrations of 1.25, 2.5, and 5 μM (Fig. S1). The results indicate that Hc-CATH shows inhibitory effect against ZIKV infection in Vero cells.

Hc-CATH decreases the susceptibility of host cells to ZIKV
To understand the anti-ZIKV mechanism of Hc-CATH, we first investigated whether Hc-CATH decreases the susceptibility of host cells. Vero cells were incubated with Hc-CATH or PBS before ZIKV infection as indicated in Fig. 2A (Hc-CATH-cell-pre). As shown in Fig. 2, B-G, the incubation of Vero cells with Hc-CATH for 2 h significantly reduced the intracellular ZIKV RNA (Fig. 2B), NS3 protein (Fig. 2, C and D), E protein (Fig. 2E), and extracellular ZIKV particles (Fig. 2, F and G) relative to PBS incubation. The incubation of Vero cells with Hc-CATH reduced about 46.1% ZIKV RNA (Figs. 2B), 50.1% ZIKV NS3 protein (Fig. 2, C and D), and 62.2% ZIKV particles (Fig. 2, F and G). The results indicate that incubation of Vero cells with Hc-CATH decreases the susceptibility of host cells to ZIKV.

Hc-CATH downregulates AXL in host cells
It is well known that several ZIKV entry factors, such as AXL, TIM-1, and TYRO3, can change the susceptibility of host cells to ZIKV (15). In order to identify whether Hc-CATH decreases the susceptibility of host cells by downregulating these factors, we performed experiment as indicated in Fig. 3A. As shown in Fig. 3B, Hc-CATH reduced the level of AXL in Vero cells relative to PBS after incubation for 24 h. Immunofluorescence (IF) staining confirmed that Hc-CATH did reduce the level of AXL in Vero cells after incubation for 24 h (Fig. 3C). While Hc-CATH did not obviously influence the other ZIKV entry factors (TYRO3 and TIM-1) at 6, 12, and 24 h post Hc-CATH addition (Fig. 3B). We next tested the effect of Hc-CATH on AXL in Vero cells upon ZIKV infection as indicated in Fig. 3D. At 24 h post Hc-CATH addition, Hc-CATH also decreased the level of AXL in Vero cells relative to PBS upon ZIKV infection (Fig. 3E). We then tested the effect of Hc-CATH on AXL in A549 cells and U251 cells as indicated in Fig. 4A and Fig. 5A, respectively. The results demonstrated that Hc-CATH effectively decreased the level of AXL in A549 cells (Fig. 4, B and C, E and F) and U251 cells (Fig. 5, B and C, E and F). Besides, Hc-CATH also decreased the susceptibility of A549 cells (Fig. 4, E and G) and U251 cells (Fig. 5, E and G) to ZIKV. The data suggest that Hc-CATH decreases the susceptibility of host cells to ZIKV by downregulating AXL.
Cyclo-oxygenase-2/prostaglandin E2/adenylyl cyclase/PKA pathway is involved in Hc-CATH-mediated AXL downregulation As described previously, cycloo-xygenase-2 (COX-2) can regulate the synthesis of prostaglandin E 2 (PGE 2 ), and PGE 2 can activate the expression of downstream signal molecules that make AXL stably express on cell surface (38). We were interested to explore whether this signaling pathway is involved in Hc-CATH-mediated AXL downregulation. We found that Hc-CATH did not influence the expression of COX-2 ( Fig. 6A), but it significantly inhibited the enzyme activity of COX-2 in a dose-dependent manner (Fig. 6B). At concentration of 10 μM, the inhibitory rate of Hc-CATH against COX-2 activity could reach about 80%. As expected, the addition of Hc-CATH significantly reduced the synthesis of PGE 2 in a dose-dependent manner (Fig. 6C). It was reported that PGE 2 can lead to the activation of adenylyl cyclase (AC) and an increase in the cAMP. Subsequently, cAMP can activate PKA (39). To see if these downstream signal molecules are involved in Hc-CATH-induced AXL downregulation, we added forskolin (FSK, a specific activator of AC) to the cell culture. As shown in Fig. 6D, the addition of FSK effectively counteracted the Hc-CATH-induced AXL downregulation. However, a specific PKA kinase inhibitor, H89, significantly facilitated Hc-CATH-mediated AXL downregulation (Fig. 6E). These results indicate that COX-2/PGE 2 /AC/PKA pathway is involved in Hc-CATH-mediated AXL downregulation. Hc-CATH-induced AXL downregulation reverses the negative regulation of AXL on type I IFN signaling Several studies have shown that AXL is an important negative regulator of type I IFN signaling and facilitate ZIKV infection by antagonizing type I IFN (40). We next tested whether Hc-CATH-induced AXL downregulation reverses the negative regulation of AXL on type I IFN as indicated (Fig. 7A). We found that Hc-CATH significantly increased the levels of type I IFN genes (Fig. 7B). Meanwhile, Hc-CATH also upregulated the levels of type I IFN genes in response to ZIKV infection (Fig. 7C). In contrast, type I IFN genes were only slightly activated or, in some cases, downregulated in response to ZIKV infection without Hc-CATH treatment (Fig. 7C). As described previously, ZIKV has evolved multiple molecular mechanisms to escape type I IFN production (41). Next, we used Sendai virus (SeV) to amplify type I IFN signaling (42) and verified the effect of Hc-CATH on type I IFN response upon SeV stimulation as indicated in Fig. 7D. We found that SeV markedly induced IFN-β expression, and Hc-CATH treatment significantly increased IFN-β expression in response to SeV stimulation as compared with PBS treatment (Fig. 7E). To further explore the role of Hc-CATH in type I IFN response, we investigated its effect on IFN signaling pathway by detecting the phosphorylation of TANK-binding kinase 1 and interferon regulatory factor 3 (IRF3) in the presence or the absence of SeV stimulation. As shown in Fig. 7, F and G, Hc-CATH increased the phosphorylation of TANKbinding kinase 1 and IRF3 relative to PBS, and it also enhanced the phosphorylation of IRF3 (indicated by a triangle) in response to SeV stimulation. The data suggest that Hc-CATHinduced AXL downregulation reverses the negative regulatory effect of AXL on type I IFN response.

Hc-CATH directly inactivates ZIKV particles by disrupting viral membrane
It was reported that some anti-ZIKV peptides can directly inactivate virions by disrupting virus particles (28,43). To investigate whether Hc-CATH exhibited direct effect on ZIKV, ZIKV virions were incubated with PBS or peptide at 37 C for 2 h, and the infectivity of ZIKV virions was tested as indicated in Fig. 8A (Hc-CATH-virus-pre). As shown in Fig. 8, B-G, the incubation of ZIKV with Hc-CATH significantly decreased the intracellular ZIKV RNA ( D), E protein ( Fig. 8E), and extracellular ZIKV titer (Fig. 8, F and G) relative to PBS incubation, indicating that the incubation of Hc-CATH with ZIKV directly inactivated viral particles. To further determine the interaction of Hc-CATH with ZIKV, we tested the binding of Hc-CATH to ZIKV by ELISA. As shown in Fig. 8H, Hc-CATH had a stronger binding affinity with ZIKV as compared with bovine serum albumin, which proved that Hc-CATH could directly bind to ZIKV particles.
We next explored whether Hc-CATH disrupt ZIKV envelope by testing the potential release of viral genomic RNA from ZIKV after the incubation of ZIKV with Hc-CATH. As shown in Fig. 8I, the genomic RNA of PBS-treated virions was protected from digestion by micrococcal nuclease, which was determined as 100%. However, the genomic RNA of Hc-CATH-treated virions was digested by micrococcal nuclease. These data suggest that Hc-CATH can bind to ZIKV, disrupt

Hc-CATH does not act on the late stage of ZIKV infection
To see if Hc-CATH act on ZIKV replication stage, we conducted experiments as indicated in Fig. 9A (Hc-CATHmaint). When Hc-CATH was maintained in the cell culture after ZIKV infection, Hc-CATH did not reduce the intracellular ZIKV RNA (Fig. 9B), NS3 protein ( Fig. 9, C and D), E protein (Fig. 9E), and extracellular ZIKV titer (Fig. 9, F and G) in Vero cells relative to PBS. Hc-CATH also did not inhibit ZIKV infection in A549 cells (Fig. 9H) and U251 cells (Fig. 9I) when Hc-CATH was added to the cell culture after ZIKV infection. The results indicate that Hc-CATH does not act on the late stage of ZIKV replication.
Helix and phenylalanine residues are key structural requirements for Hc-CATH against ZIKV infection In our previous study, Hc-CATH was demonstrated to have helical structure, positively charged residues (five arginine and seven lysine residues), and aromatic residues (three phenylalanine residues) (26). In order to understand the key structural requirements of Hc-CATH against ZIKV infection, the helix of Hc-CATH was disrupted by scrambling the amino acid sequence ( Fig. 10A and S2), and the arginine, lysine, or phenylalanine residues were substituted with alanine residues (Fig. 10A), respectively. We found that Hc-CATH was unable to downregulate AXL (Fig. 10B) and decrease the susceptibility of Vero cells to ZIKV (Fig. 10C) when the helix was disrupted. Besides, Hc-CATH was unable to directly inactivate ZIKV when the helix was disrupted, or phenylalanine residues were substituted with alanine residues (Fig. 10D). The data indicate that α-helix and phenylalanine residues are key structural requirements for Hc-CATH against ZIKV infection.

Hc-CATH shows preventive and therapeutic effect against ZIKV infection in mice
Since Hc-CATH effectively inhibits ZIKV infection in vitro, we were interested to test whether Hc-CATH can resist ZIKV infection in vivo. We first evaluated whether Hc-CATH has preventive efficacy against ZIKV infection. C57BL/6J mice, IFNα/β receptor-deficient (Ifnar1 −/− ) mice, and pregnant C57BL/6J mice were intravenously administrated with Hc-CATH at 2 h before intravenous injection of ZIKV as indicated in Fig. 11A. As shown in Fig. 11, B-D, intravenous injection of Hc-CATH at 2 h before ZIKV infection significantly decreased ZIKV replication in C57BL/6J mice (Fig. 11B), Ifnar1 −/− mice (Fig. 11C), fetal placenta, and fetal mice (Fig. 11D), implying that intravenous injection of Hc-CATH at 2 h before ZIKV challenge provides prophylactic efficacy against ZIKV infection in mice. We then evaluated whether Hc-CATH has therapeutic effect against ZIKV infection. C57BL/6J mice, Ifnar1 −/− mice, and pregnant C57BL/6J mice were intravenously administrated with Hc-CATH at 2 h after intravenous injection of ZIKV as indicated in Fig. 12A. As shown in Fig. 12, B-D, intravenous injection of Hc-CATH at 2 h after ZIKV infection significantly attenuated ZIKV replication in C57BL/6J mice (Fig. 12B), Ifnar1 −/− mice (Fig. 12C), fetal placenta, and fetal mice (Fig. 12D), suggesting that intravenous injection of Hc-CATH at 2 h after ZIKV challenge provides therapeutic efficacy against ZIKV infection in mice. In addition, the anti-ZIKV efficacy of Hc-CATH seemed to be weaker than that of AC5 and LL-37 in vitro (Fig. 1), but its anti-ZIKV efficacy was comparable with or even better than that of AC5 and LL-37 in vivo (Figs. 11E and 12E).

Discussion
It has been shown that AMPs (also called host defense peptides) not only have antibacterial and immunomodulatory activities but also can be used as an important source of antiviral drug development with broad antiviral spectrum (28,29,44,45). In recent years, a series of AMPs/host defensive peptides were shown to confer protection against ZIKV infection, including human cathelicidin AMP (46) and defensin AMP (47), bovine cathelicidin AMP (46), scorpion venom peptides derived from Scorpio maurus palmatus (48) and Euscorpiops validus (49), spider venom peptide from Alopecosa nagpag (50), frog host defense peptide from Indosylvirana aurantiaca (51), and snail antibacterial peptide from Pomacea poeyana (52). We herein found that the snake venom-derived cathelicidin AMP (Hc-CATH) from H. cyanocinctus exhibited potent preventive and therapeutic efficacy against ZIKV infection in vitro and in vivo, which provides a novel anti-ZIKV peptide isolated from AMPs/hose defense peptides. Compared with the antiviral drugs based on small molecular compounds and antibodies, peptide-based antiviral drugs have attracted more and more attention because of their good safety and lower development cost and better safety (43,53).
Anti-ZIKV peptides from biological sources display multiple antiviral mechanisms against ZIKV infection. Human cathelicidin LL-37 and its derived peptide GF-17, mouse cathelicidin   , and extracellular ZIKV titer (F and G) were tested by qPCR, Western blot, immunofluorescence staining, and plaque-forming assay, respectively. The raw data files used in the creation of F are presented in Fig. S5. H, binding of Hc-CATH to ZIKV. High-affinity binding plates were coated with 0.25 μM of Hc-CATH, BSA, AC5, or LL-37. Then, 1 × 10 6 PFU of ZIKV was added and incubated. Wells were exposed to anti-ZIKV E protein antibody, HRP-labeled secondary antibody, and TMB substrate in turn. Absorbance at 450 nm was measured after reactions were stopped using 0.5 M sulfuric acid. I, leakage of ZIKV genomic RNA induced by Hc-CATH. ZIKV (about 10 5 PFU) was incubated with Hc-CATH (1, 20, and 40 μM), PBS (solvent of peptide), or 1% Triton X-100 (positive control) at 37 C for 2 h. The released genomic RNA was digested by micrococcal nuclease at 37 C for 4 h. After the residual RNase was inactivated, the undigested genomic RNA in the intact ZIKV particles was extracted and quantified by testing the genes of PrM, E, and Cap proteins, respectively. **p < 0.01 and ***p < 0.001. BSA, bovine serum albumin; FBS, fetal bovine serum; Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus; HRP, horseradish peroxidase; MOI, multiplicity of infection; PFU, plaque-forming unit; qPCR, quantitative PCR; ZIKV, Zika virus.
Hc-CATH can disrupt ZIKV particle, induce the leakage of genomic RNA, and finally inactivate virion. The direct inactivation of ZIKV by Hc-CATH is reminiscent of the mechanism of action of other biological peptides like LL-37 (28), CRAMP , and extracellular ZIKV titer (F and G) were detected by qPCR, Western blot, immunofluorescence staining, and plaque-forming assay, respectively. The raw data files used in the creation of F are presented in Fig. S6. ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus; MOI, multiplicity of infection; qPCR, quantitative PCR; ZIKV, Zika virus.
interact with lipid membranes, resulting in instability, translocation, pore formation, or cleavage of lipid membranes (43). Therefore, viral envelope can be an important target for AMPs.
To verify whether such structure is essential for the direct inactivation of ZIKV by Hc-CATH, the helix of Hc-CATH was disrupted by scrambling the amino acid sequence. We found that scrambled Hc-CATH did not directly inactivate ZIKV virion, suggesting that the helical structure is a key structural requirement for the direct inactivation of ZIKV by Hc-CATH. In addition to helical structure, aromatic residue (phenylalanine) was also shown to be critical for the direct inactivation of ZIKV by Hc-CATH. Our previous studies have shown that helical structure and aromatic residue are essential for the AMPs to directly kill bacteria by disrupting bacterial membrane (26,55). These indicate that helical structure and aromatic residues may be common structural requirements for inactivating enveloped virus and bacteria by disrupting microbial membrane. However, the substitution of positively charged residues with alanine does not inhibit the Hc-CATHmediated direct inactivation of ZIKV. While the substitution of arginine/lysine with alanine markedly attenuates the direct antibacterial activity of Hc-CATH (26). Intriguingly, Hc-CATH effectively downregulates AXL in host cells. AXL is a member of TAM receptors that play an important role in ZIKV infection (22,40). AXL exhibits dual role during ZIKV infection. On the one hand, AXL acts as a cofactor that mediates ZIKV entry (40). TAM receptors can recognize PS. Similar to other flaviviruses, the envelope of ZIKV contains PS, which facilitates the adsorption and internalization of TAM receptors. As described previously, AXL can promote ZIKV entry in human Sertoli cells (25) and human skin cells (11). On the other hand, AXL promotes ZIKV infection by antagonizing type I IFN signaling (22,40). AXL plays a pivotal role in maintaining the immunosuppressive milieu of the testis, enhancing ZIKV infection by negatively regulating antiviral immune response (25). Consistent with these findings, Hc-CATH-mediated AXL downregulation effectively decreases the susceptibility of host cells to ZIKV and reverses the negative regulation of AXL on type I IFN signaling. In addition, COX-2//PGE 2 /AC/PKA pathway is Lower panel, ratio of NS3 to β-actin analyzed by ImageJ. The α-helix of Hc-CATH was disrupted by scrambling the amino acid sequence, and the arginine, lysine, or phenylalanine residues of Hc-CATH were substituted with alanine residues, respectively. Downregulation of AXL, decrease of the susceptibility of Vero cells to ZIKV, and inactivation of ZIKV by Hc-CATH and its mutant peptides were assayed. ns, not significant, ***p < 0.001. Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus; ZIKV, Zika virus.
involved in Hc-CATH-mediated AXL downregulation. Our findings reveal another possible mechanism of biological peptides against ZIKV infection, providing new insight into the mechanism of action of anti-ZIKV peptides isolated from AMPs/host defense peptides. Innate immune response is the first line for host defense against invading viruses. In general, the innate immune response is initiated by identifying pathogen-associated molecular patterns accumulated during infection. Pathogenassociated molecular patterns bind to pattern recognition receptors in the nucleus or in the cytoplasm, resulting in the activation of intracellular cascade signals and the upregulation of a variety of innate immune molecules and cytokines (56). Type I IFNs are important cytokines for host defense against viruses. They are responsible for coordinating the antiviral state in infected cells and adjacent cells and controlling virus infection by guiding the activation or transportation of immune cells (41). Our findings have shown that Hc-CATH can reverse the negative regulation of AXL on type I IFN response, which seems to be an important antiviral mechanism of Hc-CATH against ZIKV infection. However, Hc-CATH still possesses strong antiviral efficacy against ZIKV in Ifnar1 −/− mice, implying that the anti-ZIKV effect of Hc-CATH does not depend on the production of type I IFNs induced by Hc- CATH. It is more likely that Hc-CATH has multiple antiviral mechanism, including direct inactivation of ZIKV, induction of AXL downregulation, and enhancement of type I IFN response, and the protective efficacy of Hc-CATH in Ifnar1 −/− mice may be attributed to Hc-CATH-mediated ZIKV inactivation and AXL downregulation. In this study, we claimed that Hc-CATH enhanced type I IFN response of host cells by downregulating AXL, but we cannot exclude the direct regulatory effect of Hc-CATH on type I IFN response of host cells, which need to further elucidated in future.
In summary, Hc-CATH, cathelicidin AMP identified from sea snake H. cyanocinctus, was shown to inhibit ZIKV infection in vitro and provide preventive and therapeutic efficacy against ZIKV infection in vivo. Intriguingly, Hc-CATH downregulated AXL in host cells, thus decreased the susceptibility of host cells to ZIKV. Hc-CATH also acted as an inactivator of ZIKV by disrupting viral membrane and inducing the leakage of genomic RNA. Helical structure and phenylalanine residues are key structural requirements for Hc-CATH against ZIKV infection. Our findings provide a new peptide candidate for anti-ZIKV drug development and new insight into the cathelicidin AMPs against ZIKV infection.

Experimental procedures
Key resource table
ZIKV strain PRVABC59 (GenBank number: KU501215) was originally obtained from Dr Feifei Yin at Hainan Medical College. ZIKV was propagated in Vero cells and stored at −80 C. The virus titer was determined in Vero cells using plaqueforming assay.
Synthetic peptides, including Hc-CATH, LL-37 (positive peptide, human cathelicidin), and AC5 (positive peptide, anti-ZIKV peptide from Aedes aegypti), are listed in Table S1 and purchased from SynPeptide Co Ltd. The crude peptide was purified by reversed-phase high performance liquid chromatography and analyzed by mass spectrometry to confirm the purity higher than 98%.
Mice C57BL/6J mice, pregnant C57BL/6J mice, and Ifnar1 −/− mice were housed in specific pathogen-free conditions. The C57BL/6J mice and pregnant C57BL/6J mice were ordered Anti-ZIKV peptide derived from the sea snake cathelicidin from Shanghai SLAC Laboratory Animal Co Ltd. Ifnar1 −/− mice were gifted by Dr Chunsheng Dong's laboratory at Soochow University and bred in our laboratory. Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of Soochow University, and all research protocols were approved by the Animal Ethical Committee of Soochow University. All surgeries of mice were performed under pentobarbital sodium anesthesia with minimum fear, anxiety, and pain.

Antiviral activity of Hc-CATH against ZIKV in vitro
Vero cells were seeded in 24-well plates (5 × 10 4 cells/well). After cells were adhered to plates, cells were incubated with ZIKV (multiplicity of infection [MOI] = 1) in the presence of noncytotoxic concentration of Hc-CATH, AC5 (positive control), LL-37 (positive control), or PBS (solvent of peptide). After incubation at 37 C for 2 h, the culture media were removed, cells were washed with PBS and transferred with fresh culture media containing 2% FBS, and Hc-CATH, AC5, LL-37, or PBS was supplemented to each well. After incubation at 37 C for 48 h, cytopathic effect was observed by microscopy, and Cell Counting Kit-8 was applied to determine the percent of cytopathic effect. The percent of cytopathic effect was calculated as (A sham -A treatment )/A sham × 100 (where A sham represents the absorbance of uninfected cells, A treatment represents the absorbance of PBS-or peptide-treated cells post ZIKV infection). The intracellular ZIKV RNA, NS3 protein, E protein, and extracellular ZIKV titer were tested by qPCR, WB, IF, and plaque-forming assay, respectively (28). Primers used for qPCR are listed in Table S2.
Effect of Hc-CATH on the susceptibility of host cells to ZIKV Vero, A549, or U251 cells were incubated with peptide or PBS at 37 C for 2 h. The cells were washed three times with PBS and incubated with ZIKV (MOI = 1) at 37 C for 2 h. The culture media were replaced with fresh DMEM containing 2% FBS. After incubation at 37 C for 48 h, intracellular ZIKV RNA, NS3 protein, E protein, and extracellular ZIKV titer were detected by qPCR, WB, IF, and plaque-forming assay, respectively (28).
The effect of Hc-CATH on AXL was further confirmed in A549 and U251 cells. A549 or U251 cells were incubated with Hc-CATH (1.25, 2.5, and 5 μM) or PBS at 37 C for 24 h, and the level of AXL was detected by WB and IF, respectively. Upon ZIKV challenge, A549 or U251 cells were incubated with Hc-CATH (1.25, 2.5, and 5 μM) or PBS at 37 C for 2 h. A549 and U251 cells was then washed with PBS and challenged with ZIKV (MOI = 1). After A549 and U251 cells were cultured for 24 h, AXL and NS3 protein level was tested by WB analysis.
Effect of COX-2/PGE 2 /AC/PKA pathway on Hc-CATH-induced AXL downregulation A549 cells were cultured with Hc-CATH (1.25, 2.5, and 5 μM) or PBS at 37 C for 24 h, cells were harvested for detecting the level of COX-2 by WB analysis, and the cell supernatant was collected for detecting the level of PGE 2 by ELISA.
The effect of Hc-CATH on the enzymatic activity of COX-2 was detected by enzyme activity inhibitor screening kit. Hc-CATH (1.25, 2.5, 5, and 10 μM) or PBS was incubated with COX-2. After incubation at 37 C for 10 min, the enzymatic activity of COX-2 was detected by the kit. Celecoxib (COX-2 inhibitor, 100 nM) was used as positive control.
To assay the effect of adenylate cyclase (AC) and PKA on Hc-CATH-induced AXL downregulation, we added FSK (agonist of AC, 10 μM) and H89 (inhibitor of PKA, 5 μM) to A549 cells and incubated at 37 C. After incubation for 1 h, the culture media were removed, cells were washed three times with PBS, and fresh culture media containing 2% FBS were added to cells in the presence of Hc-CATH (5 μM). After incubation for 24 h, AXL protein level was examined by WB.
Effect of Hc-CATH on the negative regulation of AXL on type I IFN signaling U251 cells were cultured with Hc-CATH (2.5 μM) or PBS at 37 C for 24 h, and cells were collected for testing type I IFN signaling gene and protein by qPCR and WB, respectively. Upon ZIKV challenge, U251 cells were incubated with Hc-CATH (2.5 μM) or PBS at 37 C for 2 h and washed three times with PBS. Cells were then incubated with ZIKV (MOI = 1) for 2 h and washed with PBS. After culture for 24 h, type I IFN signaling genes were tested by qPCR. Upon SeV challenge, U251 cells were incubated with Hc-CATH (2.5 μM) or PBS at 37 C for 12 h, cells were then stimulated with SeV or same volume of PBS for 12 h, and type I IFN signaling gene and protein were tested by qPCR and WB, respectively.

Effect of Hc-CATH on ZIKV particles
For assay of the direct inactivation of ZIKV by Hc-CATH, ZIKV (MOI = 1) was incubated with Hc-CATH (2.5 μM), AC5 (2.5 μM), LL-37 (2.5 μM), or same volume PBS at 37 C for 2 h. ZIKV-PBS mixture and ZIKV-peptide mixture were centrifugated at 100,000g for 70 min. The pellets were washed with PBS and centrifugated at 100,000g for 70 min again. The pellets were resuspended in PBS and added to Vero cells. The cells were incubated at 37 C for 2 h, washed with PBS, and transferred with fresh DMEM containing 2% FBS. Then the cells were cultured at 37 C for 48 h. The intracellular ZIKV RNA, NS3 protein, E protein, and extracellular ZIKV titer were tested by qPCR, WB, IF staining, and plaque-forming assay, respectively (28).
For assay of the binding of Hc-CATH to ZIKV, high-affinity binding plates were coated with 0.25 μM of Hc-CATH, bovine serum albumin, AC5, or LL-37. Then 1 × 10 6 plaque-forming unit (PFU) of ZIKV was added and incubated. Wells were exposed to anti-ZIKV E protein antibody, horseradish peroxidase-labeled secondary antibody, and TMB substrate in turn. Absorbance at 450 nm was measured after reactions were stopped using 0.5 M sulfuric acid (28).
For assay of the disruption of viral membrane by Hc-CATH, ZIKV (about 10 5 PFU) was incubated with Hc-CATH (1, 20, and 40 μM), PBS, or 1% Triton X-100 (positive control) at 37 C for 2 h. The released genomic RNA was digested by micrococcal nuclease at 37 C for 4 h. The residual RNase was inactivated, the undigested genomic RNA in the intact ZIKV particles was extracted and quantified by testing viral genes encoding membrane protein (PrM), E protein, and capsid (Cap) protein, respectively (43).

Effect of Hc-CATH on ZIKV replication stage
Vero cells were incubated with ZIKV (MOI = 1) at 37 C for 2 h, washed three times with PBS, and transferred with fresh DMEM containing 2% FBS and Hc-CATH (2.5 μM), AC5 (2.5 μM), LL-37 (2.5 μM), or PBS. After culture at 37 C for 48 h, intracellular ZIKV RNA, NS3 protein, E protein, and extracellular ZIKV titer were detected by qPCR, WB, IF, and plaque-forming assay, respectively. The effect of Hc-CATH on ZIKV replication stage was further tested in A549 and U251 cells under the same condition, and intracellular ZIKV NS3 protein was detected by WB.

Structure-function relationship study
To investigate the role of helical structure of Hc-CATH in its anti-ZIKV activity, the amino acid sequence of Hc-CATH was scrambled to disrupt the α-helix. To investigate the role of aromatic residues of Hc-CATH in its anti-ZIKV activity, phenylalanine residues of Hc-CATH were substituted with alanine residues. To investigate the role of positively charged residues of Hc-CATH in its anti-ZIKV activity, arginine or lysine residues of Hc-CATH were substituted with alanine residues. The anti-ZIKV activity of each mutant peptide was tested and compared with that of Hc-CATH, respectively.

Statistical analysis
Data were represented as mean ± SD. Statistical significance was determined by an unpaired two-tailed Student's t test for two-group comparison and was determined by ANOVA followed by Bonferroni post hoc analysis for multiple-group comparison. All statistical analyses were performed using GraphPad Prism software, version 5.0 (GraphPad Software, Inc).

Data availability
All data are contained within the article.
Supporting information-This article contains supporting information (Tables S1 and S2 and Fig. S1-S6).