Effect of Noradrenaline on the Facial Stimulation-Evoked Mossy Fiber-Granule Cell Synaptic Transmission in Mouse Cerebellar Cortex

Noradrenaline is an important neuromodulator in the cerebellum. We previously found that noradrenaline depressed cerebellar Purkinje cell activity and climbing fiber–Purkinje cell synaptic transmission in vivo in mice. In this study, we investigated the effect of noradrenaline on the facial stimulation-evoked cerebellar cortical mossy fiber–granule cell synaptic transmission in urethane-anesthetized mice. In the presence of a γ-aminobutyrateA (GABAA) receptor antagonist, air-puff stimulation of the ipsilateral whisker pad evoked mossy fiber–granule cell synaptic transmission in the cerebellar granular layer, which expressed stimulus onset response, N1 and stimulus offset response, N2. Cerebellar surface perfusion of 25 μM noradrenaline induced decreases in the amplitude and area under the curve of N1 and N2, accompanied by an increase in the N2/N1 ratio. In the presence of a GABAA receptor blocker, noradrenaline induced a concentration-dependent decrease in the amplitude of N1, with a half-maximal inhibitory concentration of 25.45 μM. The noradrenaline-induced depression of the facial stimulation-evoked mossy fiber–granule cell synaptic transmission was reversed by additional application of an alpha-adrenergic receptor antagonist or an alpha-2 adrenergic receptor antagonist, but not by a beta-adrenergic receptor antagonist or an alpha-1 adrenergic receptor antagonist. Moreover, application of an alpha-2 adrenergic receptor agonist, UK14304, significantly decreased the synaptic response and prevented the noradrenaline-induced depression. Our results indicate that noradrenaline depresses facial stimulation-evoked mossy fiber–granule cell synaptic transmission via the alpha-2 adrenergic receptor in vivo in mice, suggesting that noradrenaline regulates sensory information integration and synaptic transmission in the cerebellar cortical granular layer.

Noradrenaline is an important neuromodulator in the cerebellum. We previously found that noradrenaline depressed cerebellar Purkinje cell activity and climbing fiber-Purkinje cell synaptic transmission in vivo in mice. In this study, we investigated the effect of noradrenaline on the facial stimulation-evoked cerebellar cortical mossy fibergranule cell synaptic transmission in urethane-anesthetized mice. In the presence of a γ-aminobutyrate A (GABA A ) receptor antagonist, air-puff stimulation of the ipsilateral whisker pad evoked mossy fiber-granule cell synaptic transmission in the cerebellar granular layer, which expressed stimulus onset response, N1 and stimulus offset response, N2. Cerebellar surface perfusion of 25 µM noradrenaline induced decreases in the amplitude and area under the curve of N1 and N2, accompanied by an increase in the N2/N1 ratio. In the presence of a GABA A receptor blocker, noradrenaline induced a concentration-dependent decrease in the amplitude of N1, with a halfmaximal inhibitory concentration of 25.45 µM. The noradrenaline-induced depression of the facial stimulation-evoked mossy fiber-granule cell synaptic transmission was reversed by additional application of an alpha-adrenergic receptor antagonist or an alpha-2 adrenergic receptor antagonist, but not by a beta-adrenergic receptor antagonist or an alpha-1 adrenergic receptor antagonist. Moreover, application of an alpha-2 adrenergic receptor agonist, UK14304, significantly decreased the synaptic response and prevented the noradrenaline-induced depression. Our results indicate that noradrenaline depresses facial stimulation-evoked mossy fiber-granule cell synaptic transmission via the alpha-2 adrenergic receptor in vivo in mice, suggesting that noradrenaline regulates sensory information integration and synaptic transmission in the cerebellar cortical granular layer.

INTRODUCTION
The cerebellar cortex acquires information from three classes of afferents: mossy fibers (MFs), climbing fibers, and multilayered fibers, and generates motor-related output by Purkinje cells (PCs) (Haines and Dietrichs, 2002). Under in vivo conditions, granule cells (GCs) exhibit a low frequency of spontaneous firing, but they are very sensitive to sensory stimulation (van Beugen et al., 2013). This sensory stimulation induces spike firing followed by a GABAergic inhibitory response in the GCs (Eccles et al., 1967;Ito, 1984;Jakab and Hámori, 1988), which precisely encodes the sensory information (D'Angelo et al., 2005;Jörntell and Ekerot, 2006). Therefore, it has been suggested that the GCs both exhibit high-frequency and high-fidelity properties in response to sensory stimulation, which could ensure that accurate information is transmitted to PCs (Arenz et al., 2008;van Beugen et al., 2013;Bing et al., 2015), while also filtering out unassociated components (Chadderton et al., 2004).
Noradrenaline (NA) is a widely studied neuromodulator involved in the modulation of learning and memory in the central nervous system. Anatomical studies indicate that noradrenergic (NAergic) fibers originate in the locus coeruleus (LC) and distribute through the cerebellar cortex through a multilayered fiber pathway (Kimoto et al., 1978;Schroeter et al., 2000). Noradrenergic inputs of the cerebellum have been shown to be involved in cerebellum-dependent motor learning (McCormick and Thompson, 1982;Keller and Smith, 1983;Watson and McElligott, 1984;Pompeiano, 1998) and long-term depression induction at PF-PC synapses in the flocculus by activating protein kinase A (PKA) (Inoshita and Hirano, 2021). Either iontophoretic application of NA or activation of the LC-induced potentiation of GABAergic transmission at molecular layer interneurons-PC synapse results in an inhibition of the PC spontaneous simple spike activity via activation of adrenoceptors (ARs) (Mitoma and Konishi, 1999;Saitow et al., 2000).
The ARs are G-protein-coupled receptors that come in two types, α-AR and β-AR. Both α-ARs and β-ARs are present in the cerebellar cortex, including the granular layer (GL) (McCune et al., 1993). The roles of α-ARs and β-ARs in the cerebellar cortex vary. Several studies demonstrated that NA could regulate cerebellar-dependent learning tasks and long-term memory via activation of β-ARs (Cartford et al., 2004;Schambra et al., 2005;Hein, 2006). In vitro, NA facilitated mouse cerebellar parallel fiber-PC synaptic transmission via activation of β-ARs, but it suppressed synaptic transmission via α2-ARs (Lippiello et al., 2015). However, NA facilitated spontaneous inhibitory postsynaptic currents of PCs via simultaneous activation of both α1-ARs and β-ARs located at the presynaptic terminals of molecular layer interneurons, which could synergically boost GABAergic transmitter release (Hirono et al., 2014). In addition, activation of α2-ARs by NA decreased the probability of transmitter release at climbing fiber-PC synapses, which in turn suppressed the climbing fiber-evoked dendritic calcium transients and controlled the induction of synaptic plasticity at parallel fiber-PC synapses by modulating dendritic calcium influx (Carey and Regehr, 2009). We previously found that NA-activated presynaptic α2-AR regulated climbing fiber-PC synaptic transmission via the PKA signaling pathway, suggesting that the NAergic fibers from the nucleus of the LC might regulate the output behavior of PC by suppressing the information transmission from the inferior olivary nucleus to the cerebellar cortex in vivo in mice (Sun et al., 2019;Cui et al., 2020).
Taken together, the effects of NA on cerebellar cortical neuronal synaptic transmission have been well studied in vitro, but the modulatory function of NA on sensory information processing in the cerebellar GL is not well understood. Therefore, in this study, we combined electrophysiological and pharmacological approaches to investigate the effects of NA on the facial stimulation-evoked MF-GC synaptic transmission in the absence the GABAergic inhibition in urethane-anesthetized mice.

MATERIALS AND METHODS
All the experimental procedures were approved by the Animal Care and Use Committee of Yanbian University and performed in accordance with the animal welfare guide lines of the National Institutes of Health. The permit number is SYXK (Ji) 2011-006. Anesthesia and surgical procedures have been described previously (Chu et al., 2011). In brief, either male (n = 36) or female (n = 30) adult (6-8 weeks old) ICR mice were anesthetized with urethane (1.1 g/kg body weight, intraperitoneal injection, i.p). After a water tight chamber was prepared, a 1-1.5 mm craniotomy was opened to expose the cerebellar surface of Crus II. The brain surface was superfused with oxygenated artificial cerebrospinal fluid (ACSF: 125 mM NaCl, 3 mM KCl, 1 mM MgSO 4 , 2 mM CaCl 2 , 1 mM NaH 2 PO 4 , 25 mM NaHCO 3 , and 10 mM D-glucose) with a peristaltic pump (Gilson Minipulse 3; Villiers, LeBel, France). The rectal temperature was monitored, and keeped at 37.0 ± 0.2 • C.
The sensory stimulation was performed by air-puff (60 ms, 50-60 psi) of the ipsilateral whisker pad through a 12-gauge stainless steel tube connected to a pressurized injection system (Picospritzer R III; Parker Hannifin Co., Pine Brook, Fairfield, NJ, United States). The whiskers were cut off to avoid the stimulation of the whiskers. The air-puff stimuli were controlled by a personal computer and were synchronized with the electrophysiological recordings and delivered at 0.05 Hz via a Master 8 controller (A.M.P.I., Jerusalem, Israel) and Clampex 10.4 software.
Local field potential recordings from GL were performed with an Axopatch 200B amplifier (Molecular Devices, Foster City, CA, United States) under current clamp conditions (I = 0). The potentials were acquired through a Digidata 1440 series analogto-digital interface on a personal computer using Clampex 10.4 software. Recording electrodes were filled with ACSF and with resistances of 3-5 M . Air-puff (60 ms, 50-60 psi) of the ipsilateral whisker pad evoked a paired-negative components N1, N2, accompanied with a positive component P1 in the GL of cerebellar cortical folium Crus II ( Figure 1A). According to our previous studies (Wu et al., 2014;Bing et al., 2015;Ma et al., 2019), N1 and N2 were identified as MF-GC synaptic transmission which evoked by the stimulation-on (N1) and stimulation-off (N2), respectively. P1 was identified as GABAergic inhibitory components which could be abolished by GABA A receptor blocker.
Electrophysiological data were analyzed using Clampfit 10.4 software (Molecular Device, Foster City, CA, United States). The amplitude and area under the curve (AUC) of the evoked field potential responses were maintained constant for an individual experiment in treatments of ACSF, drugs and recovery. It has been suggested that changes in the N2/N1 ratio vary inversely with the presynaptic release of transmitter (Mennerick and Zorumski, 1995;Hashimoto and Kano, 1998). We calculated N2/N1 ratio to mirror the probability of vesicular release at the MF-GC synapse . All data are expressed as the mean ± SEM. Differences between the mean values recorded under control and test conditions were evaluated with the one-way ANOVA with Tukey's post-hoc test using SPSS (Chicago, IL, United States) software. P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.

DISCUSSION
In this study, we showed that cerebellar surface perfusion of NA induced a concentration-dependent depression of facial stimulation-evoked MF-GC synaptic transmission, which was reversed by additional application of an α-AR antagonist but not reversed by a β-AR antagonist. Furthermore, the NAinduced inhibition of facial stimulation-evoked MF-GC synaptic transmission was reversed by additional application of an α2-AR antagonist but not by an α1-AR antagonist. Moreover, pharmacological activation of α2-AR significantly inhibited the facial stimulation-evoked MF-GC synaptic response and overwhelmed the NA-induced depression.
In the cerebellar cortex, GCs receive excitatory inputs from MFs and inhibitory inputs from Golgi cells (Shambes et al., 1978;Bower and Woolston, 1983;Chadderton et al., 2004). For evaluating the sensory information transmitted by MF-GC synaptic transmission, we here studied the facial stimulation-evoked field potential response in the mouse cerebellar GL in the absence of GABAergic inhibitory inputs (Ma et al., 2019). Consistent with previous studies (Wu et al., 2014;Bing et al., 2015;Ma et al., 2019), air-puff stimulation of the ipsilateral whisker pad induced MF-GC synaptic transmission, which expressed stimulus onset and stimulus offset responses in the absence of GABAergic inhibition. These results indicate that tactile mechanoreceptors generate the receptor potentials at both stimulus onset and offset, which suggests that the sensory stimulation-evoked MF-GC synaptic transmission is high-fidelity and reliably reflects the encoded sensory information (Arenz et al., 2008;van Beugen et al., 2013;Bing et al., 2015).
Previous studies showed that NAergic afferents originate in the LC and distribute throughout the cerebellar cortical GL, PC, and molecular layers (Kimoto et al., 1978;Schroeter et al., 2000). Morphological studies have shown that both α-ARs and β-ARs are present in the cerebellar cortex (McCune et al., 1993). We previously found that NA regulates spontaneously complex spikes activity of cerebellar PCs via activation of α2-ARs in vivo in mice (Sun et al., 2019). Our results in this study show that cerebellar surface perfusion of NA produces a concentration-dependent inhibition of synaptic transmission convey sensory information in the cerebellar GL. The NA-induced depression of the evoked MF-GC synaptic transmission was reversed by additional application of an α2-AR antagonist and was mimicked by activation of α2-ARs. These results indicate that NA activates α2-ARs, which results in a depression of the facial stimulation-evoked MF-GC synaptic transmission in the mouse cerebellar cortex. In addition, our results show that blockade of α2-AR has less effect on the sensory stimulation-evoked MF-GC synaptic transmission, suggesting that there is less α2-AR activation under these experimental conditions. α2-Adrenoceptors are coupled to a wide variety of second messenger systems via G i/o -proteins, which negatively regulate the activity of adenylyl cyclases and inhibit voltage-gated Ca 2+ channel activity (Limbird, 1988). Activation of α2-ARs suppresses the production of cAMP-dependent protein kinase activity, leading to the activation of protein phosphatase 1, which plays an inhibitory role in synaptic transmission through modifying α-amino-3-hydroxy-5-methyl-4-isoxazole-propionica (AMPA) receptors (Mulkey et al., 1994;Yan et al., 1999). Activation of α2-ARs reduces the phosphorylation of AMPA receptors via the PKA signaling pathway, resulting in the inhibition of synaptic transmission (Yi et al., 2013). In the cerebellar cortex, α2-ARs play critical roles in information processing and motor coordination skills (Lähdesmäki et al., 2002). A previous study demonstrated that activation of α2-ARs suppresses presynaptic glutamate release from mitral cells by a G i/o -protein-mediated inhibition of Ca 2+ channels in the mouse olfactory bulb (Huang et al., 2018). We previously found that NA inhibits complex spike activity via a presynaptic PKA signaling pathway in vitro . Our results here demonstrate that NA depresses the amplitude of N1 and N2, which is accompanied by an increase in the N2/N1 ratio, suggesting that NA modulates the facial stimulation-evoked glutamate release at the MF-GC synapse. Since the N2/N1 ratio is inversely correlated with the probability of vesicular release, we proposed that the NA-induced depression of MF-GC synaptic transmission by reducing presynaptic glutamate release from mossy fiber terminals (Mennerick and Zorumski, 1995;Hashimoto and Kano, 1998). In addition, we studied the effect of NA on the facial stimulation-evoked MF-GC synaptic transmission in urethane anesthetized mice. We could not exclude the possible effect of urethane on the sensoryevoked MF-GC synaptic transmission. However, administration of urethane produces inhibition of neuronal excitability by activation of the barium-sensitive potassium leak conductance, without effects on excitatory glutamate mediated synaptic transmission (Sceniak and Maciver, 2006;Chu et al., 2011). Therefore, urethane anesthesia might produce less effect on the facial stimulation-evoked MF-GC synaptic transmission in vivo in mice.
Cellular mechanisms of motor learning in the cerebellum are long-term depression (LTD) and potentiation (LTP) at PF-PC, MF-GC, and MLI-PC synapses (Ito and Kano, 1982;Ito, 1989;Roggeri et al., 2008;Bing et al., 2015). It has been shown that tactile stimulation of the whisker pad induces long-term synaptic plasticity in MF-GC synapses in anesthetized rats, which suggests that MF-GC synaptic transmission and plasticity are critical for sensory information-dependent motor learning in rodents (Roggeri et al., 2008). Importantly, NAergic inputs to the cerebellum have been implicated in cerebellum-dependent motor learning (McCormick and Thompson, 1982;Keller and Smith, 1983;Watson and McElligott, 1984;Pompeiano, 1998). Our present results show that NA significantly depresses sensory stimulation-evoked MF-GC synaptic transmission, which suggests that cerebellar NAergic inputs modulate synaptic transmission conveying sensory information through MF-GC synapses. In addition, NAergic inputs have been found play critical roles in sensory signal processing, as well as the facilitation of motor coordination and motor learning function (McCormick and Thompson, 1982;Keller and Smith, 1983;Watson and McElligott, 1984;Pompeiano, 1998;Waterhouse and Navarra, 2019). Thus, the NA-induced depression of MF-GC synaptic transmission may directly contribute to sensory information-dependent motor tasks. Since GCs transmit sensory information to PCs through PFs (Ito and Kano, 1982), the NA-induced depression of MF-GC synaptic transmission may modulate MF-PC synaptic plasticity and motor learning by down regulating PF excitatory inputs onto PCs. While further experiments are required to further understand the effects of NAergic inputs on cerebellar physiology, our results provide important insights into the cellular and synaptic mechanisms of how NA modulates sensory information processing in the cerebellar cortex.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
All the experimental procedures were reviewed and approved by the Animal Care and Use Committee of Yanbian University and performed in accordance with the animal welfare guide lines of the National Institutes of Health.