[目的]

中枢神经系统中，胰高血糖素样肽-1（glucagon-like peptide-1, GLP-1）是一种重要的神经递质，主要由孤束核（nucleus tractus solitarius, NTS）的前胰高血糖素（preproglucagon, PPG）神经元分泌，投射到达多个脑区，如脑干、下丘脑、杏仁核和小脑等。GLP-1受体是B型G蛋白偶联受体，可以通过Gαs发挥作用，影响多种信号通路。已有研究显示，GLP-1受体在大鼠小脑中大量表达，包括小脑分子层、浦肯野细胞、颗粒层，提示GLP-1可能参与小脑神经网络的功能活动，但GLP-1对小脑皮层的神经元活动中的调节机制目前尚不清楚，因此，本实验应用分子生物学、电生理和神经药理学等技术手段，研究GLP-1受体在小鼠小脑皮层中的表达，探讨GLP-1对小鼠小脑皮层浦肯野细胞简单峰电位（simple spike, SS）放电活动的调节机制。

[方法]

1. 分子生物学实验

（1）RT-PCR

对小鼠进行断头取脑，分离小脑组织后使用RNA提取液提取小脑RNA，使用Takara反转录PCR试剂盒对目标cDNA片段进行扩增。目的cDNA制备完成后，对cDNA样品进行PCR扩增。取5 µl的PCR扩增产物，配制溴化乙锭染色的2%琼脂糖凝胶进行电泳实验，确定目标片段是否存在，通过凝胶成像系统对凝胶进行拍照保存。

（2）Western Blot (WB)

通过断头取脑，提取小鼠小脑全组织，加入RIPA裂解缓冲液，进行匀浆，在确保组织完全裂解后使用离心机离心，收集上清液，使用BCA蛋白浓度测定试剂盒测定蛋白浓度。配制20%分离胶、5%浓缩胶，进行SDS-PAGE电泳。湿转法转膜，使用磷酸盐缓冲液（phosphate buffer saline, PBS）清洗已完全转印的PVDF印迹膜，然后将印迹膜放入5%脱脂牛奶中进行封闭。加入一抗单克隆兔抗GLP-1受体抗体（1:1000），4°C条件下过夜，隔天使用PBS清洗印迹膜后加入二抗羊抗兔抗体（1:20000），室温下孵育，用PBS洗脱。在暗室中配制ECL显色液，将印迹膜夹入ECL显色液充分反应后，使用化学发光成像系统进行扫描成像并存档。

（3）免疫组织化学和共聚焦显微成像

小鼠经腹腔注射水合氯醛深度麻醉，然后经心灌注预冷的PBS和4%多聚甲醛溶液，在4°C条件下，将大脑固定48小时，用刀片分离小脑。将小脑包埋后使用冷冻切片机将其切成厚度为8 μm的切片，并保存于4°C进行免疫组化实验。小脑切片分别在一抗兔多抗GLP-1受体抗体（1:1000）、二抗Cy3标记山羊抗兔抗体（1:200）和4',6-二脒基-2-苯基吲哚（4',6-diamidino-2-phenylindole, DAPI, 1:1000）中培养。通过共焦激光扫描显微镜获得荧光图像。在尼康C2激光共聚焦系统上，用10倍物镜获得包含所需区域的大区域图像。

2. 在体电生理记录

使用乌拉坦腹腔注射麻醉成年ICR小鼠，气管插管后将小鼠固定在脑部立体定位仪上。在小脑蚓部（Vermis）区钻取小孔，暴露小脑表面。用含氧的人工脑脊液（artificial cerebrospinal fluid, ACSF）持续灌流脑表面。使用体温仪监测并维持直肠温度处于37 ± 0.2°C。

应用Axopatch-200B放大器对小脑的浦肯野细胞（Purkinje cell, PC）进行细胞贴附式记录。利用Clampex 10.4软件，通过Digidata 1440数模转换器采集浦肯野细胞自发性放电活动信号。记录电极填充ACSF，电阻3-5 MΩ。在软脑膜下进行细胞贴壁记录，并以规则的自发SS和不规则的复杂峰电位（complex spike, CS）鉴定浦肯野细胞。

3. 离体电生理记录

选用6-8周龄ICR小鼠，异氟烷吸入麻醉后断头取脑组织，在含氧ACSF中低温冷却，使用震动切片机在小脑皮质的Vermis区制备300 μm的矢状面小脑切片。将制备的脑片置于ACSF中，在室温中孵育1个小时以上，使脑片中的神经元恢复活性，之后通过全细胞膜片钳记录小脑切片中的浦肯野细胞。

使用尼康显微镜的60倍水浸透镜结合红外CCD观察、确定适合被记录的细胞，记录电极是电阻为4-6 MΩ的厚壁玻璃电极。使用膜片钳放大器MultiClamp 700B，记录膜电位和/或膜电流，以5 kHz过滤并通过Clampex 10.4软件在电脑上用Digidata 1440A数模转换器采集记录到的数据。记录时用电压钳控制细胞电压保持在-70 mV。通过施加电压脉冲监测串联电阻，只有保持稳定串联阻值的细胞才纳入分析。所有药物通过蠕动泵灌流给药。通过给予超极化电流和膜电位活化超极化活化阳离子电流（Hyperpolarization-activated current, IH）。在电压钳记录模式下，电压门控钠通道阻断剂河豚毒素（tetrodotoxin, TTX）和BaCl₂被加入到细胞外液中，用于阻断电压门控钠通道和Ba²⁺敏感钾通道，从而分离出IH电流。

4. 数据分析

电生理数据用Clampfit 10.4软件进行分析。在ACSF灌流、药物灌流和药物洗出中，单个记录神经元的所有参数均保持不变。数值表示为平均值±标准误。使用SPSS 25.0软件进行单因素方差分析，确定各组数据之间的统计显著性水平差异。低于0.05的P值被认为实验组之间存在统计上的显著差异。

[结果]

第一部分：GLP-1对在体小鼠小脑浦肯野细胞简单峰电位放电活动的影响

（1）通过RT-PCR, Western Blot和免疫荧光实验确定GLP-1受体在小脑中表达，且在小脑浦肯野细胞层高度表达，提示GLP-1可能通过激活其受体，调节小脑神经网络的活动。

（2）小脑表面灌流GLP-1显著增强在体小鼠小脑浦肯野细胞的自发性SS的放电频率，但对SS放电的变异系数（coefficient of variation, CV）没有明显影响，提示GLP-1通过GLP-1受体调节小脑皮层浦肯野细胞的自发性放电活动。

（3）GLP-1对于浦肯野细胞的SS放电频率增强作用具有浓度依赖性，其半数有效浓度（The half effective concentration, EC₅₀）为154.4 nM。

（4）在阻断GABA_A受体条件下，小脑表面灌流GLP-1不影响GLP-1导致的浦肯野细胞SS放电频率增强作用，提示γ-氨基丁酸（γ-amino butyric acid, GABA）能介导的抑制性中间神经元网络不参与GLP-1对浦肯野细胞SS放电频率的增强作用。

（5）同时阻断GABA_A受体和离子型谷氨酸受体时，GLP-1对浦肯野细胞SS放电频率的增强作用被部分减弱，但不能完全阻断，表明阻断谷氨酸受体可以部分抑制GLP-1介导的浦肯野细胞SS放电频率增强。

（6）拮抗GLP-1受体完全阻断GLP-1介导的浦肯野细胞SS放电频率增强，而激动GLP-1受体可以增强浦肯野细胞SS放电频率。在活化GLP-1受体的基础上，GLP-1不能使浦肯野细胞SS放电频率进一步升高，表明GLP-1对于浦肯野细胞SS放电频率的增强作用是通过活化GLP-1受体实现的。

第二部分：GLP-1对离体小鼠小脑皮层浦肯野细胞简单峰电位活动的影响机制

（1）在阻断谷氨酸能和GABA能突触传递的情况下，GLP-1及其类似物可以增强浦肯野细胞去极化电流诱发的SS放电频率，并且伴有膜去极化，而拮抗GLP-1受体完全阻断GLP-1对浦肯野细胞的作用，表明GLP-1通过突触后受体机制增强浦肯野细胞的SS放电频率。

（2）GLP-1显著抑制浦肯野细胞后超极化电位（after-hyperpolarization potential, AHP）和外向整流电流（rectified current, IR），导致AHP和IR的振幅和波形下面积减小。

（3）在TTX和Ba²⁺存在条件下，GLP-1显著增强超极化刺激诱发的即时膜电流、稳定膜电流、尾电流、IH电流，且增强作用都随超极化刺激强度的增大而增强，提示GLP-1可能通过活化IH通道，导致膜去极化。

（4）在阻断IH通道的条件下，GLP-1对超极化刺激诱发的浦肯野细胞膜电流增强作用被完全阻断，表明GLP-1通过其受体增强IH通道活性，增加浦肯野细胞的兴奋性。

（5）在TTX和GABA_A受体阻断剂存在条件下，GLP-1通过其受体增加浦肯野细胞微型兴奋性突触后电流（miniature excitatory postsynaptic currents, mEPSCs）的发生频率，但对mEPSCs的振幅没有明显影响，提示GLP-1显著增强浦肯野细胞接收的谷氨酸释放。

（6）在特异性蛋白激酶A（protein kinase A, PKA）抑制剂存在条件下加入GLP-1不增加mEPSCs的发生频率，提示GLP-1通过PKA信号通路增加谷氨酸释放，促进浦肯野细胞膜去极化。

[结论]

（1）GLP-1受体在小脑浦肯野细胞高度表达，活化GLP-1受体增强小鼠小脑皮层浦肯野细胞SS放电频率。

（2）GLP-1通过其受体，活化浦肯野细胞膜的IH通道，抑制外向整流钾电流，促进膜去极化。

（3）GLP-1作用于浦肯野细胞突触前的GLP-1受体，通过PKA信号途径，增加谷氨酸释放，促进膜去极化。

[关键词]

胰高血糖素样肽-1（GLP-1）；小鼠小脑皮层；浦肯野细胞（PC）；简单峰电位放电（SS）；突触传递；微型兴奋性突触后电流（mEPSCs）；超极化活化阳离子电流（IH）

[Purpose]

In the central nervous system, glucagon-like peptide-1 (GLP-1) is an important neurotransmitter that is mainly secreted by preproglucagon (PPG) neurons in the nucleus tractus solitarius (NTS) and projects to multiple brain regions, such as the brainstem, hypothalamus, amygdala, and cerebellum. GLP-1 receptor is a B-type G protein-coupled receptor that can act through Gαs and affect a variety of signaling pathways. Studies have shown that GLP-1 receptor is abundantly expressed in the cerebellum of rats, including the molecular layer, Purkinje cells, and granular layer of the cerebellum, suggesting that GLP-1 may be involved in the functional activity of the cerebellar neural network, but the regulatory mechanism of GLP-1 in the neuronal activity of the cerebellar cortex is still unclear, therefore, in this experiment, molecular biology, electrophysiology, and neuropharmacology were used to investigate the expression of GLP-1 receptor in the cerebellar cortex of mice and to investigate the regulatory mechanism of GLP-1 on the simple spike (SS) firing activity of Purkinje cells in the cerebellar cortex of mice.

[Methods]

1. Molecular biology experiments

(1) RT-PCR

Mice were decapitated and brains were removed, cerebellar RNA was extracted using RNA extracts after isolation of cerebellar tissue, and target cDNA fragments were amplified using a Takara reverse transcription-PCR kit. After cDNA preparation, the cDNA samples were amplified by PCR. 5 µl of the PCR amplification products were used to prepare ethidium bromide-stained 2% agarose gels for electrophoresis experiments to determine the presence of target fragments, and the gels were photographed and stored by the gel imaging system.

(2) Western Blot (WB)

Brains were removed by decapitation, whole mouse cerebellum tissues were extracted, RIPA lysis buffer was added, homogenized, centrifuged using a centrifuge after ensuring complete tissue lysis, supernatants were collected, and protein concentrations were determined using a BCA protein assay kit. 20% separation gel and 5% stacking gel were prepared and subjected to SDS-PAGE electrophoresis. Membranes were transferred by wet transfer, and PVDF blot membranes that had been completely transferred were washed using phosphate buffer saline (PBS), and then the blot membranes were blocked in 5% skimmed milk. Primary antibody, monoclonal rabbit anti-GLP-1 receptor antibody (1:1000) was added, incubated overnight at 4°C, and the blot membranes were washed with PBS every other day followed by secondary antibody, goat anti-rabbit antibody (1:20000), incubated at room temperature, and eluted with PBS. The ECL solution was prepared in a dark room, and after the blot membrane was clamped into the ECL solution for sufficient reaction, scanning imaging was performed using a chemiluminescence imaging system and archived.

(3) Immunohistochemistry and confocal microscopic imaging

Mice were deeply anesthetized by intraperitoneal injection of chloral hydrate, then transcardially perfused with precooled PBS and 4% paraformaldehyde solution, and brains were fixed for 48 h at 4°C and cerebella were dissected with a blade. Cerebellum was embedded and cut into 8-μm slices using a freezing microtome and stored at 4°C for immunohistochemical experiments. Cerebellar slices were cultured in primary antibody, rabbit polyclonal anti-GLP-1 receptor antibody (1:1000), secondary antibody, Cy3-labeled goat anti-rabbit antibody (1:200), and 4', 6-diamidino-2-phenylindole (DAPI, 1:1000), respectively. Fluorescence images were obtained by confocal laser scanning microscopy. Large area images containing the desired area were obtained with a 10x objective on a Nikon C2 laser confocal system.

2. In vivo electrophysiological recordings

Adult ICR mice were anesthetized using intraperitoneal injection of urethane, and mice were fixed to a brain stereotaxic apparatus after endotracheal intubation. A small hole was drilled in the Vermis area of the cerebellum to expose the cerebellar surface. Oxygen-containing artificial cerebrospinal fluid (ACSF) was continuously perfused into the surface of the brain. Rectal temperature was monitored and maintained at 37 ± 0.2°C using a thermometer.

Purkinje cells (PCs) in the cerebellum were cell-attached recorded using an Axopatch-200B amplifier. PCs spontaneous firing activity signals were collected by Digidata 1440 digital-to-analog converter and Clampex 10.4 software. Recording electrodes were filled with ACSF and had resistances of 3 – 5 MΩ. Cell attachment recordings were performed under the leptomeninges and PCs were identified by regular spontaneous SS and irregular complex spike (CS).

3. In vitro electrophysiological recordings

Six to eight weeks old ICR mice were anesthetized by isoflurane inhalation and decapitated to obtain brain tissue, which was cooled in oxygenated ACSF at low temperature, and 300 μm sagittal cerebellar slices were prepared in the Vermis area of the cerebellar cortex using a vibratome. The prepared slices were placed in ACSF and incubated at room temperature for more than 1 hour to allow the neurons in the slices to recover. Purkinje cells in cerebellar slices were recorded by whole-cell patch-clamp.

Cells suitable for being recorded were identified using a 60-fold water immersion lens of a Nikon microscope combined with infrared CCD, and recording electrodes are thick walled glass electrodes with resistances of 4-6 MΩ. Membrane potential and/or membrane current were recorded using a patch clamp amplifier MultiClamp 700B, filtered at 5 kHz and recorded data were acquired on a computer using Digidata 1440A digital-to-analog converter via Clampex 10.4 software. Voltage clamp was used to control the cell voltage at − 70 mV during recording. Series resistance was monitored by applying voltage pulses and only cells that maintained stable series resistance values were included in the analysis. All drugs were administered by peristaltic pump. Hyperpolarization-activated current (IH) were activated by giving hyperpolarizing currents and membrane potentials. In voltage-clamp recording mode, voltage-gated sodium channel blockers tetrodotoxin (TTX) and BaCl₂ were added to the extracellular fluid to block voltage-gated sodium channels and Ba²⁺ -sensitive potassium channels, thus isolating IH currents.

4. Data Analysis

Electrophysiological data were analyzed with Clampfit 10.4 software. All parameters of single recorded neurons remained unchanged in ACSF perfusion, drug perfusion, and drug washout. Values are presented as mean ± standard error. One-way ANOVA was performed using SPSS 25.0 software to determine differences in statistical significance levels between data from each group. P-values below 0.05 were considered statistically significant differences between experimental groups.

[Results]

Part I. Mechanism of GLP-1 modulate simple spike activity of Purkinje cell in mouse cerebellar cortex in vivo

(1) RT-PCR, Western blot and immunofluorescence showed that messenger RNA, protein and antigen of GLP-1 receptor were expressed in cerebellum, suggesting that GLP-1 receptor was expressed in cerebellum. GLP-1 may modulate the activity of the cerebellar neural network by activating its receptor.

(2) Cerebellar surface perfusion of GLP-1 significantly enhanced the firing frequency of spontaneous SS in cerebellar Purkinje cells of mice, but had no significant effect on the coefficient of variation (CV) of SS firing, suggesting that GLP-1 regulates the spontaneous firing activity of Purkinje cells in cerebellar cortex through GLP-1 receptor.

(3) GLP-1 showed a concentration-dependent effect on SS firing rate enhancement in Purkinje cells, with the half effective concentration (EC₅₀) of 154.4 nM.

(4) Under the condition of blocking GABA_A receptor, cerebellar surface perfusion of GLP-1 did not affect the enhancement of SS firing frequency induced by GLP-1 in Purkinje cells, suggesting that γ-aminobutyric acid (GABA) -mediated inhibitory interneuron network was not involved in the enhancement of SS firing frequency induced by GLP-1 in Purkinje cells.

(5) When both GABA_A receptor and ionotropic glutamate receptor were blocked, the enhancement of SS firing frequency by GLP-1 in Purkinje cells was partially attenuated, but not completely blocked, indicating that blocking glutamate receptor could partially inhibit GLP-1 mediated enhancement of SS firing frequency in Purkinje cells.

(6) Antagonizing GLP-1 receptor completely abolished GLP-1 mediated enhancement of SS firing frequency in Purkinje cells, while activation of GLP-1 receptor enhanced SS firing frequency in Purkinje cells. GLP-1 did not further increase Purkinje cell SS firing frequency on the basis of activation of GLP-1 receptor, indicating that GLP-1 enhanced Purkinje cell SS firing frequency through activation of GLP-1 receptor.

Part II. Mechanism of GLP-1 modulate simple spike activity of Purkinje cell in mouse cerebellar cortex in vitro

(1) GLP-1 and its analogues can enhance the SS firing rate induced by depolarizing currents in Purkinje cells with membrane depolarization under the condition of blocking glutamatergic and GABAergic synaptic transmission, while antagonizing GLP-1 receptor completely eliminates the effect of GLP-1 on Purkinje cells, indicating that GLP-1 enhances the SS firing rate of Purkinje cells through postsynaptic receptor mechanisms.

(2) GLP-1 significantly inhibited the after-hyperpolarization potential (AHP) and outward rectified current (IR) of Purkinje cells, resulting in a decrease in the amplitude and area under curve of AHP and IR.

(3) In the presence of TTX and Ba²⁺, GLP-1 significantly enhanced the immediate instant current, steady current, tail current, and IH current induced by hyperpolarized stimulation, and these enhancement effects increased with the intensity of hyperpolarized stimulation, suggesting that GLP-1 may enhance Purkinje cell excitability by up-regulating IH channel activity, resulting in membrane depolarization and increasing Purkinje cell SS firing frequency.

(4) Under the condition of blocking IH channels, the enhancement of Purkinje cell membrane current induced by hyperpolarization stimulation by GLP-1 was completely abolished, indicating that GLP-1 enhances IH channel activity through its receptor and increases Purkinje cell excitability.

(5) In the presence of TTX and GABA_A receptor blockers, GLP-1 increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in Purkinje cells through its receptor, but had no significant effect on the amplitude of mEPSCs, suggesting that GLP-1 significantly enhanced excitatory glutamate release in Purkinje cells.

(6) Addition of GLP-1 in the presence of specific protein kinase A (PKA) inhibitors did not increase the frequency of mEPSCs, suggesting that GLP-1 increases glutamate release of Purkinje cells through PKA signaling pathway and significantly increases the frequency of mEPSCs.

[Conclusions]

(1) GLP-1 receptor is expressed in cerebellar cortex, and activated GLP-1 receptor up-regulates SS firing frequency in Purkinje cells of cerebellar cortex in mice.

(2) GLP-1, through its receptor, activates IH channels in Purkinje cell membranes, induces inhibition of outward rectifying potassium currents, and promotes membrane depolarization.

(3) GLP-1 acts on presynaptic GLP-1 receptor in Purkinje cells to upregulate the release of glutamate transmitters and to promote membrane depolarization through the PKA signaling pathway.

[Key words]

glucagon-like peptide-1 (GLP-1); mouse cerebellar cortex; Purkinje cell (PC); simple spike (SS); synaptic transmission; miniature excitatory postsynaptic currents (mEPSCs); hyperpolarization-activated current (IH)