Thecerebellummodulates thirst
小脑调节口渴

Received: 9 May 2023 收到：2023 年 5 月 9 日
Accepted: 7 June 2024 接受：2024 年 6 月 7 日
Published online: 10 July 2024
在线出版：2024 年 7 月 10 日
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Ila Mishra (1) $^{1, 2, 13}$ , Bing Feng (1) $^{3, 13}$ , Bijoya Basu(1) $^{4}$ , Amanda M. Brown $^{5, 6}$ , Linda H. Kim (1) $^{5, 6}$ , Tao Lin (1) $^{5, 6}$ , Mir Abbas Raza (D) $^{7}$ , Amelia Moore $^{7}$ , Abigayle Hahn $^{7}$ , Samantha Bailey $^{7}$ , Alaina Sharp $^{7}$ , Juan C. Bournat $^{8}$ , Claire Poulton $^{1}$ , Brian Kim $^{4}$ , Amos Langsner $^{1}$ , Aaron Sathyanesan (1) $^{79}$ , Roy V. Sillitoe (1) $^{5, 6, 10, 11}$ , Yanlin He $D^{3} ⊠$ Atul R. Chopra ${^{10})}^{1, 4, 12}$
Ila Mishra (1) $^{1, 2, 13}$ , Bing Feng (1) $^{3, 13}$ , Bijoya Basu(1) $^{4}$ , Amanda M. Brown $^{5, 6}$ , Linda H. Kim (1) $^{5, 6}$ , Tao Lin (1) $^{5, 6}$ , Mir Abbas Raza (D) $^{7}$ , Amelia Moore.Kim (1) $^{5, 6}$ , Tao Lin (1) $^{5, 6}$ , Mir Abbas Raza (D) $^{7}$ , Amelia Moore $^{7}$ , Abigayle Hahn $^{7}$ , Samantha Bailey $^{7}$ , Alaina Sharp $^{7}$ , Juan C．Bournat $^{8}$ , Claire Poulton $^{1}$ , Brian Kim $^{4}$ , Amos Langsner $^{1}$ , Aaron Sathyanesan (1) $^{79}$ , Roy V. Sillitoe (1) $^{5, 6, 10, 11}$ , Yanlin He $D^{3} ⊠$ Atul R. Chopra ${^{10})}^{1, 4, 12}$

The cerebellum, a phylogenetically ancient brain region, has long been considered strictly a motor control structure. Recent studies have implicated the cerebellum in cognition, sensation, emotion and autonomic function, making it an important target for further investigation. Here, we show that cerebellar Purkinje neurons in mice are activated by the hormone asprosin, leading to enhanced thirst, and that optogenetic or chemogenetic activation of Purkinje neurons induces rapid manifestation of water drinking. Purkinje neuron-specific asprosin receptor (Ptprd) deletion results in reduced water intake without affecting food intake and abolishes asprosin’s dipsogenic effect. Purkinje neuron-mediated motor learning and coordination were unaffected by these manipulations, indicating independent control of two divergent functions by Purkinje neurons. Our results show that the cerebellum is a thirst-modulating brain area and that asprosin-Ptprd signaling may be a potential therapeutic target for the management of thirst disorders.
小脑是一个系统发育古老的脑区，长期以来一直被认为是严格意义上的运动控制结构。最近的研究表明，小脑与认知、感觉、情绪和自主神经功能有关，因此成为进一步研究的重要目标。在这里，我们发现小鼠的小脑浦肯野神经元会被阿司匹林激素激活，导致渴感增强，并且浦肯野神经元的光遗传学或化学遗传学激活会诱导快速表现出饮水。Purkinje神经元特异性asprosin受体（Ptprd）缺失会导致水摄入量减少，但不影响食物摄入量，并且会取消asprosin的致渴性效应。Purkinje神经元介导的运动学习和协调不受这些操作的影响，表明Purkinje神经元对两种不同功能的独立控制。我们的研究结果表明，小脑是一个调节口渴的脑区，阿司匹林-Ptprd 信号转导可能是治疗口渴症的潜在治疗靶点。

Restoration of homeostasis is one of the most remarkable attributes of biological lifeforms. A magnificent illustration of this process is the maintenance of fluid homeostasis by the regulation of thirst, a vegetative function necessary for the maintenance of life. The canonical thirst regulatory circuitry resides in the hypothalamus and the cortex

^{1 - 3}

.Specifically, the circumventricular organ (CVO) of the lamina terminalis is the principal brain structure responsible for sensing and regulating internal water balance. It contains three main nuclei: the subfornical organ (SFO), the organum vasculosum lamina terminalis (OVLT) and the median preoptic nucleus, all of which are anatomically interconnected

^{1 - 3}

. In addition to these hypothalamic and cortical circuits, the insular cortex and the amygdala have also been implicated
恢复平衡是生物生命形式最显著的特性之一。通过调节口渴来维持体液平衡就是这一过程的最好例证。典型的口渴调节回路位于下丘脑和大脑皮层

^{1 - 3}

。具体来说，顶叶的环状器官（CVO）是负责感知和调节体内水分平衡的主要大脑结构。它包含三个主要核团：角下器官（SFO）、末端薄层血管器官（OVLT）和视前中核，所有这些核团在解剖学上都是相互关联的

^{1 - 3}

。除了这些下丘脑和皮层回路外，岛叶皮层和杏仁核也与此有关
in the regulation of thirst

^{4, 5}

. This complex circuitry, involving multiple regions of the brain, works harmoniously to regulate fluid and electrolyte balance and ensure adequate hydration.

^{4, 5}

调节口渴。这种复杂的电路涉及大脑的多个区域，它们协调工作，调节液体和电解质平衡，确保充足的水分。

Although the cerebellum has been extensively studied for its diverse roles in motor learning, coordination, cognition, appetite, emotion and autonomic control

^{6 - 9}

, little is known about its role in the regulation of fluid homeostasis. However, cerebellar metabolic activity has been correlated with water intake

^{10}

and, reciprocally, water deprivation has been correlated with cerebellar blood flow

^{5, 11}

, suggesting a possible role in thirst regulation. Asprosin, an adipose-derived hormone, is known to regulate food intake

^{12}

by engaging its receptor, protein tyrosine phosphatase receptor type delta (Ptprd), and the small
尽管小脑在运动学习、协调、认知、食欲、情绪和自主神经控制

^{6 - 9}

等方面的作用已被广泛研究，但人们对其在调节体液平衡方面的作用却知之甚少。然而，小脑代谢活动与水摄入量相关

^{10}

，反之，缺水与小脑血流量相关

^{5, 11}

，这表明小脑可能在口渴调节中发挥作用。众所周知，阿司匹林是一种源自脂肪的激素，它可以通过与受体--蛋白酪氨酸磷酸酶受体δ型（Ptprd）和小脑的小神经细胞--的相互作用来调节食物摄入量

^{12}

。

conductance calcium-activated potassium channel 3 to regulate the activity of hypothalamic AgRP neurons

^{13, 14}

. Here, our analysis has surprisingly revealed that cerebellar Purkinje neurons, known to regulate the coordination and learning of complex movements, are activated by a peripherally generated hormone, asprosin; that Purkinje neuron activity is necessary and sufficient for the generation of thirst; and that inhibition of asprosin signaling in Purkinje neurons, while causing hypodipsia, does not affect Purkinje neuron-mediated motor coordination and learning. Our findings highlight a powerful and clinically relevant neural circuit for the modulation of thirst.

^{13, 14}

下丘脑AgRP神经元的活动受电导钙激活钾通道3的调节。在这里，我们的分析令人惊讶地发现，小脑浦肯野神经元（已知能调节复杂动作的协调和学习）能被外周产生的激素阿司匹林激活；浦肯野神经元的活动是产生口渴的必要和充分条件；抑制浦肯野神经元中的阿司匹林信号传导虽然会导致嗜睡症，但不会影响浦肯野神经元介导的运动协调和学习。我们的发现凸显了一个强大的、与临床相关的调节口渴的神经回路。

Results 成果

Asprosin regulates water intake in a Ptprd-dependent manner Genetic deficiency of plasma asprosin (

F b n 1^{NPS / +}

) and genetic loss of the asprosin receptor Ptprd (Ptprd

^{- / -}

) have been previously reported to cause leanness, hypophagia and potent protection from diet-induced obesity

^{12, 14}

. We now report that these physiological attributes are coincident with hypodipsia, whereby daily water consumption is significantly lower, along with lower urine volume, elevated urine osmolality and unaltered plasma osmolality in adult

F b n 1^{NPS / +}

mice compared to their age-matched and sex-matched littermates (Fig.1a-g and Supplementary Fig. 1a,b). Similar to Fbn1

^{NPS / +}

mice,

{Ptprd}^{- / -}

mice present with hypodipsia, concentrated and hyperosmolar urine, with unaltered plasma osmolality (Fig. 1h-n and Supplementary Fig. 1c,d). Importantly, the changes noted in urine volume and osmolality and plasma osmolality were not confounded by variance in food intake, as all mice were fasted during the course of the experiment (Fig.1e-g,l-n). Reduced urine volume and elevated urine osmolality in fasted

{Fbnn}^{NPS / +}

and

{Ptprd}^{- / -}

mice are indicative of compensatory attempts to maintain plasma osmolality when confronted with reduced water intake.
天冬氨酸以依赖 Ptprd 的方式调节水摄入量先前有报道称，血浆天冬氨酸（

F b n 1^{NPS / +}

）的遗传性缺乏和天冬氨酸受体 Ptprd（Ptprd

^{- / -}

）的遗传性缺失会导致瘦弱、食欲减退和饮食诱发肥胖的有效保护

^{12, 14}

。我们现在报告说，这些生理特性与低嗜水症同时存在，与年龄匹配和性别匹配的同窝小鼠相比，成年

F b n 1^{NPS / +}

小鼠的日饮水量显著降低，尿量减少，尿渗透压升高，血浆渗透压不变（图 1a-g 和补充图 1a,b）。与 Fbn1

^{NPS / +}

小鼠相似，

{Ptprd}^{- / -}

小鼠也出现低嗜睡、浓缩尿和高渗尿，血浆渗透压没有变化（图 1h-n 和补充图 1c,d）。重要的是，尿量和渗透压以及血浆渗透压的变化与食物摄入量的变化无关，因为所有小鼠在实验过程中都禁食（图 1e-g、l-n）。禁食

{Fbnn}^{NPS / +}

和

{Ptprd}^{- / -}

小鼠的尿量减少和尿渗透压升高表明，在水摄入量减少的情况下，小鼠会补偿性地试图维持血浆渗透压。

Hypodipsia as a secondary consequence of compromised energy accretion and leanness cannot be ruled out in genetic loss-of-function models of the asprosin pathway. Thus, to rule out the hypodipsia displayed by Fbn1

^{NPS/+}

and Ptprd

^{- / -}

mice as a ramification of leanness, low appetite and energy expenditure, we resorted to pharmacologic asprosin loss-of-function in sex-matched, age-matched and weight-matched wild-type (WT) mice. We have previously shown that anti-asprosin monoclonal antibody (mAb) treatment in lean WT mice showed a subtle effect on blood glucose and no effect at all on 24 h cumulative food intake and body weight

^{15}

. This is in contrast to the robust reductions in plasma glucose, food intake and body weight seen in obese mice

^{15}

, suggesting that factors associated with obesity are necessary prerequisites to unlocking the glucose and food intake and body weight lowering effects of asprosin neutralization. Thus, anti-asprosin mAb treatment in lean WT mice is a strategy for acute asprosin loss-of-function without the confounding effects of altered food intake and body weight. Lean WT mice treated with the anti-asprosin mAb showed a significant reduction in 24 -h water intake with a concomitant reduction in urine output (Fig. 1o,p). Moreover, this effect was observed in mice without access to food, further confirming that the effect of asprosin on water intake is not a secondary consequence of its effects on food intake and body weight. In the opposite direction, asprosin-mediated water accretion was demonstrated in mice subjected to intranasal treatment with recombinant asprosin. Intranasal asprosin led to a twofold increase in 2-h water intake in fasted mice (Fig.1q). Furthermore, mice subjected to plasma asprosin elevation

^{13, 15, 16}

using adeno-associated viral vector serotype 8 -mediated overexpression and secretion of human asprosin protein (AAV8-asprosin) transduction also led to a significant increase in water intake, irrespective of whether the mice were ad libitum fed or fasted (Fig. 1r-t), again demonstrating asprosin-mediated increase in water intake, independent of its effects on food intake. To ascertain the relevance of Ptprd to the dipsogenic function of asprosin, we subjected WT and Ptprd

^{- 1 -}

mice to plasma asprosin elevation (with adenovirus serotype 5(Ad5)-asprosin for quicker overexpression than
在天冬氨酸通路的基因功能缺失模型中，不能排除因能量积累受损和瘦弱而继发的嗜睡症。因此，为了排除 Fbn1

^{NPS/+}

和 Ptprd

^{- / -}

小鼠显示的低嗜睡是瘦弱、低食欲和能量消耗的结果，我们在性别匹配、年龄匹配和体重匹配的野生型（WT）小鼠中采用了药理学asprosin功能缺失模型。我们先前已经证明，在瘦WT小鼠中进行抗天冬氨酸单克隆抗体（mAb）治疗对血糖有微弱影响，而对24小时累积食物摄入量和体重

^{15}

没有任何影响。这与肥胖小鼠血浆葡萄糖、食物摄入量和体重的显著降低形成鲜明对比

^{15}

，表明与肥胖相关的因素是释放天冬氨酸中和作用的葡萄糖、食物摄入量和体重降低效果的必要前提。因此，对瘦WT小鼠进行抗asprosin mAb治疗是一种急性asprosin功能丧失的策略，而不会受到食物摄入量和体重改变的干扰。用抗天冬氨酸 mAb 治疗的瘦 WT 小鼠 24 小时的水摄入量显著减少，同时尿量也减少（图 1o,p）。此外，在没有食物摄入的小鼠中也观察到了这种效应，这进一步证实了天冬氨酸对水摄入量的影响并不是其对食物摄入量和体重影响的次要结果。与此相反，小鼠经重组阿斯巴嗪鼻内处理后，阿斯巴嗪介导的水分蓄积也得到了证实。在禁食的小鼠中，鼻内注射天冬氨酸可使其 2 小时的水分摄入量增加两倍（图 1q）。此外，使用血清型8腺相关病毒载体介导的人asprosin蛋白（AAV8-asprosin）过表达和分泌（图1r-t），使小鼠血浆asprosin升高

^{13, 15, 16}

，无论小鼠是自由进食还是禁食，也会导致水摄入量显著增加，这再次表明asprosin介导的水摄入量增加与其对食物摄入量的影响无关。为了确定 Ptprd 与天冬氨肽的致猝灭功能的相关性，我们让 WT 和 Ptprd

^{- 1 -}

小鼠接受血浆天冬氨肽升高（使用腺病毒血清型 5（Ad5）-天冬氨肽比 Ptprd

^{- 1 -}

小鼠更快地过表达）。

AAV-asprosin

^{14, 16}

) and evidenced an increase in water intake in WT mice. By contrast, Ptprd

^{- 1 -}

mice were completely unresponsive to the effects of asprosin, suggesting that Ptprd is essential for asprosin-mediated water intake (Fig.1u,v). Additionally, plasma levels of asprosin in lean WT mice subjected to overnight water deprivation were found to be significantly higher than in age-matched and sex-matched littermate controls with ad libitum access to water, suggesting that thirst induces plasma asprosin levels. (Fig.1w).
AAV-asprosin

^{14, 16}

），WT小鼠的水摄入量有所增加。相比之下，Ptprd

^{- 1 -}

小鼠对天冬氨酸的作用完全没有反应，这表明 Ptprd 对天冬氨酸介导的水摄入量至关重要（图 1u、v）。此外，在一夜缺水的瘦 WT 小鼠中，发现其血浆中的天冬氨酸水平显著高于自由饮水的年龄和性别匹配的同窝对照组，这表明口渴会诱导血浆中的天冬氨酸水平（图 1w）。(图 1w）。

AgRP neurons do not mediate the dipsogenic effect of asprosin
AgRP神经元不介导天冬氨酸的致溺作用

We recently showed that asprosin engages Ptprd at hypothalamic AgRP neurons for appetite stimulation

^{14}

. We tested whether Ptprd expressed by AgRP neurons also regulates water intake, under normal chow and high-fat diet conditions (Extended Data Fig.1). For this process, we generated mice with constitutive AgRP neuron-specific knockout of Ptprd. Neither male nor female AgRP-cre; Ptprd

^{floxflox}

mice on normal chow presented with hypophagia or hypodipsia (Extended Data Fig. 1a-c). We have previously shown that female

A g R P

-cre; Ptprd

^{flox/flox}

subjected to a high-fat diet show reduced food intake and are protected from diet-induced obesity

^{14}

(Extended Data Fig. 1d,e). We tested the water intake of female

AgRP

-cre; Ptprd

^{fox/flox}

mice on a high-fat diet and did not observe a deficit (Extended Data Fig.1f,g), suggesting that asprosin regulates water intake through a neural center distinct from AgRP neurons.
我们最近研究发现，阿朴素能使下丘脑AgRP神经元的Ptprd参与刺激食欲

^{14}

。我们测试了在正常进食和高脂饮食条件下，AgRP神经元表达的Ptprd是否也能调节水的摄入量（扩展数据图1）。为此，我们产生了组成型 AgRP 神经元特异性敲除 Ptprd 的小鼠。食用正常饲料的雌雄 AgRP-cre; Ptprd

^{floxflox}

小鼠均未出现食欲减退或嗜睡（扩展数据图 1a-c）。我们之前已经证明，雌性

A g R P

-cre; Ptprd

^{flox/flox}

在高脂肪饮食中会减少食物摄入量，并防止饮食引起的肥胖

^{14}

（扩展数据图 1d,e）。我们测试了雌性

AgRP

-cre; Ptprd

^{fox/flox}

小鼠在高脂饮食中的水摄入量，没有观察到缺水现象（扩展数据图 1f,g），这表明asprosin是通过一个不同于AgRP神经元的神经中枢来调节水摄入量的。

Asprosin activates cerebellar Purkinje neurons
阿司匹林激活小脑浦肯野神经元

Ptprd is extensively expressed in numerous brain regions

^{17}

. For identification of asprosin-responsive brain regions involved in the regulation of water intake, we performed a brain-wide asprosin binding study using alkaline phosphatase (AP)-tagged asprosin. This study identified the cerebellum as a site of asprosin binding in both the human and mouse brain (Fig. 2a). The binding of AP-tagged asprosin to the cerebellum could be abolished upon pre-incubation with untagged asprosin but not untagged-GFP, demonstrating competitive binding as expected for hormone-receptor interactions (Fig. 2a). High expression of the asprosin receptor Ptprd has been previously documented in the mouse

^{17}

and human cerebellum (GTEx:ENSG00000153707.16). We confirmed this with quantitative PCR (qPCR) of visually enriched Purkinje and granule neurons, the largest and the most abundant neurons of the cerebellum, respectively (Fig. 2b). This was confirmed at the protein level by immunofluorescence, with Purkinje cell protein 2 (Pcp2) coexpression demonstrating Ptprd protein in Purkinje neurons (Fig. 2c,d).
Ptprd在许多脑区广泛表达

^{17}

。为了确定参与水摄入调节的asprosin反应脑区，我们使用碱性磷酸酶（AP）标记的asprosin进行了全脑asprosin结合研究。这项研究发现小脑是人脑和小鼠大脑中阿斯巴嗪的结合位点（图 2a）。AP标记的asprosin与小脑的结合在与未标记的asprosin预孵育后会被取消，而与未标记的GFP预孵育后不会被取消，这证明了激素-受体相互作用中预期的竞争性结合（图2a）。以前曾有文献报道小鼠

^{17}

和人类小脑中高表达阿斯巴嗪受体Ptprd（GTEx:ENSG00000153707.16）。我们用定量 PCR（qPCR）对小脑最大和最丰富的神经元--视觉富集的浦肯野神经元和颗粒神经元进行了检测，证实了这一点（图 2b）。免疫荧光在蛋白水平上也证实了这一点，普肯叶细胞蛋白 2（Pcp2）共表达显示普肯叶神经元中含有 Ptprd 蛋白（图 2c，d）。

Using slice electrophysiology, we tested granule and Purkinje neuron response to asprosin treatment. Interestingly, granule neurons, despite expressing Ptprd, were unresponsive to asprosin treatment (Extended Data Fig. 2a,b).Ptprd displays many splice variants that affect the extracellular ligand binding domain

^{18, 19}

, and it is possible that the predominant variant in granule neurons precludes asprosin responsiveness. By contrast, Purkinje neurons from tdTOMATO/Pcp2-cre mice responded to recombinant asprosin with increased firing frequency and resting membrane potential (Fig.2e,f). Given the complex distribution pattern of Purkinje neuron activity and projections

^{20 - 22}

, we tested Purkinje neuron responsiveness to asprosin by targeting lobes II, III, IV, V, VI, VII, VIII and IX using anteroposterior sampling (Fig. 2e,f) and by targeting lobes V and VI using mediolateral sampling (Extended Data Fig. 2c-e). Two distinct Purkinje neuron baseline firing frequencies were noted (Extended Data Fig. 2c, d). Irrespective of baseline firing frequency range, sampling direction (mediolateral versus anteroposterior) or cerebellar lobes targeted, all tested Purkinje neurons responded to recombinant asprosin with increased firing frequency and resting membrane potential (Fig. 2e,f and Extended Data Fig. 2d,e), suggesting that asprosin-mediated Purkinje neuron activation is a pan-cerebellar property. Furthermore, Purkinje neurons from both male and female mice could be activated by asprosin, suggesting that asprosin-mediated
我们利用切片电生理学测试了颗粒神经元和浦肯野神经元对阿司匹林处理的反应。有趣的是，尽管颗粒神经元表达了 Ptprd，但它们对阿司匹林处理没有反应（扩展数据图 2a,b）。Ptprd 有许多剪接变体，这些变体会影响细胞外配体结合域

^{18, 19}

，颗粒神经元中的主要变体可能排除了对阿司匹林的反应。相比之下，tdTOMATO/Pcp2-cre小鼠的Purkinje神经元对重组阿司匹林的反应是发射频率和静息膜电位增加（图2e,f）。鉴于浦肯野神经元活动和投射

^{20 - 22}

的复杂分布模式，我们通过使用前胸取样（图 2e,f）和使用内外侧取样（扩展数据图 2c-e）靶向叶 II、III、IV、V、VI、VII、VIII 和 IX 测试了浦肯野神经元对阿司匹林的反应性。我们注意到两种不同的 Purkinje 神经元基线发射频率（扩展数据图 2c、d）。无论基线发射频率范围、取样方向（内外侧与前胸）或所针对的小脑叶如何，所有测试的普肯列神经元都对重组阿司匹林做出了反应，发射频率和静息膜电位都有所提高（图 2e、f 和扩展数据图 2d、e），这表明阿司匹林介导的普肯列神经元激活是一种泛小脑特性。此外，雄性和雌性小鼠的浦肯野神经元都能被阿司匹林激活，这表明阿司匹林介导的浦肯野神经元激活具有泛小脑特性。

Fig.

1 ∣

Genetic and pharmacological asprosin inhibition is associated with hypodipsia. a-d, Cumulative water intake and average daily ( 24 h ) water intake of WT

{FbnI}^{+ / +}

and

F b n 1^{NPS / +}

male (a,b) and female (c,d) mice (

n = 7

WT and

n = 6

F b n 1^{NPS / +})

.e, Plasma osmolality of overnight fasted

F b n 1^{+ / +} (WT; n = 16)

and Fbn

1^{NPS / +} (n = 17)

female mice.f,g, Osmolality

(n = 14 WT

and

n = 16

Fbn

1^{NPS / +})

and volume (

n = 17

per group) of 24 h urine output of fasting WT and

F b n 1^{NPS / +}

female mice. h-k, Cumulative water intake and average daily ( 24 h ) water intake of

{Ptprd}^{+ / +} (WT)

and Ptprd

^{- / -}

male

(h, i)

and female

(j, k)

mice measured over 3 days (male,

n = 18 WT

and

n = 14 {Ptprd}^{- / -}

; female,

n = 14 WT

and

n = 10

Ptprd

^{- / -}

). 1, Plasma osmolality of overnight fasted control WT

(n = 8)

and Ptprd

^{- 1 -} (n = 9)

female mice.

m, n

, Osmolality (

n = 9

per group) and volume (

n = 8

per group) of 24-h urine output of fasting WT and Ptprd

^{- 1 -}

female mice. o,p, 24-h water intake and urine output measured after a single dose of anti-asprosin mAb (

250 μ g

per mouse) or IgG in fasting male C57BL/6 mice (

n = 8

IgG treated and

n = 7

mAb treated mice). q, 2-h water intake measured after intranasal treatment of
图：

1 ∣

遗传和药理asprosin抑制与偏食有关。a-d，WT

{FbnI}^{+ / +}

和

F b n 1^{NPS / +}

雄性（a,b）和雌性（c,d）小鼠（

n = 7

WT和

n = 6

F b n 1^{NPS / +})

.e, 隔夜禁食的

F b n 1^{+ / +} (WT; n = 16)

和Fbn

1^{NPS / +} (n = 17)

雌性小鼠的血浆渗透压。f,g,空腹 WT 和

F b n 1^{NPS / +}

雌性小鼠 24 小时尿量的渗透压

(n = 14 WT

和

n = 16

Fbn

1^{NPS / +})

及尿量（

n = 17

每组）。h-k，3 天内测量的

{Ptprd}^{+ / +} (WT)

和 Ptprd

^{- / -}

雄性

(h, i)

和雌性

(j, k)

小鼠（雄性，

n = 18 WT

和

n = 14 {Ptprd}^{- / -}

；雌性，

n = 14 WT

和

n = 10

Ptprd

^{- / -}

）的累积水摄入量和平均日（24 h）水摄入量。1, 过夜禁食对照 WT

(n = 8)

和 Ptprd

^{- 1 -} (n = 9)

雌性小鼠的血浆渗透压。

m, n

、空腹 WT 和 Ptprd

^{- 1 -}

雌性小鼠 24 小时尿量的渗透压（

n = 9

每组）和尿量（

n = 8

每组）。o,p,空腹雄性 C57BL/6 小鼠（

n = 8

IgG 处理小鼠和

n = 7

mAb 处理小鼠）单剂量服用抗天冬氨酸 mAb（

250 μ g

每只小鼠）或 IgG 后测定的 24 小时水摄入量和尿量。
recombinant asprosin (rAsprosin) (

2 μ g

15 μ l

saline,

n = 6

) or vehicle (

15 μ l

saline,

n = 5

) in male C57BL/6 mice.

r

-t, Daily food and water intake under ad libitum food and water access (

r, s)

and water intake under fasting conditions (t), measured on days 66-69 after male C57BL/6J male mice were tail-veininjected with AAV8-empty or AAV8-asprosin (

n = 8

of AAV8-empty and

n = 10

of AAV8-asprosin in ® and

n = 9

of AAV8-empty and

n = 11

of AAV8-asprosin) viral vector. u-v, Cumulative water intake of normal chow-fed WT and Ptprd

^{- / -}

male mice tail-vein transduced with Ad5-empty or Ad5-Asprosin viruses, days 9-12 post adenoviral vector transduction (

n = 6

per group except

n = 5

in Ptprd

^{- 1 -}

mice + Ad5-empty in (

v

w

, Plasma asprosin detection with sandwich ELISA in lean male mice subjected to overnight ( 16 h ) water deprivation (

n = 15

per group). Error bars, s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

and

^{* * * *} P < 0.0001

; by Student’s

t

-test (b, d-g, i,

k - t

and

v, w)

, ‘effect of genotype’ determined by two-way ANOVA

(a, c, h, j

and

u)

. Also see Supplementary Fig. 1 for individual data plot and Source Data for raw values and details of statistical analysis.
在雄性 C57BL/6 小鼠中使用重组天蚕素（rAsprosin）（

2 μ g

在

15 μ l

生理盐水中，

n = 6

）或载体（

15 μ l

生理盐水，

n = 5

）。

r

-t，自由进食和饮水条件下的每日食物和水摄入量（

r, s)

，空腹条件下的水摄入量（t）、C57BL/6J雄性小鼠尾静脉注射AAV8-empty或AAV8-asprosin病毒载体后第66-69天的测量结果（AAV8-empty的

n = 8

和AAV8-asprosin的

n = 10

在®中和AAV8-empty的

n = 9

和AAV8-asprosin的

n = 11

）。u-v，正常饲料喂养的 WT 和 Ptprd

^{- / -}

雄性小鼠尾静脉转导 Ad5-empty 或 Ad5-Asprosin 病毒后第 9-12 天的累计水摄入量（除 Ptprd

^{- 1 -}

小鼠中的

n = 5

和 (

v

中的 Ad5-empty 外，每组均为

n = 6

）。

w

，用夹心酶联免疫吸附法检测一夜（16 h）缺水的瘦雄性小鼠（每组

n = 15

）的血浆天冬氨酸。b, d-g, i,

k - t

和

v, w)

, "基因型的影响 "由双向方差分析确定

(a, c, h, j

和

u)

。单个数据图见补充图 1，原始数据和统计分析细节见原始数据。

Purkinje neuron activation is not a sexually dimorphic phenomenon (Fig. 2f). Importantly, Purkinje neurons were activated by asprosin even when all synaptic input was blocked by treatment with bicuculline
Purkinje神经元的激活并不是一种性双态现象（图 2f）。重要的是，即使在使用双谷氨酸阻断所有突触输入的情况下，Purkinje 神经元也能被阿司匹林激活。
(antagonist of the GABA receptor), tetrodotoxin (sodium channel blocker), (2R)-amino-5-phosphonovaleric acid (NMDA receptor antagonist) and cyanquixaline (a competitive AMPA/kainate receptor
(GABA 受体拮抗剂）、河豚毒素（钠通道阻滞剂）、(2R)-氨基-5-膦酰戊酸（NMDA 受体拮抗剂）和氰喹喔啉（一种竞争性 AMPA/kainate 受体）。

antagonist), suggesting that asprosin-mediated Purkinje neuron activation is probably a cell-autonomous effect (Fig. 2g,h).
拮抗剂），这表明阿司匹林介导的 Purkinje 神经元激活可能是一种细胞自主效应（图 2g,h）。

Purkinje neuron activity modulation alters water intake
浦肯野神经元活动调节改变了水的摄入量

Having established Purkinje neuron activation by asprosin, we sought to manipulate Purkinje neurons in a manner that is selective, rapid
在确定了阿司匹林对 Purkinje 神经元的激活作用后，我们试图以一种选择性、快速、有效的方式操纵 Purkinje 神经元。
and reversible, and would provide insight, both qualitative and quantitative, into the function of these neurons. To achieve this goal, we used two state-of-the-art methods: chemogenetic stimulation and inhibition (using designer receptors exclusively activated by designer drugs; DREADD)

^{23}

and optogenetic

^{24}

(using channelrhodopsin (Chr2)) stimulation of Purkinje neurons. For chemogenetic manipulation, we
而且是可逆的，并能从定性和定量两方面深入了解这些神经元的功能。为了实现这一目标，我们采用了两种最先进的方法：化学刺激和抑制（使用专门由设计药物激活的设计受体；DREADD）

^{23}

以及光遗传

^{24}

（使用通道型发光素（Chr2））刺激普肯耶神经元。对于化学遗传操作，我们

Fig. 2 |Asprosin activates cerebellar Purkinje neurons. a, Representative brain sections from mouse (left) and human (right) brains pre-incubated with GFP (top) or free asprosin (bottom) subjected to competitive binding assay with AP-tagged asprosin. Scale bars,

10 μ m . b, qPCR

results showing relative mRNA levels of Ptprd from visually enriched tdTOMATO expressing Purkinje neurons (tdTOMATO-Pcp2-cre), granule neurons and liver from male mice (

n = 10

neurons per biological replicate;

n = 6

biological replicates per Purkinje group,

n = 4

biological replicates per granule neuron;

n = 4

biological liver replicates). Error bars, s.e.m.c-d, Representative image of immunostaining of Pcp2 (red) and Ptprd (green) of cerebellum of adult male mouse using fluorescence microscopy. e, Representative image of recorded Pcp2 neurons under brightfield and fluorescence microscopy and representative action potential firing traces of Pcp2

^{+}

neurons after puff treatment of control GFP or recombinant asprosin. f, Data analysis of action potential firing frequency and membrane potential
图 2 |天冬氨酸激活小脑浦肯野神经元。a, 小鼠（左）和人（右）大脑的代表性切片，与 GFP（上）或游离天冬氨酸（下）预孵育，并与 AP 标记的天冬氨酸进行竞争性结合试验。标尺条，

10 μ m . b, qPCR

结果显示雄性小鼠视觉富集的表达tdTOMATO的Purkinje神经元（tdTOMATO-Pcp2-cre）、颗粒神经元和肝脏中Ptprd的相对mRNA水平（

n = 10

每个生物重复的神经元；

n = 6

每个Purkinje组的生物重复；

n = 4

每个颗粒神经元的生物重复；

n = 4

肝脏的生物重复）。c-d，用荧光显微镜观察成年雄性小鼠小脑 Pcp2（红色）和 Ptprd（绿色）免疫染色的代表图像。e，在明视野和荧光显微镜下记录的 Pcp2 神经元的代表图像，以及 Pcp2

^{+}

神经元在粉扑处理对照 GFP 或重组阿朴素后的动作电位发射轨迹。
in Pcp2

^{+}

neurons post GFP or 30 nM recombinant mammalian asprosin puff treatment from male (top) and female (bottom) mice (

n = 9 - 16

in each region from five different mice and sex:

n = 16

resting membrane potential and

n = 7

firing frequency of males and females treated with asprosin,

n = 9

for resting membrane potential and

n = 5

firing frequency of males and females treated with GFP). g, Representative resting membrane potential trace of Pcp2

^{+}

neurons in response to 30 nM recombinant mammalian asprosin or GFP in the presence of a cocktail of synaptic blockers including

1 μ M

tetrodotoxin (TTX),

30 μ M

AP - 5, 30 μ M

CNQX and

50 μ M

bicuculline. h , Data analysis of resting membrane potential in

{Pcp}^{+}^{+}

neurons post GFP or 30 nM recombinant mammalian asprosin puff treatment in the presence of cocktail synaptic blockers including

1 μ M

TTX,

30 μ MAP - 5, 30 μ MCNQX

and

50 μ M

bicuculline (

n = 14

in each group from three different male mice).

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

and

^{* * * *} P < 0.0001

by Student’s

t

-test (f and

h

). Details of statistical analysis are in Source Data.
雄性小鼠（上）和雌性小鼠（下）的 Pcp2

^{+}

神经元在经过 GFP 或 30 nM 重组哺乳动物阿司匹林粉扑处理后（每个区域的

n = 9 - 16

均来自五只不同性别的小鼠：

n = 16

静息膜电位和

n = 7

用阿司匹林处理的雄性和雌性小鼠的发射频率，

n = 9

静息膜电位和

n = 5

用 GFP 处理的雄性和雌性小鼠的发射频率）。g, Pcp2

^{+}

神经元对 30 nM 重组哺乳动物阿司匹林或 GFP 的反应的代表性静息膜电位轨迹，突触阻断剂包括

1 μ M

河豚毒素 (TTX)、

30 μ M

AP - 5, 30 μ M

CNQX 和

50 μ M

bicuculline。h , 在鸡尾酒突触阻断剂（包括

1 μ M

TTX、

30 μ MAP - 5, 30 μ MCNQX

和

50 μ M

bicuculline）存在的情况下，

{Pcp}^{+}^{+}

神经元在 GFP 或 30 nM 重组哺乳动物天冬氨素粉扑处理后的静息膜电位数据分析（每组中的

n = 14

来自三只不同的雄性小鼠）。

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

和

^{* * * *} P < 0.0001

经学生

t

检验（f和

h

）。统计分析详情见《原始数据》。

As a second strategy for testing Purkinje neuron-mediated regulation of water intake, we resorted to optogenetics with Cre-recombinase-dependent AAV expressing Chr2 used to target Purkinje neurons of Pcp2-cre mice. Control Pcp2-cre mice were treated with a Cre-recombinase-dependent AAV expressing GFP (Fig. 3k,I). Photostimulation of Chr2 expressing Purkinje neurons (Chr2

^{Pcp 2}

) with blue light led to a marked increase in Purkinje neuron firing frequency and resting membrane potential, concomitant with an increase in water intake (Fig.

3 m - q

and Supplementary Videos 1 and 2). No increase in food intake was noted (Fig. 3q). Importantly, photostimulation did not affect the water intake of Pcp2-cre mice not expressing Chr2 (Fig. 30), suggesting specificity in the optogenetic methodology.
作为测试普肯列神经元介导的水摄入调控的第二种策略，我们利用表达 Chr2 的 Cre 重配酶依赖性 AAV 靶向 Pcp2-cre 小鼠的普肯列神经元进行光遗传学研究。对照组 Pcp2-cre 小鼠用表达 GFP 的 Cre 重配酶依赖性 AAV 处理（图 3k，I）。用蓝光刺激表达 Chr2 的浦肯野神经元（Chr2

^{Pcp 2}

）会导致浦肯野神经元的发射频率和静息膜电位显著增加，同时水摄入量也会增加（图

3 m - q

和补充视频 1 和 2）。食物摄入量没有增加（图 3q）。重要的是，光刺激不会影响不表达 Chr2 的 Pcp2-cre 小鼠的摄水量（图 30），这表明光遗传学方法具有特异性。

Purkinje neuron-specific Ptprd deletion results in hypodipsia
浦肯野神经元特异性 Ptprd 缺失会导致嗜睡症

To elucidate the relevance of Purkinje neuron-specific Ptprd in the regulation of water intake, mice with genetic loss of Ptprd from Purkinje neurons (Pcp2-cre; Ptprd

^{flox/flox}

) were generated by crossing Ptprd

d^{floxflox}

mice with mice expressing Cre recombinase under the control of the Purkinje cell protein 2 (Pcp2) promoter. To test whether this strategy led to the successful deletion of Ptprd from Purkinje neurons, Pcp2-cre; Ptprd

^{flox f^{' l o x}}

and Pcp2-cre; Ptprd

^{+ + ∣}

mice were mated with Cre-responsive

t d T O M A T O

mice. Double-fluorescence labeling of tdTOMATO and Ptprd in Pcp2-cre/tdTomato

^{+}

Ptprd

^{flox^{flox}}

mice compared to control Pcp2-cre/tdTomato

^{+}

Ptprd

^{+ + +}

littermates confirmed successful knockdown of Ptprd from Pcp2

^{+}

neurons (Supplementary Fig. 3a,b). We tested whether Ptprd loss affects the morphology of Purkinje neurons by assessing five parameters of Purkinje neuron morphology: cell number, cell body diameter, fiber length, synapse and dendritic density. We found no impact of Ptprd loss on the morphology of Purkinje neurons (Supplementary Fig.3c,d). We then measured Ptprd protein expression across the cerebellum using immunofluorescence in control and Purkinje neuron-specific Ptprd knockout (Pcp2-cre; Ptprd

^{flox / flox)}

mice (Supplementary Fig. 4). In concordance with previous work

^{17}

, we noted Ptprd protein expression in the granule layer, Purkinje layer and the molecular layer of the control mice.
为了阐明Purkinje神经元特异性Ptprd在水摄入调节中的相关性，通过将Ptprd

d^{floxflox}

小鼠与在Purkinje细胞蛋白2（Pcp2）启动子控制下表达Cre重组酶的小鼠杂交，产生了从Purkinje神经元中遗传性缺失Ptprd的小鼠（Pcp2-cre; Ptprd

^{flox/flox}

）。为了检验这种策略是否能成功地从普肯列神经元中删除 Ptprd，Pcp2-cre; Ptprd

^{flox f^{' l o x}}

和 Pcp2-cre; Ptprd

^{+ + ∣}

小鼠与 Cre 反应型

t d T O M A T O

小鼠交配。与对照组 Pcp2-cre/tdTomato

^{+}

Ptprd

^{+ + +}

小鼠相比，Pcp2-cre/tdTomato

^{+}

Ptprd

^{flox^{flox}}

小鼠tdTOMATO 和 Ptprd 的双重荧光标记证实了 Ptprd 从 Pcp2

^{+}

神经元中成功敲除（补充图 3a,b）。我们通过评估 Purkinje 神经元形态的五个参数：细胞数、细胞体直径、纤维长度、突触和树突密度，检测了 Ptprd 缺失是否会影响 Purkinje 神经元的形态。我们发现 Ptprd 缺失对 Purkinje 神经元的形态没有影响（补充图 3c,d）。然后，我们在对照组和Purkinje神经元特异性Ptprd基因敲除（Pcp2-cre；Ptprd

^{flox / flox)}

）小鼠中使用免疫荧光测量了整个小脑中Ptprd蛋白的表达（补充图4）。与之前的研究

^{17}

一致，我们注意到对照组小鼠的颗粒层、浦肯野层和分子层都有 Ptprd 蛋白表达。
post i.p. injection of CNO or saline in Pcp2-cre mice stereotaxically injected with Cre-dependent AAV expressing hSyn-DIO-hM4Di-mCherry in lobes V-VI of the cerebellum (

n = 4

per group).

k

, Schematic experimental strategy using the Cre-dependent AAV expressing ChR2-EYFP to activate Purkinje neurons of the Pcp2-cre mice. I. Representative image of recorded Pcp2-cre neurons expressing ChR2-EYFP under brightfield and fluorescence microscopy. m,n, Data analysis of action potential firing frequency and membrane potential in Pcp2-ChR2EYFP+ neurons post blue or yellow light stimulation (

n = 8

from two different male mice in

m

and

n = 10

from two different male mice in

n

o - q, 1 h

water drinking and feeding behavior in Pcp2-GFP or Pcp2-ChR2-EYFP mice post yellow light (

598 nm, 5 Hz, 3 s

on and 3 s off) or blue light (

473 nm, 5 Hz, 3 s

on and 3 s off) stimulation (

n = 5

per group in

o

and

n = 6

per group in

p

and

q

). Also see Supplementary Videos 1 and 2. Error bars, s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

and

^{* * * *} P < 0.0001

; by Student’s

t

-test (

c - g, i, j

and

m - q)

. Also see Extended Data Fig. 3 and Source Data for raw values and details of analysis.

n = 4

，在小脑第V-VI叶立体定向注射表达hSyn-DIO-hM4Di-mCherry的Cre依赖性AAV的Pcp2-cre小鼠（

n = 4

每组），静注CNO或生理盐水后。

k

，使用表达 ChR2-EYFP 的 Cre 依赖性 AAV 激活 Pcp2-cre 小鼠 Purkinje 神经元的实验示意图。I.在明视野和荧光显微镜下记录的表达 ChR2-EYFP 的 Pcp2-cre 神经元的代表性图像。m,n, Pcp2-ChR2EYFP+ 神经元在蓝光或黄光刺激后的动作电位发射频率和膜电位的数据分析（

m

中的

n = 8

来自两只不同的雄性小鼠，

n

中的

n = 10

来自两只不同的雄性小鼠）。

o - q, 1 h

Pcp2-GFP 或 Pcp2-ChR2-EYFP 小鼠在黄光（

598 nm, 5 Hz, 3 s

开和 3 秒关）或蓝光（

473 nm, 5 Hz, 3 s

开和 3 秒关）刺激后的饮水和摄食行为（

o

中每组的

n = 5

和

p

和

q

中每组的

n = 6

）。另见补充视频 1 和 2。误差条，s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

和

^{* * * *} P < 0.0001

；经学生

t

检验（

c - g, i, j

和

m - q)

。原始数据和分析详情另见扩展数据图 3 和原始数据。

Our analysis suggests that Ptprd protein is expressed in Purkinje neuron cell bodies located in the Purkinje layer and dendritic trees traversing the molecular layer. The molecular layer signal appears to recede upon Purkinje neuron-specific Ptprd deletion, suggesting that molecular layer interneurons express little Ptprd. However, this finding needs to be corroborated with an independent study using genetically altered mice expressing molecular layer interneuron fluorescence
我们的分析表明，Ptprd 蛋白表达于位于浦肯野层的浦肯野神经元细胞体和穿过分子层的树突树。Purkinje神经元特异性Ptprd缺失后，分子层信号似乎减弱，这表明分子层中间神经元几乎不表达Ptprd。然而，这一发现还需要使用表达分子层中间神经元荧光的基因改变小鼠进行独立研究来证实。
markers. By contrast, the granule layer signal persists upon Purkinje neuron-specific Ptprd deletion, suggesting independent Ptprd expression in granule neurons (Supplementary Figs. 3 and 4).
标记。相比之下，Purkinje 神经元特异性 Ptprd 缺失后，颗粒层信号仍然存在，这表明颗粒神经元中 Ptprd 的表达是独立的（补充图 3 和 4）。

A thorough phenotypic analysis in the Promethion metabolic caging system revealed that 8-week-old Pcp2-cre; Ptprd

^{flox/flox}

male mice were hypodipsic, with nearly a

1 - 1.5 ml

deficit in daily water intake (Fig. 4b and Supplementary Fig. 5a) compared to the control
在 Promethion 代谢笼养系统中进行的全面表型分析表明，与对照组相比，8 周大的 Pcp2-cre; Ptprd

^{flox/flox}

雄性小鼠嗜水性低，每日摄水量几乎为

1 - 1.5 ml

(图 4b 和补充图 5a)。
a AAV-hSyn-DIO-hM3Dq-mCherry

h AAV-hSyn-DIO-hM4Di-mCherry

k AAV-hSyn-ChR2-EYFP

q AAV-hSyn-ChR2-EYFP

Fig.

4 ∣

Purkinje neuron-specific Ptprd deletion leads to hypodipsia.
图：

4 ∣

Purkinje 神经元特异性 Ptprd 缺失导致嗜睡症。
a, Body weight of 8-week-old control mice (Pcp2-cre; Ptprd

^{+ / +}

and Ptprd

^{flox/flox}

) and mice with Purkinje neuron-specific knockout of Ptprd (Pcp2-cre;Ptprd

^{flox/flox}

) maintained on ad libitum normal chow diet (Pcp2-cre;Ptprd

^{+ / +} n = 14

, Ptprd

^{flox flox}

n = 12

and Pcp2-cre; Ptprd

^{flox/flox} n = 18

). b, Cumulative water intake of 8 -week-old Pcp2-cre;Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{flox/flox}

(KO) male mice on normal chow over 3 days using the Promethion metabolic system (Pcp2-cre; Ptprd

^{+ + ∣} n = 9

, Ptprd

^{flox/flox} n = 12

and Pcp2-cre; Ptprd

^{fox/flox} n = 18

). c, 24-h water intake of 12-weekold Pcp2-cre; Ptprd

^{+ +}

, Ptprd

d^{flox/flox}

and Pcp2-cre;Ptprd

^{flox/flox}

male mice under conditions of ad libitum access to food and water, 24 h fasting, refeeding post 24 h fast and re-access to water after overnight water withholding (Pcp2-cre; Ptprd

^{+ / +} n = 5

per experimental paradigm, Ptprd

^{flox/flox} n = 8

per experimental paradigm and

Pcp 2 -cre; Ptprd^{flox/flox} n = 10

per experimental paradigm).
a, 8 周龄对照组小鼠（Pcp2-cre; Ptprd

^{+ / +}

和 Ptprd

^{flox/flox}

）和Purkinje神经元特异性敲除Ptprd的小鼠（Pcp2-cre；Ptprd

^{flox/flox}

）的小鼠（Pcp2-cre;Ptprd

^{+ / +} n = 14

, Ptprd

^{flox flox}

n = 12

和 Pcp2-cre;Ptprd

^{flox/flox} n = 18

）。b、使用 Promethion 代谢系统（Pcp2-cre; Ptprd

^{+ + ∣} n = 9

, Ptprd

^{flox/flox} n = 12

和 Pcp2-cre; Ptprd

^{fox/flox} n = 18

），8 周龄 Pcp2-cre;Ptprd

^{+ / +}

、Ptprd

^{flox/flox}

和 Pcp2-cre;Ptprd

^{flox/flox}

（KO）雄性小鼠 3 天内正常饲料的累积饮水量。c, 12 周龄的 Pcp2-cre; Ptprd

^{+ +}

, Ptprd

d^{flox/flox}

和 Pcp2-cre;Ptprd

^{flox/flox}

雄性小鼠在自由取食和饮水、24 小时禁食、24 小时禁食后再进食和过夜不给水后再进水条件下的 24 小时饮水量（Pcp2-cre；Ptprd

^{+ / +} n = 5

每个实验范式、Ptprd

^{flox/flox} n = 8

每个实验范式和

Pcp 2 -cre; Ptprd^{flox/flox} n = 10

每个实验范式）。
d-f, Cumulative food intake, hourly energy expenditure and respiratory exchange ratio of 8-week-old male Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox / flox}

and Pcp2-cre; Ptprd

^{flox/flox}

mice on normal chow over 3 days using the Promethion metabolic system (Pcp2-cre;Ptprd

^{+ / +} n = 10

, Ptprd

^{flox/flox} n = 11

and Pcp2-cre; Ptprd

^{flox/flox} n = 18

). g,h, Osmolality and volume of 24 h urine output of 12-week-old Pcp2-cre;Ptprd

^{+ / +}

and Pcp2-cre; Ptprd

^{floxflox}

female mice under fasting conditions with ad libitum access to water (

n = 9

per group). i, Plasma osmolality of 12-week-old Pcp2cre; Ptprd

^{+ / +}

and Pcp2-cre; Ptprd

^{floxfflox}

female mice after an overnight fast (

n = 9

per group, except

n = 8

Pcp2-cre; Ptprd

^{flox/flox})

.Error bars, s.e.m.

^{*} P < 0.05,^{* *} P < 0.01

^{* * *} P < 0.001

and

^{* * *} P < 0.0001

; by Student’s

t

-test (g,

h

and

i

), by one-way ANOVA followed by Dunnett’s multiple comparisons test (

a, c)

and ‘effect of genotype’ determined by two-way ANOVA (b, d-f). Also see Supplementary Fig. 5 for raw data plot and Source Data for raw data values and details of the analysis.
d-f, 8 周龄雄性 Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox / flox}

和 Pcp2-cre；Ptprd

^{flox/flox}

小鼠使用 Promethion 代谢系统（Pcp2-cre;Ptprd

^{+ / +} n = 10

, Ptprd

^{flox/flox} n = 11

和 Pcp2-cre;Ptprd

^{flox/flox} n = 18

）进行为期 3 天的正常进食。g,h, 12 周龄 Pcp2-cre; Ptprd

^{+ / +}

和 Pcp2-cre; Ptprd

^{floxflox}

雌性小鼠在禁食和自由饮水条件下 24 小时尿液的渗透压和排尿量（每组

n = 9

）。i, 12 周龄 Pcp2cre; Ptprd

^{+ / +}

和 Pcp2-cre; Ptprd

^{floxfflox}

雌性小鼠在一夜禁食后的血浆渗透压（

n = 9

每组，

n = 8

Pcp2-cre; Ptprd

^{flox/flox})

除外）。

^{*} P < 0.05,^{* *} P < 0.01

、

^{* * *} P < 0.001

和

^{* * *} P < 0.0001

；通过学生

t

检验（g、

h

和

i

），通过单因素方差分析，然后通过邓尼特多重比较检验（

a, c)

，通过双因素方差分析确定 "基因型的影响"（b、d-f）。原始数据图见补充图 5，原始数据值和分析详情见原始数据。

Ptprd

^{floxfllox}

and Pcp2-cre; Ptprd

^{+ / +}

littermates. This deficit in water intake was evident irrespective of whether the mice were fasted, refed post fasting or given re-access to water after water deprivation (Fig. 4c and Supplementary Fig. 5b). Similar to male Pcp2-cre; Ptprd

^{flox/flox}

mice, 8-week-old female mice with Purkinje neuron-specific Ptprd deletion also showed a deficit in daily water intake compared to the age-matched and sex-matched control littermates (Extended Data Fig. 4a,b). Importantly, the hypodipsia caused by Purkinje neuron-specific Ptprd deletion was not confounded by metabolic
Ptprd

^{floxfllox}

和 Pcp2-cre; Ptprd

^{+ / +}

幼鼠。无论小鼠是禁食、禁食后再进食还是禁水后再进食，这种水摄入不足都很明显（图 4c 和补充图 5b）。与雄性 Pcp2-cre；Ptprd

^{flox/flox}

小鼠类似，与年龄和性别匹配的对照同窝鼠相比，Purkinje 神经元特异性 Ptprd 缺失的 8 周大雌性小鼠也表现出每日摄水量不足（扩展数据图 4a,b）。重要的是，Purkinje 神经元特异性 Ptprd 缺失导致的低嗜水症并没有受到新陈代谢的影响。
deficits in either of the sexes (Extended Data Fig. 4c-f, Supplementary Figs. 5c-e and Fig. 4d-f). There was no difference in body weight between control and knockout mice of either sex on normal chow (Fig. 4a and Extended Data Fig. 4c) or on a high-fat diet (Extended Data Fig. 5a,b). Daily food intake, energy expenditure and respiratory exchange quotient were unaffected by Purkinje neuron-specific Ptprd deletion. As seen in mice with plasma asprosin deficiency (

F b n 1^{NPS / +}

) and global Ptprd knockout (Ptprd

^{- - 1}

), Purkinje neuron-specific Ptprd deletion resulted in lower urine volume, higher urine osmolality and
(扩展数据图 4c-f，补充图 5c-e 和图 4d-f)。正常饲料（图 4a 和扩展数据图 4c）或高脂肪饮食（扩展数据图 5a,b）下，对照组小鼠和基因敲除小鼠的体重没有差异。每日食物摄入量、能量消耗和呼吸交换商不受普肯列神经元特异性 Ptprd 缺失的影响。与血浆asprosin缺乏症小鼠（

F b n 1^{NPS / +}

）和整体Ptprd基因敲除小鼠（Ptprd

^{- - 1}

）的情况一样，Purkinje神经元特异性Ptprd缺失导致尿量减少、尿渗透压升高和尿液浓缩。
maintenance of plasma osmolality (Fig. 4g-i), suggesting compensatory renal activity to counter reduced water intake.
维持血浆渗透压（图 4g-i），这表明肾脏有代偿活动来抵消水摄入量的减少。

It is of note that there was no difference in lick frequency upon being given water access after overnight water deprivation, suggesting an absence of a deficit in rhythmic oromotor movements

^{25}

(Extended Data Fig.

4 g, h

). Interestingly, the deficit in drinking behavior in mice with Purkinje neuron-specific Ptprd deletion was not restricted to water intake. Compared to control mice, mice with Purkinje neuron-specific Ptprd deletion also consumed less isotonic and hypertonic saline over 48 h (Extended Data Fig. 4i,j).
值得注意的是，在一夜断水后给小鼠喂水时，舔食频率没有差异，这表明小鼠的节律性口器运动

^{25}

没有缺陷（扩展数据图

4 g, h

）。有趣的是，Purkinje神经元特异性Ptprd缺失小鼠的饮水行为缺陷并不局限于水的摄入。与对照组小鼠相比，Purkinje 神经元特异性 Ptprd 缺失的小鼠在 48 小时内消耗的等渗和高渗盐水也较少（扩展数据图 4i,j）。

Purkinje-specific deletion of Ptprd does not result in motor deficits
Purkinje特异性缺失Ptprd不会导致运动障碍

Purkinje neurons are well known to have pivotal roles in the coordination and control of complex movements

^{26, 27}

. In particular, swallowing is a complex activity requiring a sophisticated system of neurological control from neurons within the brainstem, cerebral cortex and the cerebellum

^{28}

.Similar food intake and lick frequency (Fig. 4 d and Extended Data Fig.

4 g, h

) between the controls (Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{+ / +}

) and the Pcp2-cre; Ptprd

^{flox/flox}

mice ruled out dysphagia, overt oromotor deficits or an inability to access food or water as a potential explanation for the observed hypodipsia. Nonetheless, we subjected mice to an array of motor coordination and learning assays to rule out potential deficits as a confounder of the observed hypodipsia upon Purkinje neuron-specific Ptprd deletion.Pcp2-cre; Ptprd

^{flox^{- 10 x}}

mice did not show a deficit in pedestrian activity or wheel-running activity recorded over 4 days (Fig. 5a,b and Supplementary Fig. 6a,b). We subjected mice to constant speed rotarod for 3,5 and 7 min , wherein all groups successfully completed the task, irrespective of the genotype (Fig. 5c and Supplementary Fig. 6c). Furthermore, Pcp2-cre; Ptprd

^{fox^{'} flox}

mice did not show any neuromuscular abnormalities, with similar strength to controls on the grip test and successful completion of the inverted wire-hang test despite a prolonged hold time of 5 min (Fig. 5d-f). Similar immobility time in a tail-hang assay was also noted (Fig. 5g). No deficit in fine-motor function was noted in the adhesive tape removal assay (Fig. 5h). Furthermore, no coordination deficits were noted when descending down a vertical pole or climbing in a vertical climb assay (Fig. 5i,j).
众所周知，浦肯野神经元在协调和控制复杂动作

^{26, 27}

中起着举足轻重的作用。特别是，吞咽是一项复杂的活动，需要脑干、大脑皮层和小脑内神经元的复杂神经控制系统

^{28}

。<对照组（Ptprd

^{flox/flox}

和 Pcp2-cre；Ptprd

^{+ / +}

）与 Pcp2-cre；Ptprd

^{flox/flox}

小鼠之间相似的食物摄入量和舔食频率（图 4 d 和扩展数据图）排除了吞咽困难、明显的运动障碍或无法获取食物或水作为所观察到的嗜食欲减退的潜在解释。尽管如此，我们对小鼠进行了一系列运动协调和学习测定，以排除潜在的缺陷作为普肯列神经元特异性 Ptprd 缺失后观察到的嗜睡症的混淆因素。Pcp2-cre; Ptprd

^{flox^{- 10 x}}

小鼠在 4 天内记录的步行活动或车轮运行活动中没有显示出缺陷（图 5a,b 和补充图 6a,b）。我们让小鼠进行了3、5和7分钟的匀速转动，所有组别都成功完成了任务，与基因型无关（图5c和补充图6c）。此外，Pcp2-cre; Ptprd

^{fox^{'} flox}

小鼠没有表现出任何神经肌肉异常，其握力测试强度与对照组相似，尽管保持时间延长了 5 分钟，但仍成功完成了倒挂钢丝测试（图 5d-f）。在尾悬试验中也发现了类似的不动时间（图 5g）。在撕除胶带试验中没有发现精细运动功能障碍（图 5h）。此外，在垂直杆下降或垂直攀爬试验中也没有发现协调障碍（图 5i,j）。

Fig.

5 ∣

Purkinje-specific Ptprd deletion does not affect motor learning and coordination. a,b, Cumulative pedestrian and wheel-running activity of 8-week-old Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox / f fox}

and Pcp2-cre; Ptprd

^{floxflox}

male mice on normal chow (Pcp2-cre; Ptprd

^{+ / +} n = 10;

Ptprd

^{flox/flox} n = 12

and Pcp2-cre; Ptprd

^{flox/flox}

n = 10

). c, Time latency on a constant speed rotarod over the course of three trials of 16-20-week-old Pcp2-cre; Ptprd

^{+ + ⟩}

, Ptprd

^{flox fiox}

and Pcp2-cre; Ptprd

^{floxffox}

male mice (Pcp2-cre; Ptprd

^{+ / +} n = 11, P t p r d^{flox/flox} n = 12

and Pcp2-cre; Ptprd

^{flox/flox} n = 13

). d-e, Forelimb (two paws) and hindlimb (two paws) grip force measurements of 16-week-old Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{flox/flox}

male mice (Forelimb grip assay: three trials per mouse; Pcp2-cre; Ptprd

^{+ + ∣} n = 12

, Ptprd

^{flox}^{flox}

n = 11

and Pcp2-cre; Ptprd

^{flox/flox} n = 12

; hindlimb grip assay:Pcp2-cre; Ptprd

^{+ +} n = 13

, Ptprd

^{flox / f lox} n = 10

and Pcp2-cre; Ptprd

^{flox/flox} n = 13) . f

, Wire-hanging latency, tested for up to 5 min in 20-week-old Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

and Pcp2-cre;Ptprd

^{flox/flox}

male mice. Each data point represents the time taken averaged across two trials for each mouse. Note that a failure rate of zero represents successful wire-hanging latency of a minimum of 3 min out of 5 min for all mice tested (Pcp2-cre; Ptprd

^{+ / +}

n = 13

, Ptprd

d^{flox / f l lox} n = 10

and Pcp2-cre; Ptprd

^{flox/flox} n = 13

). g, The total amount of immobility time (defined as the time during which the animal is hanging passively and motionless) for 20-week-old Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{flox/flox}

male mice subjected to tail-hang test (Pcp2-cre; Ptprd

^{+ +} n = 12

, Ptprd

^{flox/flox} n = 8

and Pcp2-cre; Ptprd

^{flox/flox} n = 13

). h, Number of trials (number of paw-to-mouth contacts or paw shakes) and time taken to remove the adhesive tape by the 20-week-old Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{flox/flox}

male mice. Each data point represents the time taken averaged from adhesive tape removal from the left paw and right paw of each mouse (Pcp2-cre; Ptprd

^{+ / +}

n = 12, {Ptprd}^{flox/flox} n = 10

and Pcp2-cre; Ptprd

^{flox/flox} n = 13

). Note that a failure rate of zero represents the successful completion of the task by all mice tested. i, Time taken by 18-week-old Pcp2-cre; Ptprd

^{+ + ρ}

, Ptprd

^{flox / fox}

and Pcp2-cre; Ptprd

^{flox / fox}

male
图：

5 ∣

Purkinje 特异性 Ptprd 缺失不影响运动学习和协调。a,b,8周大的 Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox / f fox}

和 Pcp2-cre; Ptprd

^{floxflox}

雄性小鼠在正常饲料（Pcp2-cre; Ptprd

^{+ / +} n = 10;

Ptprd

^{flox/flox} n = 12

和 Pcp2-cre; Ptprd

^{flox/flox}

n = 10

）下的累积步行和轮跑活动。c, 16-20 周龄 Pcp2-cre; Ptprd

^{+ + ⟩}

, Ptprd

^{flox fiox}

和 Pcp2-cre; Ptprd

^{floxffox}

雄性小鼠（Pcp2-cre; Ptprd

^{+ / +} n = 11, P t p r d^{flox/flox} n = 12

和 Pcp2-cre; Ptprd

^{flox/flox} n = 13

）三次试验过程中匀速转动的时间潜伏期。d-e，16 周龄 Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

和 Pcp2-cre; Ptprd

^{flox/flox}

雄性小鼠的前肢（两爪）和后肢（两爪）握力测量（前肢握力测定，每只小鼠三次；Pcp2-cre; Ptprd

^{+ / +} n = 11, P t p r d^{flox/flox} n = 12

和 Pcp2-cre; Ptprd

^{flox/flox} n = 13

）：Ptprd

^{+ + ∣} n = 12

, Ptprd

^{flox}^{flox}

n = 11

and Pcp2-cre; Ptprd

^{flox/flox} n = 12

; 后肢抓握试验：Ptprd

^{flox/flox} n = 13) . f

, 20 周龄 Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

和 Pcp2-cre; Ptprd

^{flox/flox}

雄性小鼠吊线潜伏期，测试时间长达 5 分钟。每个数据点代表每只小鼠两次试验的平均用时。请注意，失败率为零代表所有受试小鼠（Pcp2-cre；Ptprd

^{+ / +}

n = 13

、Ptprd

d^{flox / f l lox} n = 10

和Pcp2-cre；Ptprd

^{flox/flox} n = 13

）的成功挂线潜伏期为 5 分钟中的至少 3 分钟。 g, 20 周龄 Pcp2-cre；Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

和 Pcp2-cre; Ptprd

^{flox/flox}

雄性小鼠进行尾悬试验（Pcp2-cre; Ptprd

^{+ +} n = 12

, Ptprd

^{flox/flox} n = 8

和 Pcp2-cre; Ptprd

^{flox/flox} n = 13

）。h, 20 周龄 Pcp2-cre; Ptprd

^{+ / +}

, Ptprd

^{flox/flox}

和 Pcp2-cre; Ptprd

^{flox/flox}

雄性小鼠的试验次数（爪对嘴接触或摇爪次数）和去除胶带所需的时间。每个数据点代表从每只小鼠（Pcp2-cre；Ptprd

^{+ / +}

n = 12, {Ptprd}^{flox/flox} n = 10

和 Pcp2-cre；Ptprd

^{flox/flox} n = 13

）左爪和右爪撕下胶带所需的平均时间。请注意，失败率为零代表所有受测小鼠都成功完成了任务。i, 18 周大的 Pcp2-cre; Ptprd

^{+ + ρ}

, Ptprd

^{flox / fox}

和 Pcp2-cre; Ptprd

^{flox / fox}

雄性所需的时间

Furthermore, Purkinje neuron function is necessary for adaptive motor learning in addition to motor coordination

^{29}

, and altered Purkinje neuron function can result in motor coordination and learning deficits in challenging tasks

^{30, 31}

. To rule out the potential effect of Purkinje neuron-specific Ptprd deletion on motor coordination and learning, we used the ErasmusLadder to quantify gait and absolute learning in a cerebellum-dependent task (Fig. 5k). We found no differences in gait patterns on the ErasmusLadder with both control and Pcp2-cre; Ptprd

^{flox/flox}

mice, which showed similar percentages of short steps (Fig. 51 and Supplementary Fig. 6d) and long steps (Fig. 5m and Supplementary Fig. 6e). Pcp2-cre; Ptprd

^{flox/flox}

mice performed as well as control mice in terms of percentage of missteps committed on the ErasmusLadder (Fig. 5n and Supplementary Fig. 6f). Importantly, cerebellum-dependent learning was not affected in Pcp2-cre; Ptprd

^{loxfflox}

mice (Fig. 5o and Supplementary Fig. 6g). Post-perturbation step times during paired trials (conditioning stimulous + unconditioned stimulous) in the challenge sessions (sessions three to five) were not significantly different between groups, indicating normal adaptive motor learning responses in Pcp2-cre; Ptprd

^{fox / f flox}

mice. Pre-perturbation step times; that is, basal step-time measurements before obstacle presentation, were also not significantly different between groups, indicating normal baseline stepping dynamics in Pcp2-cre; Ptprd

^{flox / f l o x}

mice. Thus, ErasmusLadder experiments demonstrate that Purkinje neuron-specific Ptprd deletion does not impair Purkinje neuron-mediated coordination and learning of complex movements. Blind analysis of these ten motor assays ruled out potential motor deficits as a cause of the observed hypodipsia in Pcp2-cre; Ptprd

^{flox/flox}

mice.
此外，除了运动协调

^{29}

之外，Purkinje神经元功能对于适应性运动学习也是必要的，Purkinje神经元功能的改变会导致在挑战性任务中出现运动协调和学习障碍

^{30, 31}

。为了排除Purkinje神经元特异性Ptprd缺失对运动协调和学习的潜在影响，我们使用ErasmusLadder对小脑依赖性任务中的步态和绝对学习进行了量化（图5k）。我们发现，对照组和 Pcp2-cre; Ptprd

^{flox/flox}

小鼠在 ErasmusLadder 上的步态模式没有差异，短步（图 51 和补充图 6d）和长步（图 5m 和补充图 6e）的百分比相似。Pcp2-cre; Ptprd

^{flox/flox}

小鼠在伊拉斯谟阶梯（ErasmusLadder）上的错步百分比表现与对照组小鼠相同（图 5n 和补充图 6f）。重要的是，Pcp2-cre; Ptprd

^{loxfflox}

小鼠的小脑依赖性学习不受影响（图 5o 和补充图 6g）。在挑战环节（第三至第五环节）的配对试验（条件刺激+非条件刺激）中，各组之间的扰动后步进时间没有显著差异，这表明 Pcp2-cre; Ptprd

^{fox / f flox}

小鼠的适应性运动学习反应正常。扰动前步进时间（即障碍出现前的基础步进时间测量值）在组间也无显著差异，表明 Pcp2-cre; Ptprd

^{flox / f l o x}

小鼠的基线步进动力学正常。因此，ErasmusLadder 实验证明，Purkinje 神经元特异性 Ptprd 缺失不会损害 Purkinje 神经元介导的复杂运动的协调和学习。对这十项运动测定的盲法分析排除了潜在的运动障碍可能是导致Pcp2-cre; Ptprd

^{flox/flox}

小鼠观察到的偏食症的原因。

Purkinje-specific deletion of Ptprd renders mice unresponsive to dipsogenic effect of asprosin
Purkinje特异性缺失Ptprd会导致小鼠对阿司匹林的致浸剂效应反应迟钝

Water deprivation, which induces plasma asprosin (Fig.1w), was also found to enhance Purkinje neuron activity (Fig. 6), and this effect was completely abolished upon Purkinje neuron-specific Ptprd deletion (Fig. 6). Additionally, we found that Purkinje neuron-specific Ptprd deletion completely abolished asprosin-mediated Purkinje neuron activation using slice electrophysiology (Fig. 7a-c). By contrast, Purkinje neuron-specific Ptprd deletion had no impact whatsoever
缺水会诱导血浆阿司匹林（图 1w），缺水也会增强 Purkinje 神经元的活性（图 6），这种效应在 Purkinje 神经元特异性 Ptprd 缺失后完全消失（图 6）。此外，我们还发现，利用切片电生理学，Purkinje 神经元特异性 Ptprd 缺失完全取消了阿司匹林介导的 Purkinje 神经元激活（图 7a-c）。相比之下，Purkinje 神经元特异性 Ptprd 缺失没有任何影响
mice climb vertically up the wire mesh (Pcp2-cre; Ptprd

^{+ / +} n = 12

, Ptprd

^{flox / f l lox} n = 10

and Pcp2-cre; Ptprd

^{flox / f l o x} n = 11

). Note that no foot slips were observed while mice climbed up the wire mesh.j, Time taken (s) and the number of failures (number of falls) by the 16-20-week-old Pcp2-cre; Ptprd

^{+ +}

, Ptprd

^{flox/flox}

and Pcp2-cre;Ptprd

^{flox/flox}

male mice to descend from a vertical pole. Each data point represents the time taken averaged across two trials for each mouse (Pcp2-cre; Ptprd

^{+ / +} n = 12

, Ptprd

^{flox/flox} n = 10

and Pcp2-cre; Ptprd

^{flox/flox} n = 12

). Note that a full number of zeros represents zero failure rate across the tested genotypes. k, Schematic of ErasmusLadder experimental paradigm showing sessions one and two: ‘training’ during which mice are trained to walk across the ErasmusLadder, a horizontal ladder apparatus with pressure-sensitive stepping rungs (blue, default stepping rungs) and lower rungs that capture missteps (gray rungs). During sessions three to five, mice are challenged with a cerebellum-dependent conditioned learning paradigm in which obstacle rungs are presented (red, unconditioned stimulus (US)) during paired trials preceded by a conditioning stimulus (CS) tone separated by an interstimulus interval (ISI) of 250 ms. I-n, Stepping pattern (short steps, long steps and missteps) of Pcp2-cre; Ptprd

^{f l o x f l o x}

mice (

n = 8

) (and Pcp2-cre;

{Ptprd}^{+ +}

) male mice (

n = 9

). o, Cerebellum-dependent motor learning as measured by absolute learning step times on the ErasmusLadder during paired trials in challenge sessions with Pcp2-cre; Ptprd

^{f l o x / f o x}

mice

(n = 8)

and Pcp2-cre; Ptprd

^{+ / +}

male

(n = 9)

mice. Dashed lines represent pre-perturbation step times. Error bars, s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

and

^{* * * *} P < 0.0001

; repeated measures ANOVA (in a-c and I-o), one-way ANOVA (in d-j). Nonsignificant statistical result (NS) refers to the result of Šídák’s multiple comparisons between groups per session followed by two-way repeated measures ANOVA (in I-n) and of mixed-effects analysis followed by Tukey’s multiple comparisons test between groups per session (in 0). Also see Supplementary Fig. 6 for individual data plot and Source Data for raw values and details of analysis.
小鼠垂直爬上金属网（Pcp2-cre；Ptprd

^{+ / +} n = 12

、Ptprd

^{flox / f l lox} n = 10

和 Pcp2-cre；Ptprd

^{flox / f l o x} n = 11

）。j，16-20 周龄的 Pcp2-cre; Ptprd

^{+ +}

, Ptprd

^{flox/flox}

和 Pcp2-cre;Ptprd

^{flox/flox}

雄性小鼠从垂直杆下降所需的时间（秒）和失败次数（跌倒次数）。每个数据点代表每只小鼠（Pcp2-cre; Ptprd

^{+ / +} n = 12

, Ptprd

^{flox/flox} n = 10

和 Pcp2-cre; Ptprd

^{flox/flox} n = 12

）两次试验的平均时间。k, ErasmusLadder 实验范式示意图，显示了第一和第二阶段："训练 "期间，训练小鼠走过 ErasmusLadder，这是一种水平梯子装置，带有压力敏感的梯级（蓝色，默认梯级）和捕捉失足的较低梯级（灰色梯级）。在第三至第五次训练中，小鼠接受小脑依赖性条件学习范式的挑战，在配对试验中出现障碍梯级（红色，非条件刺激（US）），在此之前出现条件刺激（CS）音调，刺激间歇（ISI）为 250 毫秒。I-n，Pcp2-cre；Ptprd

^{f l o x f l o x}

小鼠（

n = 8

）（和 Pcp2-cre；

{Ptprd}^{+ +}

）雄性小鼠（

n = 9

）的步态（短步、长步和错步）。o，在 Pcp2-cre; Ptprd

^{f l o x / f o x}

小鼠

(n = 8)

和 Pcp2-cre; Ptprd

^{+ / +}

雄性小鼠

(n = 9)

的挑战训练中，通过配对试验中 ErasmusLadder 上的绝对学习步进时间测量的小脑依赖性运动学习。虚线代表扰动前的步长时间。误差条，s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

和

^{* * * *} P < 0.0001

；重复测量方差分析（a-c 和 I-o），单因素方差分析（d-j）。不显著的统计结果（NS）是指每节课各组之间的希达克多重比较结果，然后进行双向重复测量方差分析（在 I-n 中），以及每节课各组之间的混合效应分析结果，然后进行图基多重比较检验（在 0 中）。另见补充图 6 的单个数据图和原始数据的原始值和分析详情。
on norepinephrine-induced Purkinje neuron activation

^{32}

(Fig. 7d-f), indicating maintained health of the neurons and specificity of the perturbation for asprosin and water deprivation.

^{32}

（图 7d-f），这表明神经元的健康状况得到了维持，而且天麻素和缺水的扰动具有特异性。

Next, given that Purkinje neuron activity in vivo involves two distinct types of action potentials, simple and complex spikes

^{33}

, we sought to determine the effect of genetic loss of Ptprd on Purkinje neuron
接下来，考虑到体内浦肯野神经元的活动涉及两种不同类型的动作电位（简单尖峰和复杂尖峰）

^{33}

，我们试图确定遗传性 Ptprd 缺失对浦肯野神经元的影响。

k ErasmusLadder k 伊拉斯谟阶梯
Sessions 1-2, training 第 1-2 节，培训

I Short steps I 短暂的步骤

m Long steps m 长台阶

n Missteps n 失误

Absolute learning 绝对学习

Fig.

6 ∣

Purkinje neuron-specific Ptprd deletion abolishes water-deprivationinduced Purkinje neuron activation. a, Representative action potential firing traces of Purkinje neurons from Pcp2-cre; Ptprd

^{+ / +}

and Pcp2-cre; Ptprd

^{flox/flox}

male mice subjected to overnight water deprivation. b-c, Data analysis of action potential firing frequency and resting membrane potential in

Pcp 2 - {Cre}^{+}

neurons from Pcp2-cre; Ptprd

^{+ / +}

and Pcp2-cre; Ptprd

^{flox/flox}

male mice (three mice per group;

n = 5

readings from fed Pcp2-cre; Ptprd

^{+ + ∣}

and Pcp2-cre; Ptprd

^{flox/flox}

males,

n = 10

readings from water-deprived Pcp2-cre; Ptprd

^{+ / +}

and

n = 6

readings from water-deprived Pcp2-cre; Ptprd

^{flox/flox}

males in

b

and

n = 5

readings from fed Pcp2-cre; Ptprd

^{+ + †}

and Pcp2-cre; Ptprd

^{flox/flox}

males,

n = 14

readings from fed Pcp2-cre; Ptprd

^{+ +}, n = 15

readings from fasted Pcp2-cre; Ptprd

^{+ +}

and fed Pcp2-cre; Ptprd

^{flox/flox}

males,

n = 17

from water-deprived

P c p 2 - c r e; P t p r d^{flox/flox}

males in c). Error bars, s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

and

^{* * * *} P < 0.0001

; by Student’s

t

-test (in b and

c

) NS. Also see Source Data for raw data and statistical values.
a, Pcp2-cre；Ptprd

^{+ / +}

和 Pcp2-cre；Ptprd

^{flox/flox}

雄性小鼠的普肯列神经元的代表性动作电位发射轨迹。b-c，Pcp2-cre；Ptprd

^{+ / +}

和 Pcp2-cre；Ptprd

^{flox/flox}

雄性小鼠

Pcp 2 - {Cre}^{+}

神经元动作电位发射频率和静息膜电位的数据分析（每组三只小鼠；和 Pcp2-cre；Ptprd

^{flox/flox}

雄性，

n = 10

缺水 Pcp2-cre；Ptprd

^{+ / +}

和

n = 6

缺水 Pcp2-cre；Ptprd

^{flox/flox}

雄性，

b

和

n = 5

从喂养的Pcp2-cre读数；Ptprd

^{+ + †}

和Pcp2-cre；Ptprd

^{flox/flox}

雄性，

n = 14

从喂养的Pcp2-cre读数；Ptprd

^{+ +}, n = 15

来自禁食 Pcp2-cre；Ptprd

^{+ +}

和喂养 Pcp2-cre；Ptprd

^{flox/flox}

雄性，

n = 17

来自缺水

P c p 2 - c r e; P t p r d^{flox/flox}

雄性（c）。误差条，s.e.m.

^{*} P < 0.05,^{* *} P < 0.01,^{* * *} P < 0.001

和

^{* * * *} P < 0.0001

；经学生

t

检验（b和

c

）为NS。原始数据和统计值另见原始数据。
activity using in vivo electrophysiology. We implanted Pcp2-cre;Ptprd

^{++}

and Pcp2-cre; Ptprd

^{flox/flox}

mice with head plates and performed a craniotomy to allow recordings in cerebellar lobules IV, V and VI of the vermis and adjacent paravermis regions from awake mice (Extended Data Fig. 6a-c). The pattern of activation was determined using two different measurements, the coefficient of variation (CV) and CV2 (ref. 34). CV measures the irregularity of interspike intervals over the entire analyzed recording period, and therefore predominantly captures an overall global irregularity of the firing rate or a bursting quality in the spike pattern. CV2 measures the irregularity of one interspike interval to the next and thus captures how erratic the pattern of firing is locally. We detected no significant difference in firing rate and CV2 (Extended Data Fig. 6d-i). By contrast, a significant reduction in CV (irregularity of interspike intervals) for Purkinje neuron complex spikes of Pcp2-cre; Ptprd

^{floxflox}

mice was noted (Extended Data Fig. 6h), indicating that the pattern of complex spikes is more regular upon Purkinje neuron-specific Ptprd deletion. To record Purkinje neuron response to asprosin in live, awake and behaving mice, we performed stereotaxic injection of Cre-responsive AAV-hSyn-FLEX-GCaMP and placed an optical fiber in cerebellar lobe IV in controls and mice with Purkinje neuron-specific Ptprd deletion (Fig. 7g,h). At 40 days
活动。我们给 Pcp2-cre;Ptprd

^{++}

和 Pcp2-cre;Ptprd

^{flox/flox}

小鼠植入了头板，并进行了开颅手术，以记录清醒小鼠的蚓部小脑第四、第五和第六小叶以及相邻副蚓部区域的活动（扩展资料图 6a-c）。激活模式是通过变异系数（CV）和 CV2（参考文献 34）这两种不同的测量方法确定的。CV 测量整个分析记录期间尖峰间期的不规则性，因此主要捕捉发射率的整体不规则性或尖峰模式的突发性。CV2 测量的是一个尖峰间期与下一个尖峰间期之间的不规则性，因此可以捕捉局部发射模式的不规则性。我们检测到发射率和 CV2 没有明显差异（扩展数据图 6d-i）。相比之下，我们注意到 Pcp2-cre; Ptprd

^{floxflox}

小鼠的普肯列神经元复合尖峰的 CV（尖峰间隔的不规则性）显著降低（扩展数据图 6h），这表明普肯列神经元特异性 Ptprd 缺失后，复合尖峰的模式更加规则。为了记录活的、清醒的和有行为的小鼠的Purkinje神经元对阿司匹林的反应，我们对Cre反应性AAV-hSyn-FLEX-GCaMP进行了立体定向注射，并在对照组和Purkinje神经元特异性Ptprd缺失的小鼠的小脑第四叶中放置了一根光纤（图7g,h）。40天后
after AAV-hSyn-FLEX-GCaMP treatment, we intracerebroventricularly (i.c.v.) or intravenously (i.v.) injected recombinant asprosin and recorded Purkinje neuron activity (Fig. 7g,h). Asprosin elicited an increase in Purkinje neuron activity within 10-15 min of treatment in control mice (Fig. 7i-p and Supplementary Fig. 7). This effect was completely abolished in mice with Purkinje neuron-specific Ptprd deletion (Fig. 7j-n and Supplementary Fig. 7). Moreover, control mice i.c.v. injected with asprosin exhibited hyperdipsia and hyperphagia (Fig. 7o,p). Importantly, asprosin failed to elicit hyperdipsia in mice with Purkinje neuron-specific Ptprd deletion while eliciting hyperphagia normally (Fig. 7o,p), demonstrating with exquisite specificity that asprosin-Ptprd signaling in Purkinje neurons is limited to the regulation of water intake.
AAV-hSyn-FLEX-GCaMP处理后，我们在小鼠脑室内（i.c.v.）或静脉内（i.v.）注射重组阿司匹林并记录Purkinje神经元活动（图7g,h）。在对照组小鼠中，阿司匹林在处理后 10-15 分钟内引起浦肯野神经元活动增加（图 7i-p 和补充图 7）。这种效应在 Purkinje 神经元特异性 Ptprd 缺失的小鼠中完全消失（图 7j-n 和补充图 7）。此外，静脉注射阿司匹林的对照组小鼠表现出多饮和多食（图 7o,p）。重要的是，asprosin 在 Purkinje 神经元特异性 Ptprd 缺失的小鼠中不能引起高嗜水症，而能正常引起高吞咽症（图 7o,p）。

Lastly, we tested Purkinje neuron response to traditional dipsogenic triggers such as acute hyperosmolality and hypovolemia. We recorded Purkinje neuron GCaMP fluorescence responses to 3 M NaCl , 2 M mannitol and 30% polyethylene glycol (PEG). The SFO and OVLT are well established in the regulation of water intake in response to these dipsogenic challenges

^{1, 35, 36}

. Although osmolar stress induced by hypertonic saline or mannitol is known to dose-dependently activate SFO neurons within a few minutes

^{35}

, no such activation was observed in Purkinje neurons for up to 35 min (Extended Data Fig. 7). Furthermore, Purkinje neurons were also not responsive to PEG, which induces hypovolemia, at a dose and time frame known to be sufficient to activate SFO neurons

^{35}

. This unresponsiveness of Purkinje neurons to hyperosmolality and hypovolemia establishes a distinct modus operandi of cerebellar Purkinje neurons from that of the CVO for the regulation of fluid homeostasis. Moreover, Purkinje neuron-specific Ptprd deletion had no impact on the ability of mice to consume significantly more water when subjected to dipsogenic triggers, again suggesting that Purkinje neurons exert tonic control over water intake but are not necessary for responding to hyperosmolality and hypovolemia, instead responding to a peripherally generated hormone.
最后，我们测试了Purkinje神经元对急性高渗和低血容量等传统致浸诱因的反应。我们记录了Purkinje神经元对3 M NaCl、2 M甘露醇和30%聚乙二醇（PEG）的GCaMP荧光反应。SFO和OVLT在调节水摄入量以应对这些致渗挑战

^{1, 35, 36}

方面已得到公认。虽然已知高渗盐水或甘露醇诱导的渗透压应激会在几分钟内按剂量激活 SFO 神经元

^{35}

，但在长达 35 分钟的时间里，Purkinje 神经元没有观察到这种激活（扩展数据图 7）。此外，Purkinje 神经元对 PEG 也没有反应，PEG 可诱导低血容量，其剂量和时间足以激活 SFO 神经元

^{35}

。Purkinje神经元对高渗透压和低血容量反应迟钝，这证明小脑Purkinje神经元在调节体液稳态方面的工作方式与CVO不同。此外，Purkinje神经元特异性Ptprd缺失对小鼠在摄入更多水分的能力没有影响，这再次表明Purkinje神经元对水分摄入进行强直性控制，但不是对高渗和低血容量做出反应所必需的，而是对外周产生的激素做出反应。

Discussion 讨论

The cerebellum has long been known to be involved in the regulation of movement, balance and coordination

^{9}

. In addition to sensorimotor and vestibular control, the cerebellum also contributes to cognition, emotion, memory, autonomic function, satiety and meal termination

^{6 - 8, 37 - 39}

. Functional imaging studies have reported increased cerebellar activity with thirst in human subjects

^{5, 11, 40}

, and a murine study has correlated pan-cerebellar and midbrain metabolic activity with water intake

^{10}

. However, whether the cerebellum can directly modulate fluid homeostasis has remained unknown, largely owing to the complex nature of functional parcillation

^{41}

, cellular diversity

^{42}

and spatially diverse anatomical connections

^{21, 22}

of the cerebellar cortex. The present study directly implicates cerebellar Purkinje neurons in the regulation of thirst (Extended Data Fig. 8). Notably, through the combined use of chemogenetic and optogenetic manipulation of Purkinje neurons, bidirectional changes in water intake, without corresponding changes in food consumption, were observed. Furthermore, our results-demonstrating asprosin-mediated activation of Purkinje neurons in vivo and ex vivo, irrespective of the basal firing rate, and from extensive anteroposterior and mediolateral sampling of the cerebellar cortex from both sexes-strongly implicate Purkinje neurons as a target of a peripherally generated hormone.
人们很早就知道小脑参与调节运动、平衡和协调

^{9}

。除了感觉运动和前庭控制外，小脑还对认知、情绪、记忆、自律神经功能、饱腹感和进餐终止做出贡献

^{6 - 8, 37 - 39}

。功能成像研究报告称，在人类受试者口渴时小脑活动增加

^{5, 11, 40}

，一项小鼠研究将小脑和中脑代谢活动与水摄入量相关联

^{10}

。然而，小脑是否能直接调节体液稳态一直是个未知数，这主要是由于小脑皮层的功能旁路

^{41}

、细胞多样性

^{42}

和空间解剖连接

^{21, 22}

的复杂性。本研究将小脑浦肯野神经元与口渴的调节直接联系起来（扩展数据图 8）。值得注意的是，通过联合使用化学遗传学和光遗传学对普肯列神经元进行操作，观察到了水摄入量的双向变化，而食物消耗量没有相应的变化。此外，我们的研究结果证明了阿朴素介导的体内和体外普肯列神经元激活（与基础发射率无关），以及对两性小脑皮层前胸和内外侧的广泛取样，都有力地证明了普肯列神经元是外周激素的靶点。

Whether circulating concentrations of asprosin are experimentally decreased (genetic depletion in Fbn1

^{NS / +}

mice, or acute removal by immunologic neutralization in WT mice) or increased (AAV-mediated or Ad5-mediated overexpression and secretion, intranasal or i.c.v. delivery of recombinant asprosin), the result is a corresponding consistent change in water intake. Importantly, hyperdipsia in fasted mice subjected to plasma asprosin elevation (AAV-asprosin), and hypodipsia in fasted mice subjected to plasma asprosin neutralization (anti-asprosin mAb) demonstrate regulation of water intake
无论实验中降低（Fbn1

^{NS / +}

小鼠的遗传耗竭，或 WT 小鼠通过免疫中和急性去除）或增加（AAV 或 Ad5 介导的过表达和分泌，鼻内或静脉注射重组天冬氨酸）天冬氨酸的循环浓度，结果都会导致摄水量发生相应的一致变化。重要的是，血浆天冬氨酸升高（AAV-天冬氨酸）导致的禁食小鼠多尿，以及血浆天冬氨酸中和（抗天冬氨酸 mAb）导致的禁食小鼠少尿，都证明了水摄入量的调节作用

independent of asprosin-mediated food intake and body weight regulation. Ptprd-mediated signaling seems to be central to these effects, as the genetic loss of Ptprd ( Ptprd

^{- / -}

) rendered mice unresponsive
与阿司匹林介导的食物摄入量和体重调节无关。Ptprd 介导的信号传导似乎是这些效应的核心，因为遗传性 Ptprd 缺失（Ptprd

^{- / -}

）会使小鼠对这些效应不敏感。
to asprosin’s dipsogenic effects. Published in situ hybridization and immunohistochemistry analysis showed that Ptprd is expressed in the cerebellum, including Purkinje neurons

^{17}

. We verified this with
的作用。已发表的原位杂交和免疫组化分析显示，Ptprd在小脑中表达，包括浦肯野神经元

^{17}

。我们用

Fig. 7 |Asprosin activates Purkinje neurons in vivo, and Purkinje neuronspecific Ptprd deletion renders mice unresponsive to the dipsogenic effects of asprosin. a, Representative action potential firing traces of Purkinje neurons from Pcp2-cre (control, WT) and Pcp2-cre; Ptprd

^{flox/flox} (KO)

mice, treated with recombinant asprosin. b-c, Data analysis of action potential firing frequency and membrane potential of Purkinje neurons from control (Pcp2-cre) and Pcp2cre; Ptprd

^{flox/flox}

(KO) mice, post treatment with recombinant asprosin (

n = 15

b

and

n = 17

c

; from three different mice each group).

d

, Representative action potential firing traces of Purkinje neurons from Pcp2-cre (control, WT) and Pcp2-cre; Ptprd

^{flox/flox}

(KO) mice, treated with norepinephrine (NE).
a, Pcp2-cre（对照，WT）和 Pcp2-cre；Ptprd

^{flox/flox} (KO)

小鼠的 Purkinje 神经元的代表性动作电位发射轨迹，用重组阿司匹林处理。b-c，对照组（Pcp2-cre）和 Pcp2cre; Ptprd

^{flox/flox}

(KO)小鼠的浦肯野神经元的动作电位发射频率和膜电位的数据分析，用重组阿司匹林处理后（

b

中的

n = 15

和

c

中的

n = 17

；每组来自三只不同的小鼠）。

d

，Pcp2-cre（对照组，WT）和Pcp2-cre；Ptprd

^{flox/flox}

(KO)小鼠的Purkinje神经元的代表性动作电位发射轨迹，用去甲肾上腺素（NE）处理。
e-f, Data analysis of action potential firing frequency and membrane potential of Purkinje neurons from control (Pcp2-cre) and Pcp2-cre; Ptprd

^{flox/flox}

(KO) mice, post treatment with NE (

n = 12

e

and

n = 15

f

; from three different mice each group). (g) Schematic experimental strategy using the Cre-dependent AAV expressing hSyn-FLEX-GCaMP7f for optical recording of activated Purkinje neurons of the Pcp2-cre (control) and Pcp2-cre; Ptprd

^{flox/flox}

(KO) mice subjected to i.c.v. or i.v. injection of GFP or recombinant asprosin. h, Representative image of GCaMP7 expressing Pcp2 neurons in lower magnification

\times 2

of the coronal section of the cerebellum. i-j, GCaMP7 fluorescent response in Pcp2

^{+}

neurons from control (Pcp2-cre) and KO (Pcp2-cre; Ptprd

^{flox/flox}

) mice post i.v. injection of
e-f，对照组（Pcp2-cre）和 Pcp2-cre；Ptprd

^{flox/flox}

（KO）小鼠经 NE 处理后（

e

中为

n = 12

，

f

中为

n = 15

；每组三只不同小鼠）普肯野神经元的动作电位发射频率和膜电位数据分析。(g）使用表达 hSyn-FLEX-GCaMP7f 的 Cre 依赖性 AAV 光学记录 Pcp2-cre（对照组）和 Pcp2-cre；Ptprd

^{flox/flox}

（KO）小鼠的活化普肯耶神经元的实验策略示意图。i-j，对照组（Pcp2-cre）和 KO 组（Pcp2-cre; Ptprd

^{flox/flox}

）小鼠小脑冠状切片中 Pcp2

^{+}

神经元的 GCaMP7 荧光反应。
recombinant asprosin or saline vehicle (

n = 6

Pcp2-cre; Ptprd

^{+ + ∣} +

saline and Pcp2-cre;Ptprd

^{+ / +} +

asprosin,

n = 4

Pcp2-cre; Ptprd

^{flox/flox} +

saline and Pcp2-cre; Ptprd

^{flox/flox} +

asprosin). k-I, Data analysis of GCaMP7 fluorescent response in Pcp2

^{+}

neurons from control (Pcp2-cre) and Pcp2-cre; Ptprd

^{flox/flox}

mice at every 5-min interval for 30 min , post i.v. injection of saline or asprosin (

n = 6

Pcp2cre; Ptprd

^{+ / +} +

saline and Pcp2-cre;Ptprd

^{+ / +} +

asprosin,

n = 4

Pcp2-cre; Ptprd

^{flox/flox} +

saline and Pcp2-cre; Ptprd

^{flox/flox} +

asprosin). m, GCaMP7 fluorescent response in Pcp2

^{+}

neurons from control (Pcp2-cre) and KO (Pcp2-cre;Ptprd

^{flox/flox}

) mice post i.c.v. injection of GFP or recombinant asprosin (

n = 4

Pcp2-cre; Ptprd

^{+ / +} +

asprosin,

n = 5

Pcp2-cre;Ptprd

^{+ / +} +

GFP and

n = 6

Pcp2-cre;Ptprd

^{flox/flox} +

asprosin). n, Data analysis of GCaMP7 fluorescent response in Pcp2

^{+}

neurons from control (Pcp2-cre) and Pcp2-cre; Ptprd

^{flox/flox}

mice at

0, 5, 10

and 15 min post i.c.v. injection of GFP or asprosin (

n = 4

Pcp2-cre; Ptprd

^{+ / +} +

asprosin,

n = 5

Pcp2-cre; Ptprd

^{+ / +} +

GFP and

n = 6

Pcp2-cre; Ptprd

^{flox/flox} +

asprosin). o-p, 24-h water drinking and food intake of Pcp2-cre (control, WT) and Pcp2-cre; Ptprd

^{flox/flox}

mice after i.c.v. injection of GFP or recombinant asprosin (

n = 4

per group). Error bars, s.e.m.

^{*} P < 0.05,^{* *} P < 0.01

^{* * *} P < 0.001

and

^{* * * *} P < 0.0001

by Student’s

t

-test (b, c, e,

f, o

and

p)

and two-way ANOVA followed by post hoc Šídák’ tests (

k, I

and

n

). Also see Supplementary Fig. 7 for raw data plots and Source Data for raw and statistical values.
Ptprd

^{+ / +} +

asprosin,

n = 4

Pcp2-cre; Ptprd

^{flox/flox} +

saline and Pcp2-cre; Ptprd

^{flox/flox} +

asprosin）。k-I，对照组（Pcp2-cre）和 Pcp2-cre；Ptprd

^{flox/flox}

小鼠 Pcp2

^{+}

神经元中 GCaMP7 荧光反应的数据分析。Ptprd

^{+ / +} +

saline and Pcp2-cre;Ptprd

^{+ / +} +

asprosin,

n = 4

Pcp2-cre; Ptprd

^{flox/flox} +

saline and Pcp2-cre; Ptprd

^{flox/flox} +

asprosin）。m，对照组（Pcp2-cre）和 KO 组（Pcp2-cre; Ptprd

^{flox/flox}

）小鼠 Pcp2

^{+}

神经元的 GCaMP7 荧光反应。注射 GFP 或重组天冬氨酸（

n = 4

Pcp2-cre; Ptprd

^{+ / +} +

asprosin,

n = 5

Pcp2-cre;Ptprd

^{+ / +} +

GFP 和

n = 6

Pcp2-cre;Ptprd

^{flox/flox} +

asprosin ）。n，对照组（Pcp2-cre）和 Pcp2-cre；Ptprd

^{flox/flox}

小鼠 Pcp2

^{+}

神经元中 GCaMP7 荧光反应的数据分析。Ptprd

^{+ / +} +

asprosin,

n = 5

Pcp2-cre; Ptprd

^{+ / +} +

GFP 和

n = 6

Pcp2-cre; Ptprd

^{flox/flox} +

asprosin）。o-p，Pcp2-cre（对照组，WT）和 Pcp2-cre；Ptprd

^{flox/flox}

小鼠在静脉注射 GFP 或重组天冬氨酸（每组

n = 4

）后 24 小时的饮水量和食物摄入量。误差条，s.e.m。

^{*} P < 0.05,^{* *} P < 0.01

、

^{* * *} P < 0.001

和

^{* * * *} P < 0.0001

经Student's

t

检验（b、c、e、

f, o

和

p)

和双向方差分析后经post hoc Šídák'检验（

k, I

和

n

）。原始数据图见补充图 7，原始数据和统计值见原始数据。
increased hyperpolarization of inferior olive neurons

^{46}

. Cerebellar nuclei neurons can also shape the pattern of complex spikes

^{47}

. Additionally, Purkinje neurons themselves have some intrinsic control over the generation of complex spikes

^{48}

. As the knockout of Ptprd is specific to Purkinje neurons in the present study, it is within reason to speculate that it originates as a consequence of the Purkinje neuron manipulation.
下橄榄神经元的超极化增强

^{46}

。小脑核神经元也能塑造复杂尖峰的模式

^{47}

。此外，浦肯野神经元本身对复杂尖峰的产生也有一定的内在控制

^{48}

。在本研究中，由于 Ptprd 的敲除是 Purkinje 神经元特有的，因此有理由推测它是 Purkinje 神经元操纵的结果。

Collectively, our findings have defined a conserved thirst center in the cerebellum that responds to a peripherally generated hormonal signal to modulate thirst in either direction commensurate with the needs of the organism. This has the potential to serve as an important therapeutic target for the management of thirst disorders like polydipsia, hypodipsia and adipsia.
总之，我们的研究结果确定了小脑中一个保守的口渴中枢，它能对外周产生的激素信号做出反应，根据机体的需要向两个方向调节口渴。这有可能成为治疗多尿症、少尿症和肥胖症等口渴症的重要治疗靶点。

Online content 在线内容

Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41593-024-01700-9IF: 21.2 Q1 .

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Methods 方法

Mice 小鼠

WT C57BL/6 mice (JAX no. 000664), Rosa26-LSL-tdTOMATO (JAX no. 007914), Rosa26-eGFP/RpI10a (JAX no. 024750), B6.129-Tg(Pcp2-cre)2Mpin/J (Pcp2-cre;JAX no. 004146) and Fbn1

^{NPS / +}

(C57BL/6- C57BL/6-Fbn1

^{em1Chop/J;}

JAX no. 033548) were purchased from The Jackson Laboratory. Two different strains of Ptprd mice were used in this work and were maintained as heterozygous, as previously described

^{13, 14}

. B6;129-Ptprd < tm1Yiw > mice were purchased from the RIKEN BioResource Center. Mice with Ptprd cKO potential (RBRC04925; C57BL/6NA<tm1Brd>Ptprd<tm2a(KOMP)Wtsi>/ WtsiOrl; MEXY mice) was purchased from Wellcome Trust Sanger Institute and crossed to

{Flpase}^{+}

mice (JAX no. 003946) to remove the neomycin selection cassette and LacZ reporter, thereby making a conditional allele, as described previously

^{14}

. Thereafter, homozygous conditionally ready floxed mice (Ptprd tm2c(KOMP)Wtsi) were mated with Pcp2-cre mice to create Purkinje neuron-specific Ptprd knockout (Pcp2-cre; Ptprd

^{flox/flox}

). In addition, homozygous conditionally ready floxed mice (Ptprd tm2c(KOMP)Wtsi) were mated with AgRP-IRES-Cre (C57BL/6-Agrptm1(cre)Lowl; JAX no. 012899) to create AgRP neuron-specific Ptprd knockout, as previously described

^{14}

. For tdTOMATO or GFP labeling of Purkinje neurons, Rosa26-LSL-tdTOMATO mice (JAX no. 007914) or Rosa26-eGFP/ Rpl10a (JAX no. 024750) were mated with the above-described Pcp2-cre and Pcp2-cre; Ptprd

^{flox/flox}

mice for the generation of Pcp2-cre; Rosa26-LSL-tdTOMATO mice, Pcp2-cre-eGFP-Rpl10a mice and Pcp2-cre; Ptprd

^{flox/flox}

; Rosa26-LSL-tdTOMATO, respectively. For generation of tissue-specific knockout mouselines, male and female mice aged

8 - 52

weeks were used.
WT C57BL/6 小鼠（JAX 编号 000664）、Rosa26-LSL-tdTOMATO（JAX 编号 007914）、Rosa26-eGFP/RpI10a（JAX 编号 024750）、B6.129-Tg(Pcp2-cre)2Mpin/J（Pcp2-cre;JAX 编号 004146）和 Fbn1

^{NPS / +}

（C57BL/6- C57BL/6-Fbn1

^{em1Chop/J;}

JAX 编号 033548）。004146）和 Fbn1

^{NPS / +}

（C57BL/6- C57BL/6-Fbn1

^{em1Chop/J;}

JAX no.这项工作中使用了两种不同品系的 Ptprd 小鼠，并按照先前的描述

^{13, 14}

将其维持为杂合型。B6;129-Ptprd < tm1Yiw > 小鼠购自理化学研究所生物资源中心。具有 Ptprd cKO 潜力的小鼠（RBRC04925；C57BL/6NAPtprd/ WtsiOrl；MEXY 小鼠）购自 Wellcome Trust Sanger 研究所，并与

{Flpase}^{+}

小鼠（JAX 编号：003946）杂交，去除新霉素选择盒和 LacZ 报告基因，从而形成条件等位基因，如前所述

^{14}

。此后，将同源的条件准备好的floxed小鼠（Ptprd tm2c(KOMP)Wtsi）与Pcp2-cre小鼠交配，产生普肯列神经元特异性Ptprd基因敲除（Pcp2-cre；Ptprd

^{flox/flox}

）。此外，同卵条件准备好的浮性小鼠（Ptprd tm2c(KOMP)Wtsi）与 AgRP-IRES-Cre 交配（C57BL/6-Agrptm1(cre)Lowl；JAX no.012899）交配，以产生 AgRP 神经元特异性 Ptprd 基因敲除，如前

^{14}

所述。为了对浦肯野神经元进行tdTOMATO或GFP标记，Rosa26-LSL-tdTOMATO小鼠（JAX no.024750）与上述 Pcp2-cre 和 Pcp2-cre; Ptprd

^{flox/flox}

小鼠交配，分别产生 Pcp2-cre; Rosa26-LSL-tdTOMATO 小鼠、Pcp2-cre-eGFP-Rpl10a 小鼠和 Pcp2-cre; Ptprd

^{flox/flox}

; Rosa26-LSL-tdTOMATO。为了产生组织特异性基因敲除小鼠，使用了

8 - 52

周龄的雌雄小鼠。

Littermates from in-house mating served as controls in all experiments, except for WT lean mice that were bought from the Jackson Laboratory and used for experimentation after acclimation to the mouse housing facility. Mice were housed in microventilator cages on a 12-h light cycle in an animal facility maintained at

20 - 25^{\circ} C

and

40 - 60 %

humidity. Mice had ad libitum access to water and normal chow (5V5R, Lab Supply), dustless pellet diet (F0173, Bio-Serv) or Teklad High Fat Diet (Envigo; TD.06414), unless otherwise specified.
除了从杰克逊实验室购买并在适应小鼠饲养设施后用于实验的 WT 瘦小鼠外，所有实验均以内部交配的同窝小鼠作为对照。小鼠被饲养在微通风笼中，光照周期为12小时，动物饲养设施的湿度保持在

20 - 25^{\circ} C

和

40 - 60 %

。除非另有说明，小鼠可自由取用水和普通饲料（5V5R，Lab Supply）、无尘颗粒饲料（F0173，Bio-Serv）或Teklad高脂饲料（Envigo；TD.06414）。

Animal housing, husbandry and killing were conducted under animal protocols approved by the Case Western Reserve University (CWRU) Institutional Animal Care and Use Committee (protocol no. 2018-0042) and Pennington Biomedical Research Center Institutional Animal Care andUse Committee (protocol no. PBRC21119). The general health of mice was monitored by the CWRU animal resource center and PBRC animal resource center.
动物饲养、管理和捕杀均按照凯斯西储大学（CWRU）机构动物护理和使用委员会（协议编号：2018-0042）和彭宁顿生物医学研究中心机构动物护理和使用委员会（协议编号：PBRC21119）批准的动物协议进行。小鼠的总体健康状况由CWRU动物资源中心和PBRC动物资源中心监控。

Validation of Ptprd deletion in Purkinje neurons
在浦肯野神经元中进行 Ptprd 缺失验证

Validation of Ptprd deletion was done as previously described

^{13}

.Inbrief, at 09:00 h, 12-week-old Pcp2-cre; Ptprd

^{floxflox}

; Rosa26-LSL-tdTOMATO and control Pcp2-cre; Rosa26-LSL-tdTOMATO male mice were anesthetized with inhaled isoflurane and perfused with saline followed by

10 %

formalin. Brain sections (

25 μ m

in thickness) were collected and then subjected to immunofluorescence for Ptprd and tdTOMATO. In brief, one series of the brain sections was blocked (

5 %

normal donkey) for 1 h . Then, the brain sections were incubated with rabbit anti-Ptprd antibody (1:1,000 dilution; A15713, ABclonal) on a shaker at

4^{\circ} C

overnight. The next day, the brain sections were incubated by the donkey anti-rabbit Alexa Fluor 488 (1:500; A21206, Invitrogen) for 2 h . Sections were mounted on slides and coverslipped with DAPI mounting medium. Fluorescence images were taken using the Leica TCS SP5 fluorescence microscope with OptiGrid structured illumination. Purkinje neurons coexpressed by tdTOMATO and Ptprd were counted and averaged in at least four consecutive coronal brain sections from each mouse, and these data were treated as one biological sample. Data from four different mice were used in statistical analyses.
简言之，在9:00 h，用吸入异氟醚麻醉12周龄的Pcp2-cre; Ptprd

^{floxflox}

; Rosa26-LSL-tdTOMATO 和对照Pcp2-cre; Rosa26-LSL-tdTOMATO雄性小鼠，然后用生理盐水和

10 %

福尔马林灌注。收集脑切片（

25 μ m

厚），然后对Ptprd和tdTOMATO进行免疫荧光。简言之，将一系列脑切片（

5 %

正常驴）阻断 1 小时。然后，用兔抗 Ptprd 抗体（1:1,000 稀释度；A15713，ABclonal）在

4^{\circ} C

摇床上孵育脑切片过夜。第二天，用驴抗兔 Alexa Fluor 488（1:500；A21206，Invitrogen 公司）孵育脑切片 2 小时。将切片安装在载玻片上，用 DAPI 安装介质盖玻片。使用带有 OptiGrid 结构照明的 Leica TCS SP5 荧光显微镜拍摄荧光图像。对每只小鼠至少四个连续冠状脑切片中tdTOMATO和Ptprd共表达的浦肯野神经元进行计数并取平均值，这些数据被视为一个生物样本。统计分析使用了四只不同小鼠的数据。

Assessment of Purkinje neuron morphology
评估浦肯野神经元形态

Brain sections from 12-week-old Pcp2-cre; Ptprd

^{flox / f l o x};

Rosa26-LSL-tdTOMATO and control Pcp2-cre; Rosa26-LSL-tdTOMATO male mice were used for the assessment of Purkinje neuron morphology. The sections were coverslipped with Vectashield Mounting Medium (H-1000, Vector Laboratories) and imaged by a confocal laser scanning microscope(Leica DMI6000 B, Leica Microsystems). NIS-Elements-AR (Nikon) software was used for the analysis of Purkinje neuron morphology parameters, including cell number, cell body (soma) diameter, fiber length, synapse density and dendritic density.
用12周龄的Pcp2-cre; Ptprd

^{flox / f l o x};

Rosa26-LSL-tdTOMATO雄性小鼠和对照组Pcp2-cre; Rosa26-LSL-tdTOMATO雄性小鼠的脑切片评估浦肯野神经元形态。切片用 Vectashield Mounting Medium（H-1000，Vector Laboratories）盖玻片，并用激光共聚焦扫描显微镜（Leica DMI6000 B，Leica Microsystems）成像。NIS-Elements-AR (Nikon) 软件用于分析浦肯野神经元形态参数，包括细胞数、细胞体（体节）直径、纤维长度、突触密度和树突密度。

AP staining AP 染色

Human asprosin was cloned into pAPtag-5 (AP-TAG Kit B, GenHunter Corporation;Q202). HEK293T cells were grown up in 10 cm dishes and transfected with

15 μ g

of pAPtag-5_Asprosin according to the manufacturer’s protocol (FuGENE HD, Promega E2311). At 16 h after transfection, the media was replaced with serum-free DMEM media. AP-tagged asprosin was secreted into the media and collected over the course of 4 days. Media was concentrated to

< 500 μ

using 15 ml Amicon centrifugal filters. Brains of adult WT C57BL/6 mice were dissected and frozen in OCT and sectioned coronally. Frozen sections were washed with HBS buffer and then rinsed with HBAH buffer (GenHunter). AP-tagged asprosin protein was added to slides and incubated for 90 min at room temperature

(20 - 25^{\circ} C)

in a moist chamber. Sections were washed with HBAH buffer and then fixed for 15 s with acetone-formaldehyde fixative. Sections were then washed with HBS twice before being incubated in HBS at

65^{\circ} C

for 15 min to inactivate endogenous phosphatases. Sections were then stained with AP assay reagent S (GenHunter) at room temperature before stopping the reaction with PBS-10 mM EDTA.
人asprosin被克隆到pAPtag-5（AP-TAG Kit B，GenHunter Corporation；Q202）中。将 HEK293T 细胞培养在 10 cm 的平皿中，并按照生产商的方案（FuGENE HD, Promega E2311）用

15 μ g

pAPtag-5_Asprosin 转染。转染 16 h 后，将培养基更换为无血清 DMEM 培养基。AP 标记的asprosin分泌到培养基中，并在 4 天内收集。使用 15 ml Amicon 离心过滤器将培养基浓缩至

< 500 μ

。解剖WT C57BL/6成年小鼠的大脑并在OCT中冷冻，然后进行冠状切片。用 HBS 缓冲液清洗冷冻切片，然后用 HBAH 缓冲液（GenHunter）冲洗。在切片中加入 AP 标记的asprosin 蛋白，并在室温

(20 - 25^{\circ} C)

下于潮湿室中孵育 90 分钟。用 HBAH 缓冲液清洗切片，然后用丙酮-甲醛固定液固定 15 秒。然后用 HBS 冲洗切片两次，再在

65^{\circ} C

HBS 中孵育 15 分钟以灭活内源性磷酸酶。然后在室温下用 AP 检测试剂 S（GenHunter）对切片进行染色，最后用 PBS-10 mM EDTA 停止反应。

Metabolic caging, water and food intake assessment
代谢笼、水和食物摄入量评估

Metabolic data were recorded for 12-14-month-old

F b n 1^{+ / +}

and

F b n 1^{NPS / +}

mice, 12-week-old Ptprd

^{+ / +}

and Ptprd

^{- / -}

mice, 8-week-old Pcp2-cre; Ptprd

^{flox / f l o x}

and Pcp2-cre; Ptprd

^{+ + +}

mice and 16-week-old AgRP -cre

^{-}

;
记录了 12-14 个月大的

F b n 1^{+ / +}

和

F b n 1^{NPS / +}

小鼠、12 周大的 Ptprd

^{+ / +}

和 Ptprd

^{- / -}

小鼠、8 周大的 Pcp2-cre; Ptprd

^{flox / f l o x}

和 Pcp2-cre; Ptprd

^{+ + +}

小鼠以及 16 周大的 AgRP -cre

^{-}

小鼠的代谢数据；

ments were performed at the Cardiovascular Research Institute Mouse Metabolic and Phenotyping Core at CWRU (IACUC no. 2019-0029). Mice were housed with a 12-h light-dark cycle (7:00-19:00 h) at

22^{\circ} C

with controlled humidity. Metascreen software (v.2.3.15.11) controlled system data acquisition. Respirometry

({VO}_{2}, {VCO}_{2}, H_{2} O

vapor), activity and ad libitum food and water intake measures were collected individually using a Promethion metabolic cage system (Sable Systems). Gas analyzers were calibrated before each run. Gas measurements were multiplexed over eight cages and baselined to a cage-equivalent volume of room air twice per

5 - \min

cycle while maintaining a

21 \min^{- 1}

per cage, negative pressure-derived flow rate. Acquired data was processed using Macro Interpreter (v.2.34) running Macro v.2.33.3slice1hr. Energy expenditure was calculated using the Weir equation

^{49}

.
实验在CWRU心血管研究所小鼠代谢和表型核心进行（IACUC编号：2019-0029）。小鼠饲养在12小时光暗周期（7:00-19:00 h）、湿度可控的

22^{\circ} C

环境中。Metascreen 软件（v.2.3.15.11）控制系统数据采集。使用 Promethion 新陈代谢笼系统（Sable Systems）单独收集呼吸测量

({VO}_{2}, {VCO}_{2}, H_{2} O

蒸气）、活动和自由进食和饮水测量数据。每次运行前都对气体分析仪进行校准。气体测量在八个笼子中复用，并在每个

5 - \min

周期内两次与笼子等体积的室内空气进行基线连接，同时保持每个笼子

21 \min^{- 1}

的负压流速。获得的数据使用运行 Macro v.2.33.3slice1hr 的 Macro Interpreter (v.2.34) 进行处理。能量消耗用韦尔方程

^{49}

计算。

In some experiments, for manual measurement of food intake, 16-week-old diet-induced obese (DIO) mice were acclimated to a crushed high-fat diet (

60 %

calories from fat; TD.06414, Envigo Teklad) in solitary housing for 3 days. The diet was replenished, weighed and re-weighed every 24 h to establish food intake. In the chronic asprosin overexpression study, lean WT mice were acclimated to a dustless pellet diet (F0173, Bio-Serv) for 4 days in solitary housing, after which the food intake was measured. For manual measurement of water intake, lean WT mice were acclimated to sipper water bottles for up to 4 days in solitary housing. The 24 -h water intake was measured at baseline, during fasting and refeeding, and water re-access after overnight water deprivation. For assessment of isotonic ( 150 mM ) and hypertonic ( 500 mM ) saline intake, mice were acclimated to sipper bottles (with water) for 4 days, followed by a 7 -day paradigm consisting of sequential ad libitum access to isotonic saline (for 2 days), water (for 2 days), hypertonic saline (for 1 day) and water (for 2 days). Post acclimation, 48-h intake of isotonic and hypertonic saline was recorded. Additionally,
在一些实验中，为了手动测量食物摄入量，16周大的饮食诱导肥胖（DIO）小鼠在单独饲养条件下适应粉碎的高脂肪饮食（

60 %

来自脂肪的卡路里；TD.06414，Envigo Teklad）3天。每隔 24 小时补充食物、称重和复称一次，以确定食物摄入量。在慢性asprosin过表达研究中，瘦WT小鼠在单独饲养条件下适应无尘颗粒食物（F0173，Bio-Serv）4天，之后测量食物摄入量。为了手动测量水的摄入量，瘦WT小鼠在单独饲养条件下适应了长达4天的自吸式水瓶。在基线期、空腹和进食期间以及隔夜断水后再次进水时测量 24 小时的水摄入量。为了评估等渗（150毫摩尔）和高渗（500毫摩尔）生理盐水的摄入量，小鼠先在啜饮瓶（装有水）中适应4天，然后进行为期7天的范例实验，包括依次自由摄入等渗生理盐水（2天）、水（2天）、高渗生理盐水（1天）和水（2天）。适应后，记录 48 小时等渗和高渗盐水的摄入量。此外、
the water intake of mice was recorded in response to i.p. injection of hypertonic saline ( 3 M NaCl ), isotonic saline ( 154 mM ), mannitol (2 M) and

30 %

PEG as previously described

^{35}

. Then,

300 μ

PEG (

30 %

in PBS vehicle) was administered subcutaneously, and NaCl and mannitol (total volume,

150 μ l

) were administered by i.p.injection. Water intake was recorded for 2 h post isotonic saline, mannitol and hypertonic saline treatment and for 48 h post PEG treatment. Manual measurement of water intake was done in 8-week-old Pcp2-cre; Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{+ / +}

mice in all experiments. Manual food intake was done for AAV-transduced mice (21-week-old C57BL/6J mice) and 16-week-old

A g R P

-cre

^{-}; P t p r d^{flox / f l o x}

and

A g R P

-cre

^{+}; P t p r d^{flox / f l o x}

mice. For video analysis for lick frequency, 8-week-old control (Pcp2-cre, Ptprd

^{+ +}

) and knockout mice (Pcp2-cre; Ptprd

^{flox/flox)}

) were allowed to adapt to the water bottle and food in the testing cage for 2 days before experiments and given 5 min to acclimate to the environment before the measurement. Water availability was restricted overnight before the lick frequency measurement. On the day of the experiment, mice were given access to water in the testing cage and recorded on video for 20 min . Water licks were counted every minute to calculate the lick frequency per 1 min or per 5 min .
如前

^{35}

所述，记录小鼠在静脉注射高渗盐水（3 M NaCl）、等渗盐水（154 mM）、甘露醇（2 M）和

30 %

PEG时的水摄入量。然后，皮下注射

300 μ

PEG（

30 %

PBS载体）和氯化钠和甘露醇（总量，

150 μ l

）。记录等渗盐水、甘露醇和高渗盐水处理后 2 小时以及 PEG 处理后 48 小时的摄水量。在所有实验中，对 8 周龄的 Pcp2-cre; Ptprd

^{flox/flox}

和 Pcp2-cre; Ptprd

^{+ / +}

小鼠的摄水量进行人工测量。AAV转导的小鼠（21周大的C57BL/6J小鼠）和16周大的

A g R P

-cre

^{-}; P t p r d^{flox / f l o x}

和

A g R P

-cre

^{+}; P t p r d^{flox / f l o x}

小鼠的手动食物摄入量是通过手动操作完成的。为了对舔食频率进行视频分析，实验前让 8 周龄对照组（Pcp2-cre、Ptprd

^{+ +}

）和基因敲除组（Pcp2-cre；Ptprd

^{flox/flox)}

）小鼠适应测试笼中的水瓶和食物 2 天，并在测量前给它们 5 分钟适应环境。在舔食频率测量前，限制水的供应一夜。实验当天，让小鼠在测试笼中饮水，并用视频记录20分钟。每分钟对舔水次数进行计数，以计算每 1 分钟或每 5 分钟的舔水频率。

Overexpression of asprosin and recombinant asprosin treatment
天冬氨酸的过表达和重组天冬氨酸的处理

To test for overexpression of asprosin,12-14-week-old normal chow-fed lean Ptprd

^{- -}

and Ptprd

^{+ + +}

(WT) littermate male mice were i.v.injected in the tail vein with Ad5 and AAV as previously described

^{13 - 16}

. Mice injected with Ad5-empty (

5 \times 10^{10}

pfu per mouse) served as controls for experimental mice that received Ad5-IL-2-asprosin (

5 \times 10^{10}

pfu per mouse) containing an amino-terminal His-tagged human asprosin coding region preceded by an IL-2 signal peptide, under control of an EF1 promoter. The 12-week-old normal chow-fed lean WT (C57BL/6J) male mice were i.v. injected in the tail vein with AAV8 as previously described

^{15}

. Mice injected with AAV8-empty (

1 \times 10^{12} GC

per mouse) served as controls for experimental mice that received AAV8-IL-2-asprosin (

1 \times 10^{12}

GC per mouse) containing an N-terminal His-tagged human asprosin coding region preceded by an IL-2 signal peptide, under control of an EF1 promoter. The 12-week-old normal chow-fed lean WT littermate male mice were intranasally treated with

2 μ g

recombinant asprosin (in

15 μ l

saline; BioLegend, 761902), and water intake was manually recorded for 2 h post treatment.
为了检测天冬氨酸的过表达，按照之前描述的方法

^{13 - 16}

，在 12-14 周大的正常饲料喂养的瘦 Ptprd

^{- -}

和 Ptprd

^{+ + +}

(WT) 同窝雄性小鼠的尾静脉注射 Ad5 和 AAV。注射Ad5-empty（每只小鼠

5 \times 10^{10}

pfu）的小鼠作为实验小鼠的对照，接受Ad5-IL-2-asprosin（每只小鼠

5 \times 10^{10}

pfu）的小鼠在EF1启动子的控制下接受实验。按照先前的描述

^{15}

，12周大的正常饲料喂养的瘦WT（C57BL/6J）雄性小鼠尾静脉注射AAV8。注射了AAV8-empty（

1 \times 10^{12} GC

每只小鼠）的小鼠作为实验小鼠的对照，实验小鼠接受了AAV8-IL-2-asprosin（

1 \times 10^{12}

每只小鼠的GC），该AAV8-IL-2-asprosin含有N端His标记的人asprosin编码区，编码区前面是IL-2信号肽，受EF1启动子控制。用

2 μ g

重组asprosin（在

15 μ l

生理盐水中；BioLegend，761902）鼻内处理12周龄正常饲料喂养的瘦WT同窝雄性小鼠，并人工记录处理后2小时的水摄入量。

DREADD manipulation of Purkinje neurons
DREADD 操纵浦肯野神经元

To examine the metabolic effects of Purkinje neurons on water drinking, male Pcp2-cre mice were bilaterally injected with 200 nl of AAV-hSyn-DIO-hM3Dq-mCherry (RRID: Addgene_44361) or AAV-hSyn-DIO-hM4Di-mCherry (RRID: Addgene_50475)

^{23}

into the cerebellum lobes (V-VI: anteroposterior, -6.12 mm , mediolateral,

\pm 1.67 mm

, dorsoventral,

- 2.61 mm; V - VI

: anteroposterior, -6.62 mm , mediolateral,

\pm 2.07 mm

, dorsoventral, -2.70 mm ;VI-VII: anteroposterior, -7.02 mm , mediolateral,

\pm 1.77 mm

, dorsoventral, -2.96 mm ; VII-VIII: anteroposterior, -7.42 mm , mediolateral,

\pm 1.77 mm

, dorsoventral, -3.12 mm ) at 10 weeks of age. As a control, male Pcp2-cre mice were bilaterally injected with 200 nl of AAV-hSyn-DIO-mCherry into the cerebellum lobe VI-VII (anteroposterior, -7.02 mm , mediolateral,

\pm 1.77 mm

, dorsoventral, -2.96 mm ). After a 2-week recovery phase,

CNO (3 mg kg

) was i.p. injected in control (mCherry), activation (hM3Dq) and inactivation (hM4Di) mice at 09:30 h and 17:00 h , respectively. Food intake and water intake were monitored using the BioDAQ system. After a3-day rest period, both control and experimental mice were subjected to the same procedure but received an i.p. injection of saline. After we finished all the experiments, all mice were perfused with

10 %

formalin, and brains were dissected, sectioned and mounted. The mCherry signals were monitored under a fluorescent microscope for validation of injection accuracy. Only those mice with mCherry signals exclusively in the cerebellum lobes were included in analyses for feeding and drinking behavior.
为了研究浦肯野神经元对饮水的代谢影响，雄性Pcp2-cre小鼠双侧注射200 nl的AAV-hSyn-DIO-hM3Dq-mCherry（RRID：Addgene_44361）或AAV-hSyn-DIO-hM4Di-mCherry（RRID：Addgene_50475）

^{23}

到小脑叶（V-VI：前胸，-6.12毫米，内外侧，

\pm 1.67 mm

，背腹，

- 2.61 mm; V - VI

：前胸，-6.62毫米，内外侧，

\pm 2.07 mm

，背腹，-2.70毫米；VI-VII：前胸，-7.02毫米，内外侧，

\pm 1.77 mm

，背腹，-2.96毫米；VII-VIII：前胸，-7.42毫米，内外侧，

\pm 1.77 mm

，背腹，-3.12毫米）。作为对照，雄性Pcp2-cre小鼠在小脑VI-VII叶（前胸，-7.02毫米，内外侧，

\pm 1.77 mm

，背腹，-2.96毫米）双侧注射200毫升AAV-hSyn-DIO-mCherry。经过 2 周的恢复阶段后，分别于 09:30 h 和 17:00 h 向对照组（mCherry）、激活组（hM3Dq）和失活组（hM4Di）小鼠肌注

CNO (3 mg kg

)。使用 BioDAQ 系统监控食物摄入量和水摄入量。休息 3 天后，对照组和实验组小鼠接受相同的实验，但都要接受生理盐水的静脉注射。完成所有实验后，用

10 %

福尔马林对所有小鼠进行灌注，并对大脑进行解剖、切片和装片。在荧光显微镜下监测 mCherry 信号，以验证注射的准确性。只有在小脑叶中有mCherry信号的小鼠才被纳入进食和饮水行为的分析中。

The expression efficiency of DREADD AAV vectors was validated by immunohistochemical analysis of mCherry and Pcp2-GFP colocalization.
通过对 mCherry 和 Pcp2-GFP 共定位的免疫组化分析，验证了 DREADD AAV 载体的表达效率。

Optogenetic manipulation of Purkinje neurons
浦肯野神经元的光遗传学操作

To examine the acute effects of Purkinje neurons on water drinking, male Pcp2-cre mice were bilaterally injected with 200 nL of AAV-hSyn-ChR2-EYFP (RRID: Addgene_139283) into the cerebellum lobe (V-VI: anteroposterior, -6.62 mm , mediolateral,

\pm 2.07 mm

, dorsoventral, -2.70 mm ) at 10 weeks of age. As a control, male Pcp2-cre mice were bilaterally injected with 200 nl of AAV-hSyn-GFP (RRID: Addgene_50465) into the cerebellum lobe V-VI (anteroposterior, -6.62 mm , mediolateral,

\pm 2.07 mm

, dorsoventral, -2.70 mm ). In the same surgery, a dual-channel wireless optogenetic device (Neurolux) was implanted over lobe V-VI (refs. 24,50). After a 1-week recovery phase, mice with Pcp2 expressing ChR2 and GFP were monitored for food and water intake after yellow (

598 nM, 5 Hz, 3 s

on and 3 s off) or blue light (

473 nM, 5 Hz, 3 s

on and 3 s off) stimulation, respectively. Food intake and water intake were measured manually. Mice were allowed to adapt to the water bottle and food in the testing cage for 2 days before experiments and given 5 min to acclimate to the environment before light stimulation. After we finished all the experiments, all mice were perfused with

10 %

formalin and brains were dissected, sectioned and mounted. The YFP signals were monitored under a fluorescent microscope for validation of injection accuracy. Only those mice with YFP signals exclusively in the cerebellum lobes were included in analyses for feeding and drinking behavior. The expression efficiency of AAV-ChR2 vectors was validated by immunohistochemical analysis of YFP and Pcp2-tdTOMATO colocalization.
为了研究Purkinje神经元对饮水的急性影响，雄性Pcp2-cre小鼠在10周龄时双侧小脑叶（V-VI：前胸，-6.62 mm，内外侧，

\pm 2.07 mm

，背腹，-2.70 mm）注射200 nL AAV-hSyn-ChR2-EYFP（RRID：Addgene_139283）。作为对照，雄性Pcp2-cre小鼠在小脑V-VI叶（前胸，-6.62毫米，内外侧，

\pm 2.07 mm

，背腹，-2.70毫米）双侧注射200毫升AAV-hSyn-GFP（RRID：Addgene_50465）。在同一手术中，V-VI叶上植入了双通道无线光遗传装置（Neurolux）（参考文献 24,50）。经过 1 周的恢复阶段后，分别在黄光（

598 nM, 5 Hz, 3 s

亮起，3 秒后熄灭）或蓝光（

473 nM, 5 Hz, 3 s

亮起，3 秒后熄灭）刺激后监测表达 ChR2 和 GFP 的 Pcp2 小鼠的进食量和饮水量。食物摄入量和水摄入量由人工测量。实验前两天让小鼠适应试验笼中的水瓶和食物，并在光刺激前给小鼠5分钟适应环境。完成所有实验后，用

10 %

福尔马林对所有小鼠进行灌注，并对大脑进行解剖、切片和装片。在荧光显微镜下监测 YFP 信号，以验证注射的准确性。仅在小脑叶中有YFP信号的小鼠才被纳入进食和饮水行为分析。通过对YFP和Pcp2-tdTOMATO共定位的免疫组化分析，验证了AAV-ChR2载体的表达效率。

qPCR

For assessment of Ptprd expression in Purkinje and granule neurons, individual neurons from Pcp2-cre; Rosa26-LSL-tdTOMATO16-week-old mice were manually picked up by the pipette and ten neuron cells were combined for RNA extraction and reverse transcription using the Ambion Single-Cell-to-CT Kit (Ambion, Life Technologies) according to the manufacturer’s instruction. In brief, 10 ml Single Cell Lysis solution with DNase I was added to each sample, and then the entire cell contents were used for cDNA synthesis

(25^{\circ} C

for

10 \min, 42^{\circ} C

for 60 min and

85^{\circ} C

for 5 min ). Real-time qPCR was performed using iTaq Universal Probes Supermix (Bio-Rad) and the Bio-Rad CFX96 Real-Time system (Bio-Rad). Target gene primer sets were designed to be compatible with the Universal ProbeLibrary (Roche). The following primers were used:Ptprd-Fwd:5’-TCTGAGGCCAGGAACTGTTT-3’, Ptprd-Rev: 5’-TGGAACCCTTTTAGAGCTTGC-3’; Gapdh-Fwd: 5’-GG GTTCCTATAAATACGGACTGC-3’, Gapdh-Rev: 5’-CCATTTTGTC TACGGGACGA-3’.
为了评估Ptprd在浦肯野神经元和颗粒神经元中的表达，用移液管从Pcp2-cre; Rosa26-LSL-tdTOMATO16周龄小鼠中人工拾取单个神经元，并按照制造商的说明使用Ambion Single-Cell-to-CT Kit（Ambion，Life Technologies）合并10个神经元细胞进行RNA提取和反转录。简言之，每个样本加入 10 ml 含有 DNase I 的单细胞裂解液，然后将整个细胞内容物用于 cDNA 合成

(25^{\circ} C

for

10 \min, 42^{\circ} C

for 60 min and

85^{\circ} C

for 5 min )。使用 iTaq Universal Probes Supermix (Bio-Rad) 和 Bio-Rad CFX96 Real-Time 系统 (Bio-Rad) 进行实时 qPCR 分析。目的基因引物组的设计与通用探针库（Universal ProbeLibrary，Roche）兼容。使用的引物如下：Ptprd-Fwd:5'-TCTGAGGCCAGGAACTGTTT-3', Ptprd-Rev: 5'-TGGAACCCTTTTAGAGCTTGC-3'; Gapdh-Fwd：5'-GG GTTCCTATAAATACGGACTGC-3'，Gapdh-Rev：5'-CCATTTTGTC TACGGGACGA-3'。

In vivo anti-asprosin mAb treatment
体内抗天冬氨酸 mAb 治疗

WT (C57BL/6J) male mice (14 weeks old) were acclimated to metabolic cages with ad libitum food and water for 2 days before the experiment. The 24 -h water intake and urine output were recorded in fasting mice after an i.p. treatment of either anti-asprosin mouse mAb or the mouse IgG control ( 250 mg in

250 μ I

USP-grade saline per mouse).
实验前将 WT（C57BL/6J）雄性小鼠（14 周大）放入代谢笼中，自由进食和饮水 2 天。空腹小鼠在静脉注射抗天冬氨酸小鼠 mAb 或小鼠 IgG 对照组（每只小鼠 250 毫克，加入

250 μ I

USP 级生理盐水中）后，记录 24 小时的水摄入量和尿量。

Plasma and urine parameters
血浆和尿液参数

The Texas A&M Rodent Preclinical Phenotyping Core was used to determine plasma and urine osmolality. Osmolality was tested on the plasma and urine collected from overnight fasted single-housed mice with ad libitum access to water. Plasma and urine were centrifuged at

17, 000 g

for 2 min at room temperature (

20 - 25^{\circ} C

). Osmolality was measured using an OsmoPRO Multi-Sample Micro-Osmometer (Advanced Instruments). For urine volume and osmolality determination, mice were subjected to 24 h housing in metabolic cages under fasting conditions, with ad libitum access to water. Urine volume, plasma and urine
德克萨斯农工大学啮齿动物临床前表型核心用于测定血浆和尿液渗透压。检测渗透压的方法是从一夜禁食、自由饮水的单饲养小鼠身上收集的血浆和尿液。血浆和尿液在室温下以

17, 000 g

离心2分钟（

20 - 25^{\circ} C

）。使用 OsmoPRO 多样品微渗透压仪（Advanced Instruments）测量渗透压。为了测定尿量和渗透压，小鼠在禁食条件下在代谢笼中饲养 24 小时，自由饮水。尿量、血浆和尿液
osmolality were measured in 14 -week-old

F b n 1^{+ / +}

and

F b n 1^{NPS/+}

female mice, 14-month-old Ptprd

^{+ / +}

and Ptprd

^{- 1 -}

female mice and 12-week-old Pcp2-cre; Ptprd

^{flox/flox}

and Pcp2-cre; Ptprd

^{+ +}

mice.
测量了 14 周大的

F b n 1^{+ / +}

和

F b n 1^{NPS/+}

雌性小鼠、14 个月大的 Ptprd

^{+ / +}

和 Ptprd

^{- 1 -}

雌性小鼠以及 12 周大的 Pcp2-cre; Ptprd

^{flox/flox}

和 Pcp2-cre; Ptprd

^{+ +}

小鼠的渗透压。

A custom-built sandwich ELISA was used for measuring plasma asprosin in 14-week-old lean mice subjected to overnight water deprivation. Asprosin in

50 μ l

plasma was captured using a fully human anti-asprosin mAb ( 100 ng per well), developed and generated from a naive human phage display antibody library by panning against recombinant full-length human asprosin (Texas Therapeutics Institute at the University of Texas Health Science Center at Houston

^{15}

). A mouse anti-asprosin mAb, against human asprosin amino acids 106-134 (human profibrillin amino acids 2,838-2,865) served as the capture antibody ( 100 ng per well)

^{15}

. An anti-mouse secondary antibody linked to

HRP (1 : 10, 000)

was used to generate a signal, and mammalian-cell-produced recombinant mouse asprosin (BioLegend 762002) was used to generate a standard curve.
使用定制的夹心酶联免疫吸附法测定14周大瘦弱小鼠一夜缺水后的血浆天冬氨酸含量。血浆中的

50 μ l

rosprosin是用全人类抗asprosin mAb（每孔100纳克）捕获的，这种mAb是针对重组全长人类asprosin（休斯顿得克萨斯大学健康科学中心得克萨斯治疗研究所

^{15}

），从天真人类噬菌体展示抗体库中开发和生成的。小鼠抗asprosin mAb，针对人asprosin氨基酸106-134（人profibrillin氨基酸2,838-2,865）作为捕获抗体（每孔100纳克）

^{15}

。用与

HRP (1 : 10, 000)

连接的抗小鼠二抗产生信号，用哺乳动物细胞生产的重组小鼠asprosin（BioLegend 762002）产生标准曲线。

Motor function assays 运动功能检测

The Promethion metabolic cage system (Sable Systems), as described above, was used to record hourly pedestrian locomotor activity and wheel-running activity.
如上所述，Promethion 代谢笼系统（Sable Systems）用于记录行人每小时的运动活动和车轮运行活动。

{Cre}^{+}

control,

{Flox}^{+}

control and knockout mice (16-21 weeks old) were subjected to eight motor function assays (constant speed rotarod, forelimb grip strength, hindlimb grip strength, pole test, adhesive removal assay, vertical climb test, wire-hang test and tail-hang test) in a blinded fashion, as described previously

^{51 - 56}

. In addition, 26-week-old

{Cre}^{+}

control and knockout mice were subjected to the ErasmusLadder assay

^{30}

. In brief, the constant Speed Rotarod assay was performed using a Rotarod Rotamax Machine, and 16-20-week-old mice were placed at a constant speed of 4.0 r.p.m. Time latency over the course of three trials was recorded whereby mice were placed on a constant rotating rod for

3 \min (t r i a l 1), 5 \min

(trial 2 ) or 7 min (trial 3). Mice were given a minimum of 1 h recovery between each trial. Time latency was recorded when mice fell off the rod onto soft bedding (to prevent any injury to the mouse). A grip strength meter (Bioseb BIO-GS3) was used to measure the forelimb and hindlimb grip strength of 16 -week-old mice. Grip strength testing was repeated three times for each mouse, with a 10 min gap between the readings. The average of the three values was recorded.

{Cre}^{+}

对照组小鼠、

{Flox}^{+}

对照组小鼠和基因敲除小鼠（16-21周大）按照先前

^{51 - 56}

描述的方法，以盲法进行了八项运动功能测试（匀速转动、前肢握力、后肢握力、极点试验、去胶试验、垂直攀爬试验、线悬试验和尾悬试验）。此外，对26周大的

{Cre}^{+}

对照组和基因敲除组小鼠进行了伊拉斯谟阶梯试验

^{30}

。简而言之，恒速旋转试验是使用Rotarod Rotamax机器进行的，16-20周大的小鼠被置于4.0 r.p.m的恒定速度下，记录三次试验过程中的时间潜伏期，即小鼠被置于恒定旋转杆上

3 \min (t r i a l 1), 5 \min

（试验2）或7分钟（试验3）。每次试验之间至少给小鼠 1 小时的恢复时间。当小鼠从杆上跌落到柔软的垫料上时（以防小鼠受伤），记录下时间潜伏期。使用握力计（Bioseb BIO-GS3）测量 16 周龄小鼠的前肢和后肢握力。每只小鼠的握力测试重复三次，每次读数之间间隔 10 分钟。记录三个数值的平均值。

A tail-hang assay was performed to measure core strength and mobility

^{51}

. For the 6 -min assay, 20-week-old mice were taped by the end of their tails and recorded. The total amount of time the mouse hung passively and motionless was recorded. If mice attempted to climb their tails more than

20 %

of the total trial time they were removed from the analysis. All mice were observed afterward for any adverse effects during the test.
进行了尾悬试验，以测量核心力量和活动能力

^{51}

。在 6 -min 试验中，将 20 周大的小鼠尾部绑住并记录。记录小鼠被动悬挂不动的总时间。如果小鼠试图爬上尾巴的时间超过总试验时间的

20 %

，则将其从分析中剔除。之后观察所有小鼠在试验过程中是否有任何不良反应。

The pole test

^{56}

was used to evaluate the mouse’s ability to climb down and maneuver on a pole to descend into its home cage. A 56 cm metal pole with a diameter of 1.5 cm was wrapped in parafilm to create a grippable surface. For the task, 16-20-week-old mice were placed facing upwards on the pole, with their head 5 cm from the top of the pole. Mice then must orient themselves to face the downward direction and descend the pole. The time taken to descend two consecutive trials was recorded and averaged. The number of falls was also recorded and considered a ‘failure’ by the mouse.
杆子测试

^{56}

用于评估小鼠爬下杆子并在杆子上操作以进入笼子的能力。直径为 1.5 厘米的 56 厘米金属杆用保鲜膜包裹，以形成可抓握的表面。执行任务时，将 16-20 周大的小鼠面朝上放在杆子上，头部距离杆子顶端 5 厘米。然后，小鼠必须调整方向，使自己朝下，并从杆子上下降。记录连续两次下降所需的时间并取平均值。小鼠跌倒的次数也会被记录下来，并被视为 "失败"。

For the adhesive tape removal assay

^{54}

, a small piece of adhesive tape (

0.5 {cm}^{2}

) was placed on the left or right forepaw of 20-week-old mice and the time taken to remove the adhesive (maximum of 3 min ) and the number of efforts (mouth to foot and paw shakes) were assessed. This was then repeated on the right paw. Values were averaged between both paws.
在胶带去除试验

^{54}

中，将一小块胶带（

0.5 {cm}^{2}

）贴在 20 周龄小鼠的左前爪或右前爪上，评估去除胶带所需的时间（最长 3 分钟）和努力次数（嘴对脚和抖动爪子）。然后在右爪上重复这一过程。两个爪子的数值取平均值。

For vertical climb assay

^{55}, 18

-week-old mice were placed on a vertically hanging wire mesh ( 20 cm wide, 40 cm tall), with the bottom of the mesh hanging 30 cm above the table. After the mouse was released, the time for it to reach the top of the wire was recorded and
将一周大的小鼠放在垂直悬挂的铁丝网（宽 20 厘米，高 40 厘米）上，铁丝网底部高出桌面 30 厘米，进行垂直攀爬试验。释放小鼠后，记录小鼠爬到铁丝网顶端的时间，并将小鼠的爬行速度记录下来。
the number of foot slips were counted. A total of 120 s was given for the mouse to complete this task.
计算滑脚的次数。小鼠完成这项任务的总时间为 120 秒。

For the wire-hang assay, 20-week-old mice were placed upside down on a mesh wire and the time to fall was measured. The mesh bottom was approximately 40 cm above a soft underlay to prevent mice from jumping down but not too high that they would be harmed in the event of a fall. The latency to fall down was recorded. Wire hang latency of up to 5 min was recorded. The data point presents the time taken averaged across two trials per mouse.
在铁丝网悬挂试验中，将 20 周大的小鼠倒挂在铁丝网上，然后测量小鼠坠落的时间。网底距离软垫约 40 厘米，以防止小鼠跳下，但也不能太高，以免小鼠跌落时受到伤害。记录小鼠坠落的潜伏期。记录的挂线潜伏期最长可达 5 分钟。数据显示的是每只小鼠两次试验的平均时间。

Experiments using the ErasmusLadder (Noldus Information Technology) were performed as described previously

^{30}

. In brief, ErasmusLadder experiments were conducted over five subsequent days with one session per day and 72 trials per session. A single trial consisted of a mouse moving from one goalbox to the other, with a comprehensive list of metrics quantified during the trial including different gait patterns and cerebellum-dependent learning dynamics. Control and Pcp2-cre; Ptprd

d^{floxflix}

mice ( 26 weeks old) were trained on the ErasmusLadder for sessions one and two and then subjected to a challenge paradigm over three sessions (sessions three to five). In addition to the unperturbed trials, during the challenge paradigm, in ‘paired’ trials, one obstacle (randomly activated from a series of obstacles) is presented in the path of locomotion of the mouse (unconditioned stimulus), preceded by a high-pitched conditioning tone (conditioning stimulus) with an interstimulus interval of 250 ms . Absolute learning is determined by the post-perturbation step-time; that is, the time between the rungs just preceding the obstacle presented and the rung just after the obstacle during the paired trials (conditioning stimulus + unconditioned stimulus). Pre-perturbation step times are also measured during trials. In addition to cerebellum-dependent learning measures, the ErasmusLadder also recorded locomotor stepping or discrete gait dynamics, including short steps (step on a subsequent rung on the same side; that is

n + 1

), long steps (step over one rung on the same side; that is,

n + 2

) or missteps (step on lower, non-optimal stepping rungs).
使用 ErasmusLadder（Noldus 信息技术公司）进行的实验如前

^{30}

所述。简而言之，ErasmusLadder 实验是在随后的五天内进行的，每天一个时段，每个时段 72 次试验。单次试验由小鼠从一个目标箱移动到另一个目标箱组成，试验期间量化了一系列指标，包括不同的步态模式和依赖小脑的学习动态。对照组和Pcp2-cre; Ptprd

d^{floxflix}

小鼠（26周大）在伊拉斯谟阶梯上接受了第一和第二阶段的训练，然后在三个阶段（第三至第五阶段）接受了挑战范式的训练。在挑战范式中，除了无干扰试验外，在 "配对 "试验中，小鼠的运动路径上会出现一个障碍物（从一系列障碍物中随机激活）（非条件刺激），在此之前会出现一个高音调的条件音（条件刺激），刺激间隔为 250 毫秒。绝对学习由扰动后步进时间决定；即在配对试验（条件刺激 + 非条件刺激）中，障碍物出现前的梯级与障碍物出现后的梯级之间的时间。在试验过程中还会测量扰动前的步长时间。除了依赖小脑的学习测量外，ErasmusLadder 还记录了运动步态或离散步态动态，包括短步（踏上同一侧的后续梯级；即

n + 1

）、长步（踏过同一侧的一个梯级；即

n + 2

）或错步（踏上较低、非最佳的梯级）。

Ex vivo electrophysiology
体外电生理学

Purkinje neuron labeling and electrophysiology experiments were performed as previously described

^{57}

. In brief, for tdTOMATO or GFP labeling of Purkinje neurons, Rosa26-LSL-tdTOMATO mice or Rosa26-eGFP/Rpl10a were mated with the above-described Pcp2-cre and Pcp2-cre; Ptprd

^{flox/flox}

mice for the generation of Pcp2-cre; Rosa26-LSL-tdTOMATO mice, Pcp2-cre-eGFP-Rpl10a mice and Pcp2-cre; Ptprd

^{flox/flox};

Rosa26-LSL-tdTOMATO, respectively.
普肯列神经元标记和电生理学实验按照之前

^{57}

所述的方法进行。简言之，为了对浦肯野神经元进行tdTOMATO或GFP标记，Rosa26-LSL-tdTOMATO小鼠或Rosa26-eGFP/Rpl10a与上述Pcp2-cre和Pcp2-cre交配；Ptprd

^{flox/flox}

小鼠交配，分别产生Pcp2-cre; Rosa26-LSL-tdTOMATO小鼠、Pcp2-cre-eGFP-Rpl10a小鼠和Pcp2-cre; Ptprd

^{flox/flox};

Rosa26-LSL-tdTOMATO小鼠。

On the day of the electrophysiology recording experiment, mice were killed after being maintained under fed or water-deprived conditions. Then, the entire brains of the mice were removed and immediately submerged in an ice-cold sucrose-based cutting solution (adjusted to pH 7.3 ) containing

10 mM NaCl, 25 mM {NaHCO}_{3}

, 195 mM sucrose, 5 mM glucose,

2.5 mM KCl, 1.25 mM {NaH}_{2} {PO}_{4}, 2 mM

Na pyruvate,

0.5 mM {CaCl}_{2}

and

7 mM {MgCl}_{2}

, bubbled continuously with

95 % O_{2}

and

5 % {CO}_{2}

. The slices (

250 μ m

) were cut with sagittal and coronal planes using a Microm HM 650 V vibratome (Thermo Scientific) or VT1200 S vibratome (Leica) and recovered for 1 h at

34^{\circ} C

and then maintained at room temperature in artificial cerebrospinal fluid (aCSF, pH 7.3) containing

126 mM NaCl, 2.5 mM KCl, 2.4 mM {CaCl}_{2}

1.2 {mMNaH}_{2} {PO}_{4}, 1.2 {mMMgCl}_{2}, 11.1 mMglucose

and

21.4 mM {NaHCO}_{3}

saturated with

95 % O_{2}

and

5 % {CO}_{2}

. TOMATO

^{+}

neurons were visualized using epifluorescence and IR-DIC imaging on an upright microscope equipped with a moveable stage (MP-285, Sutter Instrument).
电生理记录实验当天，小鼠在喂食或缺水条件下被杀死。然后，取出小鼠的整个大脑，立即浸没在冰冷的蔗糖切片溶液（pH 值调至 7.3）中，该溶液含有

10 mM NaCl, 25 mM {NaHCO}_{3}

、195 mM 蔗糖、5 mM 葡萄糖、

2.5 mM KCl, 1.25 mM {NaH}_{2} {PO}_{4}, 2 mM

丙酮酸 Na 酯、

0.5 mM {CaCl}_{2}

和

7 mM {MgCl}_{2}

，并持续充泡

95 % O_{2}

和

5 % {CO}_{2}

。用 Microm HM 650 V 振动器（Thermo Scientific）或 VT1200 S 振动器（Leica）在矢状切面和冠状切面切割切片（

250 μ m

），在

34^{\circ} C

下恢复 1 小时，然后在室温下保存在含有

126 mM NaCl, 2.5 mM KCl, 2.4 mM {CaCl}_{2}

和

1.2 {mMNaH}_{2} {PO}_{4}, 1.2 {mMMgCl}_{2}, 11.1 mMglucose

的人工脑脊液（aCSF，pH 7.3），其中含有

126 mM NaCl, 2.5 mM KCl, 2.4 mM {CaCl}_{2}

、

1.2 {mMNaH}_{2} {PO}_{4}, 1.2 {mMMgCl}_{2}, 11.1 mMglucose

和

21.4 mM {NaHCO}_{3}

，饱和的

95 % O_{2}

和

5 % {CO}_{2}

。在配有可移动平台（MP-285，Sutter Instrument）的直立显微镜上，使用外荧光和红外-DIC成像技术观察番茄

^{+}

神经元。

For electrophysiological recording, brain slices were superfused at

34^{\circ} C

in oxygenated aCSF at a flow rate of

1.8 - 2 ml \min^{- 1}

. Patch pipettes with resistances of

3 - 5 M Ω

were filled with intracellular solution ( pH 7.3) containing 128 mM -gluconate,

10 mM KCl, 10 mM

HEPES, 0.1 mM EGTA,

2 mMMgCl_{2}, 0.05 mMNa

-GTP and 0.05 mMMg -ATP. Recordings were made using a MultiClamp 700B amplifier (Axon Instrument),
为了进行电生理记录，脑片在

34^{\circ} C

流速为

1.8 - 2 ml \min^{- 1}

的充氧脑脊液中超滤。在电阻为

3 - 5 M Ω

的贴片移液管中注入细胞内溶液（pH 7.3），其中含有 128 mM -葡萄糖酸盐、

10 mM KCl, 10 mM

HEPES、0.1 mM EGTA、

2 mMMgCl_{2}, 0.05 mMNa

-GTP 和 0.05 mMMg -ATP。使用 MultiClamp 700B 放大器（Axon Instrument）进行记录、
sampled using Digidata 1440A and analyzed offline with pClamp v.10.3 software (Axon Instruments). Series resistance was monitored during the recording, and the values were generally

< 10 M Ω

and were not compensated. Data were excluded if the series resistance increased dramatically during the experiment or without overshoot for the action potential. Currents were amplified, filtered at 1 kHz and digitized at 20 kHz . The current clamp was engaged to test neural firing frequency and resting membrane potential in control and Ptprd

^{Pcp2-cre}

knockout neurons after GFP (United States Biological cat. no. G8965-10E) or asprosin treatment (2-s puff, 30 nM ; BioLegend cat. no. 761902), or norepinephrine treatment (2-s puff,

5 μ M

; Cayman Chemicals cat. no.16673).
使用 Digidata 1440A 采样，并使用 pClamp v.10.3 软件（Axon Instruments）进行离线分析。在记录过程中监测串联电阻，其值通常为

< 10 M Ω

，且未进行补偿。如果串联电阻在实验过程中急剧增大或动作电位没有过冲，则排除数据。电流经过放大、1 kHz 滤波和 20 kHz 数字化处理。使用电流钳测试对照组和 Ptprd

^{Pcp2-cre}

敲除神经元在 GFP（United States Biological cat.

In another experiment, brain slices containing the Purkinje and granule neurons were visually identified and prepared using the same method described above. Purkinje and granule neuron depolarization and firing rate were recorded in response to asprosin ( 2 s puff treatment) or a physiologically irrelevant protein (GFP, 2 s puff) as described above. In some experiments, the aCSF solution also contained

1 μ M

tetrodotoxin and a cocktail of fast synaptic inhibitors, namely bicuculline (

50 μ M

; a GABA receptor antagonist) DAP-5 (

30 μ M

; an NMDA receptor antagonist) and CNQX (

30 μ M

; an NMDA receptor antagonist) to block the majority of presynaptic inputs, as described previously

^{11}

.
在另一项实验中，采用与上述相同的方法目测并制备含有浦肯野神经元和颗粒神经元的脑片。如上所述，记录普肯耶神经元和颗粒神经元对阿司匹林（2 s puff treatment）或生理无关蛋白（GFP，2 s puff）反应的去极化和发射率。在某些实验中，如前

^{11}

所述，aCSF 溶液还含有

1 μ M

河豚毒素和鸡尾酒快速突触抑制剂，即双库氨酸（

50 μ M

；一种 GABA 受体拮抗剂）、DAP-5（

30 μ M

；一种 NMDA 受体拮抗剂）和 CNQX（

30 μ M

；一种 NMDA 受体拮抗剂），以阻断大多数突触前输入。

Surgery for in vivo awake electrophysiology
体内清醒电生理学手术

Surgical procedures for headplate implant and craniotomy for awake in vivo electrophysiology in mice have been previously described

^{58}

. In brief, 5-month-old Pcp2-cre; Ptprd

^{+ / +}

and Pcp2-cre; Ptprd

^{flox/flox}

mice of both sexes were prepared for surgery with pre-emptive analgesics including slow-release buprenorphine

1 mg {kg}^{- 1}

and meloxicam

5 mg {kg}^{- 1}

. They were anesthetized with

3 %

isoflurane, and anesthesia was maintained with

2 %

isoflurane. Fur was removed from the top of the head and neck with depilatory cream (Nair). An incision was made in the skin to expose the skull anterior to bregma and posterior to the occipital bone. Bregma was identified and a craniotomy was performed 6.4 mm posterior and 1.3 mm lateral to bregma. A custom 3D-printed chamber was affixed to the skull surrounding the craniotomy and a custom headplate was affixed to the skull over bregma using

C

and

B

Metabond Adhesive Luting Cement (Parkell). The entire surface area of metabond was then covered in dental cement (A-MSystems; dental cement powder, cat. no. 525000 and solvent, cat. no. 526000). The recording chamber was filled with antibiotic ointment and sealed with a silicone cap. Mice were allowed to recover for 3 days with

5 mg {kg}^{- 1}

meloxicam for postoperative analgesics before experimentation was initiated.
用于小鼠清醒体内电生理学的头板植入术和开颅术的手术过程

^{58}

先前已有描述。简言之，5 个月大的 Pcp2-cre；Ptprd

^{+ / +}

和 Pcp2-cre；Ptprd

^{flox/flox}

雌雄小鼠在手术前都要做好镇痛准备，包括缓释丁丙诺啡

1 mg {kg}^{- 1}

和美洛昔康

5 mg {kg}^{- 1}

。用

3 %

异氟醚对它们进行麻醉，并用

2 %

异氟醚维持麻醉。用脱毛膏（Nair）去除头顶和颈部的毛发。切开皮肤，露出前囟门前和枕骨后的头骨。确定囟门后，在囟门后 6.4 毫米和外侧 1.3 毫米处进行开颅手术。使用

C

和

B

Metabond粘合剂（Parkell）将定制的3D打印室粘贴在开颅手术周围的颅骨上，并将定制的头板粘贴在前囟上方的颅骨上。然后在整个 Metabond 表面覆盖牙科水泥（A-MSystems；牙科水泥粉末，猫科编号：525000 和溶剂，猫科编号：525000）。525000 和溶剂，订货号：526000）。526000).在记录室内注入抗生素软膏，并用硅胶帽密封。小鼠术后服用

5 mg {kg}^{- 1}

美洛昔康镇痛剂恢复 3 天后再开始实验。

In vivo awake electrophysiology
体内清醒电生理学

The mice were placed on a foam wheel and the implanted headplate was affixed to a rigid frame on the day of recording. The antibiotic ointment was removed from the chamber and replaced with

0.9 %

saline solution. Tungsten electrodes of 5-8

M Ω

resistance (Thomas Recording) were lowered into the brain using a motorized micromanipulator (MP-225; Sutter Instrument) to record from individual neurons. Signals were amplified, bandpass-filtered from 0.3-13 kHz (ELC-03XS amplifier, NPI Electronic Instruments) and digitized (CED Power 1401, CED). Signals were recorded and spike sorted with Spike2 software (CED). Purkinje cells were identified by the presence of both complex spikes and simple spikes as well as distance from the surface of the brain. Recordings used in the analyses had a duration of about 30 s .
记录当天，将小鼠放在泡沫轮上，并将植入的头板固定在硬质框架上。从腔体中取出抗生素软膏，换上

0.9 %

生理盐水。使用电动微型机械手（MP-225；Sutter Instrument）将电阻为 5-8

M Ω

的钨电极（Thomas Recording）放入大脑，记录单个神经元的信号。信号经过放大、0.3-13 kHz 带通滤波器（ELC-03XS 放大器，NPI Electronic Instruments）和数字化（CED Power 1401，CED）处理。使用 Spike2 软件（CED）记录信号并进行尖峰分类。根据是否存在复杂尖峰和简单尖峰以及与大脑表面的距离来识别浦肯野细胞。分析中使用的记录持续时间约为 30 秒。

Fiber photometry 纤维光度测量

To record the activity of Purkinje neurons in freely moving mice, male Pcp2-cre or Pcp2-cre; Ptprd

^{floxfflox}

mice ( 10 weeks of age) were anesthetized by isoflurane and received stereotaxic injections of 200 nl AAV-FLEX-GCaMP7f virus (Addgene, 104492; 2.6

\times 10^{12} vg {ml}^{- 1}

) into lobe V-VI of the cerebellum (anteroposterior, -6.62 mm , mediolateral,
为了记录自由活动小鼠Purkinje神经元的活动，雄性Pcp2-cre或Pcp2-cre; Ptprd

^{floxfflox}

小鼠（10周龄）被异氟烷麻醉，并接受立体定向注射200 nl AAV-FLEX-GCaMP7f病毒（Addgene, 104492; 2.6

\times 10^{12} vg {ml}^{- 1}

）注射到小脑 V-VI 叶（前胸，-6.62 毫米，内外侧，-6.62 毫米）、
+2.07 mm , dorsoventral, -2.70 mm ) using a stereotaxic instrument with non-puncture ear bars (RWD Life Science). During the same surgery, an optical fiber (fiber core,

400 μ m; 0.39 NA; M 3

thread titanium receptacle; RWD Life Science) was implanted over lobe V-VI anteroposterior, -6.62 mm , mediolateral, +2.07 mm , dorsoventral, -2.65 mm ). During the same surgery, a stainless steel i.c.v. cannulas (RWD) were inserted into the lateral ventricles (i.c.v. coordinates without angle: anteroposterior, +0.34 mm , mediolateral, -1.00 mm , dorsoventral, -2.30 mm ). Optical fibers and cannulas were fixed to the skull by using dental acrylic. Mice were individually housed for at least 3 weeks post-surgery before acclimating to the investigator’s handling for 1 week before the recordings.
+2.07毫米，背腹侧，-2.70毫米）。在同一手术中，将一根光纤（光纤芯，

400 μ m; 0.39 NA; M 3

螺纹钛托架；RWD 生命科学公司）植入 V-VI 叶（前胸，-6.62 毫米，内外侧，+2.07 毫米，背腹，-2.65 毫米）。在同一手术中，将不锈钢静脉插管（RWD）插入侧脑室（不含角度的静脉坐标：前胸，+0.34 毫米，内外侧，-1.00 毫米，背腹，-2.30 毫米）。光纤和插管用牙科丙烯酸固定在头骨上。小鼠在手术后至少单独饲养 3 周，然后在记录前 1 周适应研究人员的操作。

Mice were allowed to adapt to the tethered patchcord for 2 days before experiments and given 5 min to acclimate to the tethered patchcord before any recording occurred. Fiber photometry recordings of Purkinje neurons were done in mice under fed conditions without food. All the control or Ptprd

^{Pcp2}

knockout mice received i.c.v. injection of GFP (as a control) and 10 ng asprosin (in

1 μ

l saline) or i.v. injection (in the lateral tail vein) of saline (

50 μ l

) and

20 μ g

asprosin (in

50 μ l

saline) on different days. There was a 1-week washout period between each injection. Each mouse was recorded for a

5 - \min

baseline, 15 min post i.c.v.injections and 30 min posti.v. injections. All mice were returned to their home cages, and food intake and water drinking were monitored for 24 h post i.c.v. injections. GCaMP7 fluorescent response of Pcp2-cre neurons was also recorded for up to 35 min in response to hypertonic stress ( 3 M NaCl and 2 M mannitol and hypovolemic stress (30% PEG).
实验前让小鼠适应系留跳线 2 天，并在记录前给小鼠 5 分钟适应系留跳线。小鼠在不进食的情况下进行Purkinje神经元的纤维光度记录。所有对照组或 Ptprd

^{Pcp2}

基因敲除小鼠都在不同的日期接受了 GFP（作为对照）和 10 ng asprosin（在

1 μ

l 生理盐水中）的静脉注射，或生理盐水（

50 μ l

）和

20 μ g

asprosin（在

50 μ l

生理盐水中）的静脉注射。每次注射之间有 1 周的冲洗期。记录每只小鼠的

5 - \min

基线、静脉注射后 15 分钟和静脉注射后 30 分钟。将所有小鼠关回笼子，监测注射后24小时的进食和饮水情况。在高渗应激（3 M NaCl和2 M甘露醇）和低血容量应激（30% PEG）下，记录Pcp2-cre神经元的GCaMP7荧光反应长达35分钟。

The fiber photometry recording was carried out using a commercial device (RWD Life Science) as previously described

^{59}

. For each recording, continuous

30 μ W

blue LED at 470 nm and

15 μ W

UVLED at 410 nm served as excitation light sources, driven by an R810 dual-color multichannel fiber photometry system (RWD Life Science). GCaMP7 calcium signals and UV autofluorescent signals were collected through the same fibers back to the R810 system. We derived the values of GCaMP fluorescence change

(Δ F / F_{n})

by calculating

(F_{470} - F_{0}) / F_{0}

, where

F_{0}

is the 5 min average baseline fluorescence of the

F_{470}

channel before i.c.v. injection. The

F_{410}

channel is used as an isosbestic fluorescence channel; we derived the values of isosbestic fluorescence change

(Δ F / F_{n})

by calculating

(F_{410} - F_{0}) / F_{0}

, where

F_{0}

is the 5 min average of the baseline fluorescence of the

F_{410}

channel before the i.c.v. injection.
光纤光度记录是使用一种商用设备（RWD 生命科学公司）进行的，如前

^{59}

所述。每次记录时，由 R810 双色多通道光纤测光系统（RWD Life Science）驱动，470 nm 的连续

30 μ W

蓝色 LED 和 410 nm 的

15 μ W

紫外 LED 作为激发光源。GCaMP7 钙信号和紫外自发荧光信号通过相同的光纤收集，并返回 R810 系统。我们通过计算

(F_{470} - F_{0}) / F_{0}

得出 GCaMP 荧光变化值

(Δ F / F_{n})

，其中

F_{0}

是静脉注射前

F_{470}

通道的 5 分钟平均基线荧光。

F_{410}

通道被用作等光荧光通道；我们通过计算

(F_{410} - F_{0}) / F_{0}

得出等光荧光变化值

(Δ F / F_{n})

，其中

F_{0}

是静脉注射前

F_{410}

通道基线荧光的 5 分钟平均值。

Quantification and statistical analysis
量化和统计分析

All results are presented as means

\pm

s.e.m. Age-matched and sex-matched mice were assigned to groups randomly for all experiments. In general, data distribution was assumed to be normal but this was not formally tested. No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications

^{12 - 16}

. Data collection was done in a blinded fashion, using pCLAMP v.10.3 for electrophysiology experiments, Spike2 v. 10 for data collection and spike sorting and Metascreen software (v.2.3.15.11) controlled system for data acquisition from Promethion Metabolic cages. Statistical significance of continuous data was tested using Student’s

t

-tests or one-way or two-way ANOVA, and mixed-effects analysis, when appropriate, followed by the Bonferroni multiple test corrections post hoc analysis, Šídák’s multiple comparisons test and Tukey’s multiple comparisons test using GraphPad Prism 8 and 10. Repeated measures analysis was used in experiments that involved multiple measures of the same variable. For in vivo electrophysiology, calculations of spike features were performed using custom Matlab scripts (Matlab R2023a), statistical tests were performed using GraphPad 8 , comparisons were made using unpaired

t

-tests with Welch’s correction and

P

values adjusted for multiple comparisons using the Bonferroni method. For all experiments, experimenters were blind to the genotype of mice while running the experiments, except in the manual food and water intake measurement experiments in which data collection and analysis were not performed blind to the conditions
所有结果均以平均值

\pm

s.e.m表示。在所有实验中，年龄匹配和性别匹配的小鼠被随机分配到各组。一般情况下，数据分布被假定为正态分布，但没有进行正式测试。没有使用统计方法预先确定样本量，但我们的样本量与之前发表的

^{12 - 16}

中报告的样本量相似。数据收集以盲法进行，使用 pCLAMP v.10.3 进行电生理实验，使用 Spike2 v. 10 进行数据收集和尖峰分类，使用 Metascreen 软件（v.2.3.15.11）控制系统从 Promethion 代谢笼中采集数据。使用 GraphPad Prism 8 和 10 进行连续数据的统计意义检验，采用学生

t

检验或单向或双向方差分析，适当时采用混合效应分析，然后进行 Bonferroni 多重检验校正事后分析、Šídák 多重比较检验和 Tukey 多重比较检验。重复测量分析用于对同一变量进行多次测量的实验。在体内电生理学实验中，尖峰特征的计算使用定制的 Matlab 脚本（Matlab R2023a）进行，统计检验使用 GraphPad 8 进行，比较使用韦尔奇校正的非配对

t

检验，

P

值使用 Bonferroni 方法进行多重比较调整。在所有实验中，实验人员在进行实验时都对小鼠的基因型视而不见，只有在手动测量食物和水摄入量的实验中，数据收集和分析不在视而不见的条件下进行。
of the experiments. Additionally, experimenters were blind to the genotype of mice during data collection from Promethion metabolic caging experiments, qPCR studies, electrophysiological recordings and behavioral studies. Power analysis was not done to predetermine the sample size; instead,

n \geq 6

mice of each sex were used for in vivo studies. The sample size was determined based on our previous publications assessing the effects of asprosin overexpression, asprosin deficiency, Ptprd genetic knockout and pharmacological silencing on the metabolism and behavior of mice

^{12 - 16}

. Data were excluded if a mouse showed signs of sickness (mange, sudden weight loss, less than normal food intake or fighting wounds in group housing). Additionally, manually determined water intake data were excluded if the sipper bottle was found to be leaky, and urine samples with contamination of feces and/or food particles were also excluded. The level of statistical significance was set at

α = 0.05

. Confirmation of successful GCaMP7 expression in Pcp2 neurons, double labeling of Pcp2 and Ptprd was performed by immunohistochemical analysis of one randomly chosen mouse per group.
在实验过程中，实验人员对小鼠的基因型视而不见。此外，实验人员在收集丙硫磷代谢笼养实验、qPCR 研究、电生理记录和行为研究的数据时，对小鼠的基因型视而不见。没有进行功率分析来预先确定样本量，而是使用

n \geq 6

种性别的小鼠进行体内研究。样本量是根据我们以前发表的评估asprosin过表达、asprosin缺乏、Ptprd基因敲除和药物沉默对小鼠

^{12 - 16}

代谢和行为影响的文章确定的。如果小鼠出现生病迹象（疥疮、体重突然下降、进食量低于正常水平或在群居环境中出现打斗伤口），数据将被排除。此外，如果发现饮水器瓶子漏水，则排除人工测定的饮水量数据；如果尿液样本受到粪便和/或食物颗粒的污染，则排除尿液样本。统计显著性水平设定为

α = 0.05

。通过对每组随机选择的一只小鼠进行免疫组化分析，确认GCaMP7在Pcp2神经元中的成功表达、Pcp2和Ptprd的双重标记。

Editing 编辑

ChatGPT3.5 was used exclusively for language refinement. The authors reviewed and edited the output as needed and take full responsibility for the content of the publication.
ChatGPT3.5 完全用于语言完善。作者根据需要对结果进行了审核和编辑，并对出版物的内容负全部责任。

Reporting summary 报告摘要

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
有关研究设计的更多信息，请参阅本文链接的《自然组合报告摘要》。

Data availability 数据可用性

All data used in the analysis are available to any researcher for the purposes of reproducing or extending the analysis in source data files. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author. Source data are provided with this paper.
分析中使用的所有数据可供任何研究人员复制或扩展源数据文件中的分析。如需了解更多信息以及索取资源和试剂，请直接联系通讯作者，通讯作者将予以满足。本文提供源数据。

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Acknowledgements 致谢

We thank members of the Chopra lab for helpful suggestions and critical reading of the manuscript. This work was supported by the National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (DK130931, DK118290), the NIH National Institute of Neurological Disorders and Stroke (NINDS) (RO1NS119301, RO1NS127435), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (P5OHD103555) for use of the Cell and Tissue Pathogenesis Core and In Situ Hybridization Core (the BCM IDDRC) and the Harrington Discovery Institute at University Hospitals, Cleveland, Ohio. The Genotype-Tissue Expression (GTEx) Project is supported by the Common Fund of the Office of the Director of the NIH (additional funds were provided by the National Cancer Institute, National Human Genome Research Institute, National Heart, Lung, and Blood Institute, National Institute on Drug Abuse, National Institute of Mental Health and NINDS). The use of the Texas A&M Rodent Preclinical Phenotyping Core is acknowledged for the determination of plasma and urine osmolality. The Cardiovascular Research Institute Mouse Metabolic and Phenotyping Core of CWRU (IACUC no. 2019-0029) is acknowledged for the use of metabolic caging.
我们感谢乔普拉实验室成员提出的有益建议和对手稿的审阅。这项工作得到了美国国立卫生研究院（NIH）国家糖尿病、消化道和肾脏疾病研究所（NIDDK）（DK130931、DK118290）、美国国立卫生研究院国家神经疾病和中风研究所（NINDS）（RO1NS119301、RO1NS127435）、美国国立卫生研究院尤尼斯-肯尼迪-施莱佛国家儿童健康和人类发展研究所（P5OHD103555）、美国国立卫生研究院尤尼斯-肯尼迪-施莱佛国家儿童健康和人类发展研究所（P5OHD103555美国国立卫生研究院尤妮斯-肯尼迪-施莱佛国家儿童健康与人类发展研究所（NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development）（P5OHD103555），用于使用细胞与组织发病机制核心和原位杂交核心（BCM IDDRC）以及俄亥俄州克利夫兰大学医院哈灵顿发现研究所（Harrington Discovery Institute at University Hospitals, Cleveland, Ohio）。基因型-组织表达（GTEx）项目由美国国立卫生研究院（NIH）院长办公室共同基金（由美国国立癌症研究所、美国国立人类基因组研究所、美国国立心肺血液研究所、美国国立药物滥用研究所、美国国立精神卫生研究所和美国国立神经疾病研究所提供额外资金）支持。感谢德克萨斯农工大学啮齿动物临床前表型核心（Texas A&M Rodent Preclinical Phenotyping Core）对血浆和尿液渗透压的测定。感谢CWRU心血管研究所小鼠代谢和表型核心（IACUC编号：2019-0029）使用代谢笼。

Author contributions 作者供稿

I.M. and B.F. performed water intake studies. B.F. performed immunohistochemistry. J.C.B. carried out the alkaline phosphatase tag staining. B.F. performed fiber photometry recording experiments. I.M., B.B., M.A.R., A.M., A.H., S.B., A. Sharp, C.P., B.K. and A.L. performed motor function and learning assays. A.M.B., L.H.K. and T.L. performed in vivo electrophysiology and MATLAB analysis. I.M., B.B. and B.F. maintained mouse colonies and conducted genotyping of mice. I.M., Y.H. and A.R.C. defined the methodology. I.M., B.F. and A. Sathyanesan conducted the analysis. I.M. and A.R.C. conceptualized the idea. I.M. wrote the original draft. A. Sathyanesan, R.V.S., Y.H. and A.R.C. supervised, provided funding and reviewed and edited the manuscript.
I.M. 和 B.F. 进行了水摄入量研究。B.F. 进行了免疫组化。J.C.B. 进行碱性磷酸酶标记染色。B.F. 进行纤维光度记录实验。I.M.、B.B.、M.A.R.、A.M.、A.H.、S.B.、A. Sharp、C.P.、B.K.和 A.L. 进行了运动功能和学习实验。A.M.B.、L.H.K.和T.L.进行了体内电生理学和MATLAB分析。I.M.、B.B.和B.F.维护小鼠群落并对小鼠进行基因分型。I.M.、Y.H. 和 A.R.C. 确定了研究方法。I.M.、B.F. 和 A. Sathyanesan 进行分析。I.M. 和 A.R.C. 构思。I.M. 撰写原稿。A. Sathyanesan、R.V.S.、Y.H. 和 A.R.C. 监督、提供资金并审阅和编辑手稿。

Competing interests 竞争利益

A.R.C. has been awarded asprosin-related patents and is a co-founder and equity holder of Aceragen and Recall Therapeutics. The other authors declare no competing interests.
A.R.C.已获得天冬氨酸相关专利，是 Aceragen 和 Recall Therapeutics 的共同创始人和股权持有人。其他作者不声明任何利益冲突。

Additional information 其他信息

Extended data is available for this paper at
本文的扩展数据见
https://doi.org/10.1038/s41593-024-01700-9IF: 21.2 Q1 .
https://doi.org/10.1038/s41593-024-01700-9 .
Supplementary information The online version
补充信息在线版本
contains supplementary material available at
载有补充材料，可在
https://doi.org/10.1038/s41593-024-01700-9IF: 21.2 Q1 .
https://doi.org/10.1038/s41593-024-01700-9 。
Correspondence and requests for materials should be addressed to Yanlin He or Atul R. Chopra.
通讯和资料索取请联系：Yanlin He 或 Atul R. Chopra。

Peer review information Nature Neuroscience thanks Albert Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
同行评议信息《自然-神经科学》感谢 Albert Chen 和其他匿名审稿人对这项工作的同行评议所做的贡献。

Reprints and permissions information is available at www.nature.com/reprints.
转载和许可信息请访问www.nature.com/reprints。

Normal chow diet 普通饲料

High fat diet 高脂肪饮食

Extended Data Fig.

1 ∣

AgRP neuron-specific Ptprd deletion does not affect water intake. (a) Body weight of 5-week-old male and female Ptprd

^{f / f}

and AgRPcre;Ptprd

^{ff}

mice maintained on ad libitum fed normal chow diet (

n = 8

males and 9 females/group). (b-c) 24 h food and water intake of 14-week-old male and 8 -weekold female Ptprd

^{f / f}

and AgRP-cre;Ptprd

^{f / f}

mice maintained on ad libitum normal chow diet (

n = 4 /

group). (d) Body weight of female Ptprd

^{f / f}

and AgRP-cre;Ptprd

^{f/f}

mice on normal chow (week

5, n = 10 /

group), and after 5 weeks of high fat diet (Week 10;

n = 8

/group). (e-g) Daily Food intake (E), cumulative water intake (F)
扩展资料图

1 ∣

AgRP 神经元特异性 Ptprd 缺失不影响水的摄入。(a) 5 周龄雄性和雌性 Ptprd

^{f / f}

及 AgRPcre;Ptprd

^{ff}

小鼠的体重（

n = 8

雄性和 9 只雌性/组）。(b-c）14 周龄雄性 Ptprd

^{f / f}

和 8 周龄雌性 AgRP-cre;Ptprd

^{f / f}

小鼠（

n = 4 /

组）24 小时的食物和水摄入量。(d）雌性 Ptprd

^{f / f}

和 AgRP-cre;Ptprd

^{f/f}

小鼠在正常饲料（

5, n = 10 /

周组）和高脂肪饲料 5 周后（第 10 周；

n = 8

/组）的体重。(e-g）每日食物摄入量（E）、累计饮水量（F）

and average daily water intake (G) of 14-week-old Ptprd

^{f/f}

and AgRP-cre;Ptprd

^{f / f}

mice, measured over 4 days using the Promethion metabolic system (

n = 6

/ group). Error bars represent mean

\pm

s.e.m.

^{*}

<0.05,

^{* *} p < 0.01,^{* * *} p < 0.001

, and

^{* * * *}

< 0.0001; by Two-way ANOVA followed by Sidak’s multiple comparison in A-E, 2-Way ANOVA (effect of genotype) in F and two tailed unpaired Student’s t -test in G. Raw data values, P values and details of statistical tests in Extended source data 1.
和 14 周大的 Ptprd

^{f/f}

和 AgRP-cre;Ptprd

^{f / f}

小鼠的平均日摄水量（G），使用 Promethion 代谢系统（

n = 6

/组）在 4 天内测定。误差条代表平均值

\pm

s.e.m.

^{*}

<0.05，

^{* *} p < 0.01,^{* * *} p < 0.001

和

^{* * * *}

<0.0001；A-E中采用双向方差分析，然后进行Sidak多重比较，F中采用双向方差分析（基因型的影响），G中采用双尾非配对学生t检验。原始数据值、P值和统计检验细节见扩展资料1。

Purkinje neuron response to asprosin
浦肯野神经元对阿司匹林的反应

E.
brain section showing Purkinje neurons recorded in different places. (d-e) Data analysis of cerebellar Purkinje neurons firing frequency and resting membrane potential in response to puff ( 2 s ) treatment of 30 nM recombinant asprosin (

n = 21

neurons with baseline firing from 3 male mice in the range of

1 - 6 Hz, n = 14

neurons from 3 male mice with baseline firing in the range of

10 - 30, n = 38

neurons from 3 male mice with baseline firing in the range of

46 - 64 Hz

^{*} p < 0.05

^{* *} p < 0.01,^{* * *} p < 0.001

, and

^{* * *} p < 0.0001

; by Two-tailed paired Student’s t-test. Raw data values, P values and details of statistical tests in Extended source data 2.
脑切片，显示在不同位置记录到的浦肯野神经元。(d-e）小脑浦肯野神经元的发射频率和静息膜电位对 30 nM 重组阿司匹林扑灭（2 秒）处理的反应的数据分析（3 只雄性小鼠的

n = 21

神经元基线发射在

1 - 6 Hz, n = 14

范围内，3 只雄性小鼠的

10 - 30, n = 38

神经元基线发射在

46 - 64 Hz

范围内）。

^{*} p < 0.05

、

^{* *} p < 0.01,^{* * *} p < 0.001

和

^{* * *} p < 0.0001

；通过双尾配对学生 t 检验。原始数据值、P 值和统计检验详情见扩展源数据 2。

Rawdatavalues Pvalues and
原始数据值 P 值和

Extended Data Fig.

2 ∣

Cerebellar Purkinje neurons, but not granule neurons, are responsive to asprosin. (a) Data analysis of cerebellar granule neurons resting membrane potential in response to puff (2s) treatment of 30 nM recombinant asprosin (

n = 19

neurons from 3 male mice). (b) Data analysis of cerebellar granule neurons resting membrane potential in response to puff ( 2 s ) treatment of 30 nM recombinant asprosin, in the presence of cocktail synaptic blockers including

1 μ M

tetrodotoxin (TTX,

30 μ MAP - 5, 30 μ MCNQX

and

50 μ M

bicuculline;

n = 17

2 ∣

小脑浦肯野神经元而非颗粒神经元对阿司匹林有反应。(a) 小脑颗粒神经元静息膜电位对 30 nM 重组阿司匹林（来自 3 只雄性小鼠的

n = 19

神经元）扑杀（2 秒）处理的响应数据分析。(b) 在鸡尾酒突触阻断剂（包括

1 μ M

河豚毒素 (TTX)、

30 μ MAP - 5, 30 μ MCNQX

和

50 μ M

双谷氨酸；来自 3 只雄性小鼠的

n = 17

神经元）存在的情况下，小脑颗粒神经元静息膜电位对 30 nM 重组阿司匹林扑粉（2 秒）处理的反应的数据分析。冠状图

CNO+ AAV-hSyn-DIO-hM3Dq-mCherry Treatment
CNO+ AAV-hSyn-DIO-hM3Dq-mCherry 处理

CNO+ AAV-hSyn-DIO-mCherry Treatment
CNO+ AAV-hSyn-DIO-mCherry 处理

Extended Data Fig.

3 ∣

Chemogenetic activation of Purkinje neurons enhances water intake without affecting food intake or body weight. (a-i) Mean

\pm

s.e.m. 24 h water intake (A,D,G) food intake (B,E,H) and body weight (C,F,I) post intraperitoneal injection of CNO (

3 mg / kg

, twice/day) or saline in Pcp2cre male mice stereotaxically injected with Cre-dependent AAV expressing hSyn-DIO-hM3Dq-mCherry in lobe IV-V, VII-VIII and VIII-IX of cerebellum (

n = 6

mice/treatment/brain site). (j-1) CNO treatment does not cause hyperdipsia
扩展数据图

3 ∣

Purkinje神经元的化学激活可提高水分摄入量，而不影响食物摄入量或体重。(a-i）平均

\pm

s.e.m.腹腔注射 CNO（

3 mg / kg

，两次/天）或生理盐水后的 24 小时摄水量（A,D,G）、摄食量（B,E,H）和体重（C,F,I）。(j-1) CNO 治疗不会导致多尿症

in absence of hSyn-DIO-hM3Dq-mCherry. Mean

\pm

s.e.m. 24 h food and water intake (number of drinking bouts, time spent drinking and water intake) post intraperitoneal injection of CNO (

3 mg / kg

, twice/day) or saline in Pcp2cre male mice stereotaxically injected with Cre-dependent AAV expressing hSyn-DIOmCherry in lobe VII-VIII of cerebellum (

n = 6

mice/treatment).

^{*} p < 0.05,^{* *} p < 0.01

^{* * *} p < 0.001

, and

^{* * * *} p < 0.0001

; by unpaired two-tailed Student’s t-test. Raw data values, P values and details of statistical tests in Extended source data 3.
在没有 hSyn-DIO-hM3Dq-mCherry 的情况下。在小脑 VII-VIII 叶立体定向注射表达 hSyn-DIOmCherry 的 Cre 依赖性 AAV 的 Pcp2cre 雄性小鼠（

n = 6

小鼠/处理），腹腔注射 CNO（

3 mg / kg

，两次/天）或生理盐水后 24 小时食物和水摄入量（饮水次数、饮水时间和水摄入量）的平均值

\pm

s.e.m. 。

^{*} p < 0.05,^{* *} p < 0.01

、

^{* * *} p < 0.001

和

^{* * * *} p < 0.0001

；采用非配对双尾学生 t 检验。原始数据值、P 值和统计检验详情见扩展源数据 3。

우

Extended Data Fig. 4|Purkinje neuron-specific Ptprd deletion affects water, isotonic and hypertonic saline intake. (a-b) Water intake of 8-week-old Pcp2cre;Ptprd

^{+ / +}

and Pcp2cre;Ptprd

^{flox/flox}

female mice maintained on normal chow diet, measured using the Promethion metabolic system (

n = 5 /

^{+ / +}

and Pcp2-cre;Ptprd

^{foxffox}

female mice maintained on normal chow diet (

n = 5 /

group). (d-f) Cumulative food intake (D), hourly energy expenditure (E) and respiratory exchange ratio (F) of 8-week-old Pcp2-cre;Ptprd

^{+ / +}

and Pcp2cre;Ptprd

^{flox/flox}

female mice maintained on normal chow diet, measured using the Promethion metabolic system (

n = 5 /

group).
扩展资料图 4|Purkinje神经元特异性Ptprd缺失影响水、等渗和高渗盐水的摄入。(a-b）使用 Promethion 代谢系统测量的正常饲料饲养的 8 周龄 Pcp2cre;Ptprd

^{+ / +}

和 Pcp2cre;Ptprd

^{flox/flox}

雌性小鼠的水摄入量（

n = 5 /

组）。Pcp2cre;Ptprd

^{+ / +}

和 Pcp2-cre;Ptprd

^{foxffox}

雌性小鼠（

n = 5 /

组）的体重。(d-f）使用 Promethion 代谢系统测量的正常饲料饲养的 8 周龄 Pcp2-cre;Ptprd

^{+ / +}

和 Pcp2cre;Ptprd

^{flox/flox}

雌性小鼠（

n = 5 /

组）的累积食物摄入量（D）、每小时能量消耗（E）和呼吸交换比（F）。

(g-h) Lick frequency (counts per first minute, and cumulative counts per first 5 minutes of water access) of Pcp2cre;Ptprd

^{+ +}

(

n = 5

per group) and Pcp2cre;Ptprd

^{flox/flox}

male (

n = 4

per group) mice given re-access to water after overnight water deprivation. (i-j) 48 h isotonic and hypertonic ( 500 mM ) saline intake of Pcp2-cre;Ptprd

^{+ / +}

and Pcp2cre;Ptprd

^{fox / flox}

male mice (

n = 4

per group). Error bars represent mean

\pm

s.e.m.

^{*}

< 0.05,^{* *}

< 0.01,^{* * *} p < 0.001

, and

^{* * * *}

< 0.0001; by Student’s t-test (B, C,G-J) and two-way ANOVA (effect of genotype) in A,D,E and F. Raw data values, P values and details of statistical tests in Extended source data 4.
(g-h）Pcp2cre;Ptprd

^{+ +}

（每组

n = 5

）和 Pcp2cre;Ptprd

^{flox/flox}

雄性（每组

n = 4

）小鼠在过夜缺水后重新获得水的舔食频率（每第一分钟的计数和每获得水的前 5 分钟的累积计数）。(i-j）Pcp2-cre;Ptprd

^{+ / +}

和 Pcp2cre;Ptprd

^{fox / flox}

雄性小鼠（每组

n = 4

）48 小时等渗和高渗（500 mM）生理盐水摄入量。误差条代表平均值

\pm

s.e.m.

^{*}

< 0.05,^{* *}

< 0.01,^{* * *} p < 0.001

, and

^{* * * *}

< 0.0001; by Student's t-test (B, C,G-J) and two-way ANOVA (effect of genotype) in A,D,E and F. Raw data values, P values and details of statistical tests in Extended source data 4.

Extended Data Fig.

5 ∣

Purkinje neuron-specific Ptprd deletion does not protect from diet induced obesity. Weekly body weight change of Pcp2cre;Ptprd

^{+ + +}

(male:

n = 8

; female:

n = 5

) and Pcp2cre;Ptprd

^{flox/flox}

(male:

n = 7

;
扩展数据图

5 ∣

Purkinje 神经元特异性 Ptprd 缺失不能防止饮食引起的肥胖。Pcp2cre;Ptprd

^{+ + +}

（雄性：

n = 8

；雌性：

n = 5

）和 Pcp2cre;Ptprd

^{flox/flox}

（雄性：

n = 7

；雌性：

n = 7

）的每周体重变化：

n = 7

；

female:

n = 5

) mice maintained on high-fat diet (HFD) from 5-weeks-of-age. NS: not significant; by two-way ANOVA (effect of genotype). Raw data values, P values and details of statistical tests in Extended source data 5.
雌性：

n = 5

）小鼠从 5 周龄开始食用高脂饮食（HFD）。NS：不显著；采用双向方差分析（基因型的影响）。原始数据值、P 值和统计检验细节见扩展源数据 5。

Hypertonic saline 高渗盐水

Extended Data Fig. 7 |Purkinje neurons are unresponsive to traditional dipsogenic stimuli. (a-c) GCaMP7 fluorescent response of Pcp2cre neurons in response to hypertonic stress ( 3 M NaCl and 2 M mannitol) and hypovolemic stress (

30 %

polyethylene glycol;PEG;

n = 5

wild type mice in each treatment). (d-f) 2 h water intake (D,E) and 48 h water intake (F) of control (Pcp2-cre;Ptprd

^{+ / +}

) and knockout (Pcp2-cre;Ptprd

^{foxffox}

) mice injected with 3M NaCl (D), 2M mannitol
扩展资料图 7 |Purkinje 神经元对传统的致浸性刺激无反应。(a-c）Pcp2cre神经元对高渗应激（3 M NaCl和2 M甘露醇）和低血容量应激（

30 %

聚乙二醇；PEG；

n = 5

野生型小鼠在每种处理中）的GCaMP7荧光反应。(d-f）对照组（Pcp2-cre;Ptprd

^{+ / +}

）和基因敲除组（Pcp2-cre;Ptprd

^{foxffox}

）小鼠注射 3M NaCl (D)、2M 甘露醇 (D,E)和低血容量应激（

30 %

聚乙二醇；PEG
(E) and 30%PEG (F).

n = 6

Pcp2-cre;Ptprd

^{+ / +}

and 11 Pcp2-cre;Ptprd

^{fox/flox}

mice in (D) or 5 Pcp2-cre;Ptprd

^{+ / +}

and 11 Pcp2-cre;Ptprd

^{fox/flox}

mice in (E) and n = 12 Pcp2-cre;Ptprd

^{+ / +}

and

n = 11

Pcp2-cre;Ptprd

^{fox/flox}

mice in (F). Error bars represent mean

\pm

s.e.m.

^{*}

< 0.05,

^{* *}

< 0.01,^{* * *}

< 0.001

, and

^{* * * *}

p 0.0001 ; by two-tailed unpaired Student’s

t

-test. Raw data values, P values and details of statistical tests in Extended source data 7.
(E）和 30%PEG （F）。(D) 中的

n = 6

Pcp2-cre;Ptprd

^{+ / +}

和 11 只 Pcp2-cre;Ptprd

^{fox/flox}

小鼠或 5 只 Pcp2-cre;Ptprd

^{+ / +}

和 11 只 Pcp2-cre；Ptprd

^{fox/flox}

小鼠（E）和 n = 12 Pcp2-cre;Ptprd

^{+ / +}

和

n = 11

Pcp2-cre;Ptprd

^{fox/flox}

小鼠（F）。误差条代表平均值

\pm

s.e.m.

^{*}

< 0.05,

^{* *}

< 0.01,^{* * *}

< 0.001

, 和

^{* * * *}

p 0.0001 ; 通过双尾非配对学生

t

检验。原始数据值、P 值和统计检验详情见扩展源数据 7。

Extended Data Fig.

8 ∣

Purkinje Neurons modulate thirst. Asprosin activates the cerebellar Purkinje neurons via the Ptprd receptor, leading to rapid manifestation of water drinking behavior.
扩展资料图

8 ∣

Purkinje 神经元调节口渴。阿司匹林通过Ptprd受体激活小脑浦肯野神经元，导致快速表现出饮水行为。

Reporting Summary 报告摘要

Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.
自然出版集团希望提高我们所出版作品的可复制性。本表提供了报告的一致性和透明度。有关自然出版集团政策的更多信息，请参阅我们的编辑政策和编辑政策清单。

Statistics 统计资料

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
对于所有统计分析，请确认图例、表格图例、正文或 "方法 "部分包含以下项目。

n/a $∣$ Confirmed 不适用 $∣$ 已确认

X

The exact sample size

(n)

for each experimental group/condition, given as a discrete number and unit of measurement

X

每个实验组/条件的确切样本量

(n)

，以离散数和计量单位表示

↘

A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

↘

说明是对不同样本进行测量，还是对同一样本进行重复测量
The statistical test(s) used AND whether they are one- or two-sided
使用的统计检验，以及是单侧检验还是双侧检验
Only common tests should be described solely by name; describe more complex techniques in the Methods section.
只有常见的测试才应仅用名称进行描述；更复杂的技术应在 "方法 "部分进行描述。

◻

A description of all covariates tested

◻

所有测试协变量的说明

↘

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons

↘

关于任何假设或校正的说明，例如正态性检验和多重比较调整
A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
完整描述统计参数，包括中心倾向（如平均值）或其他基本估计值（如回归系数）和变异（如标准差）或相关的不确定性估计值（如置信区间）

For null hypothesis testing, the test statistic (e.g.

F, t, r

) with confidence intervals, effect sizes, degrees of freedom and

P

value noted Give

P

values as exact values whenever suitable.
对于零假设检验，检验统计量（如

F, t, r

）以及置信区间、效应大小、自由度和

P

值只要合适，就把

P

值作为精确值。

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
对于贝叶斯分析，有关先验选择和马尔科夫链蒙特卡罗设置的信息
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
对于分层设计和复杂设计，确定测试的适当级别并全面报告结果
Estimates of effect sizes (e.g. Cohen’s

d

, Pearson’s

r

), indicating how they were calculated
效应大小估计值（如 Cohen's

d

, Pearson's

r

），说明计算方法
Our web collection on statistics for biologists contains articles on many of the points above.
我们为生物学家收集的统计资料中包含有关上述许多问题的文章。

Software and code 软件和代码

Policy information about availability of computer code
关于计算机代码可用性的政策信息

Data collection pCLAMP 10.3 for electrophysiological data collection, Spike2 version 10 was used for data collection and spike sorting, Metascreen software (V2.3.15.11) controlled system was used for data acquisition from Promethion Metabolic cages
数据收集 pCLAMP 10.3 用于电生理数据收集，Spike2 10 版用于数据收集和尖峰分类，Metascreen 软件（V2.3.15.11）控制系统用于采集 Promethion 代谢笼的数据。

Data analysis Graphpad Prism 8 and 10, Matlab R2023a for calculation of spike features in in vivo Electrophysiology experiments.
数据分析 Graphpad Prism 8 和 10、Matlab R2023a 用于计算体内电生理学实验的尖峰特征。

Data 数据

Policy information about availability of data
有关数据可用性的政策信息
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:
所有稿件必须包含数据可用性声明。该声明应酌情提供以下信息：

Accession codes, unique identifiers, or web links for publicly available datasets
公开数据集的存取码、唯一标识符或网络链接
A description of any restrictions on data availability
关于数据可用性限制的说明
For clinical datasets or third party data, please ensure that the statement adheres to our policy
对于临床数据集或第三方数据，请确保声明符合我们的政策

Research involving human participants, their data, or biological material
涉及人类参与者、其数据或生物材料的研究
Policy information about studies with human participants or human data. See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.
有关人类参与者或人类数据研究的政策信息。另请参阅有关性、性别（身份/表现）和性取向以及种族、民族和种族主义的政策信息。

Reporting on sex and gender 关于性和性别的报告	Not applicable. We have not used human participant data in this manuscript 不适用。本手稿未使用人类参与者数据
Reporting on race, ethnicity, or other socially relevant groupings 关于种族、民族或其他社会相关群体的报道	Not applicable. We have not used human participant data in this manuscript 不适用。本手稿未使用人类参与者数据
Population characteristics 人口特征	Not applicable. We have not used human participant data in this manuscript 不适用。本手稿未使用人类参与者数据
Recruitment 招聘	Not applicable. We have not used human participant data in this manuscript 不适用。本手稿未使用人类参与者数据
Ethics oversight 道德监督	Identify the organization(s) that approved the study protocol. 标明批准研究方案的机构。

Note that full information on the approval of the study protocol must also be provided in the manuscript.
请注意，手稿中还必须提供有关研究方案批准情况的完整信息。

Field-specific reporting 针对具体领域的报告

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
请选择以下最适合您研究的一项。如果您不确定，请在选择前阅读相应章节。

Behavioural & social sciences Ecological, evolutionary & environmental sciences
行为与社会科学生态、进化与环境科学

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf
如需所有章节的参考文件，请参阅 nature.com/documents/nr-reporting-summary-flat.pdf 。

Life sciences study design
生命科学研究设计

All studies must disclose on these points even when the disclosure is negative.
所有研究都必须披露这几点，即使披露的是负面信息。

Sample size 样本量	Power analysis was not done for predetermining the sample size, instead an $n \geq 6$ mice of each sex was always used for in vivo studies. The sample size was determined based on our previous publications assessing effects of asprosin overexpression, asprosin deficiency, Ptprd genetic knockout and pharmacological silencing on metabolism and behavior of mice (PMID: 35298903IF: 27.7 Q1 B1; PMID: 36812308IF: 11.7 Q1 B1; PMID: 33904407IF: 6.4 Q1 B1) 没有进行功率分析以预先确定样本量，而是始终使用每种性别的 $n \geq 6$ 只小鼠进行体内研究。样本量是根据我们以前发表的评估asprosin过表达、asprosin缺乏、Ptprd基因敲除和药物沉默对小鼠新陈代谢和行为的影响的文章（PMID: 35298903; PMID: 36812308; PMID: 33904407）确定的。
Data exclusions 数据排除	Data was excluded if mouse showed signs of sickness (mange, sudden weight loss, less than normal food intake, fighting wounds in group housing). Additionally, manually determined water intake data was excluded if the sipper bottle was found leaky and urine samples with contamination of feces and/or food particles were also excluded. 如果小鼠出现生病迹象（疥疮、体重突然下降、进食量低于正常水平、在群居环境中出现打斗伤口），则排除数据。此外，如果发现饮水瓶漏水，则排除人工测定的饮水量数据；如果尿样受到粪便和/或食物颗粒的污染，则排除尿样数据。
Replication 复制	Experiments were replicated at least twice in a blinded fashion. 实验在盲法下至少重复两次。
Randomization 随机化	Both sexes of the three mouselines (NPS, Ptprd knock-out, and purkinje neuron specific knock-out of Ptprd) were used in experiments. Ageand sex-matched were used for all experiments. Littermates were used as controls for all experiments. 实验中使用了三种鼠线（NPS、Ptprd 基因敲除和 Ptprd 的浦肯野神经元特异性基因敲除）的雌雄鼠。所有实验均使用年龄和性别匹配的动物。所有实验均使用同窝幼鼠作为对照。
Blinding 致盲	For execution of experiments, experimenters were blinded to the genotype of the mice. Data collection and data analysis was also done in a blinded fashion for the Promethion metabolic caging, qPCR studies, behavioral analysis studies, and urine and plasma osmolality studies. 在执行实验时，实验人员对小鼠的基因型进行了盲法处理。数据收集和数据分析也是以盲法进行的，包括丙硫磷代谢笼、qPCR 研究、行为分析研究以及尿液和血浆渗透压研究。

Reporting for specific materials, systems and methods
特定材料、系统和方法的报告

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
我们要求作者提供有关许多研究中使用的某些材料、实验系统和方法的信息。在此，请指出所列的每种材料、系统或方法是否与您的研究相关。如果您不确定列表中的某项是否适用于您的研究，请在选择答案前阅读相应章节。

Materials & experimental systems 材料与实验系统		Methods 方法
n/a 不适用	Involved in the study 参与研究	n/a 不适用	Involved in the study 参与研究
$◻$	X Antibodies X 抗体	X	$◻$ ChIP-seq $◻$ ChIP-seq
$◻$	Eukaryotic cell lines 真核细胞系	X	$◻$ Flow cytometry $◻$ 流式细胞仪
】	$◻$ Palaeontology and archaeology $◻$ 古生物学和考古学	区	$◻$ MRI-based neuroimaging $◻$ 基于磁共振成像的神经成像技术
	X Animals and other organisms X 动物和其他生物
X	$◻$ Clinical data $◻$ 临床数据
X	$◻$ Dual use research of concern $◻$ 值得关注的双重用途研究
区	$◻$ Plants $◻$ 植物

Antibodies used 使用的抗体

Validation 验证
rabbit anti-PTPRD (ABclonal A15713): 1:1000 dilution
兔抗 PTPRD（ABclonal A15713）：1:1000 稀释
Mouse anti-asprosin monoclonal antibody (This paper, available upon request from Chopra lab): 250 ug injected per mouse, 100ng/ well for ELISA
小鼠抗asprosin 单克隆抗体（本文，可向 Chopra 实验室索取）：每只小鼠注射 250 微克，用于 ELISA 时每孔 100ng
mouse IgG antibody (Southern Biotech 0107-01): 250 ug injected per mouse
小鼠 IgG 抗体（Southern Biotech 0107-01）：每只小鼠注射 250 微克
Donkey anti-rabbit Alexa Fluor488 (Invitrogen A21206): 1:500
驴抗兔子 Alexa Fluor488（Invitrogen A21206）：1:500
HRP-conjugated anti-mouse (KPL Scientific 474-1806): 1:10000
HRP 结合物抗小鼠（KPL Scientific 474-1806）：1:10000
human anti-asprosin monoclonal antibody: 100ng/well for ELISA
人类抗天冬氨酸单克隆抗体：100ng/孔，用于 ELISA 检测

Rabbit anti-PTPRD (ABclonal A15713): PMID: 36812308IF: 11.7 Q1 and A15713
兔抗 PTPRD（ABclonal A15713）：PMID: 36812308 和 A15713
Mouse anti-asprosin monoclonal antibody and human anti-asprosin monoclonal antibody: dose dependent binding to asprosin published in PMID: 33904407
《小鼠抗天冬氨肽单克隆抗体和人类抗天冬氨肽单克隆抗体：与天冬氨肽的剂量依赖性结合》发表于：PMID: 33904407IF: 6.4 Q1 IF: 6.4 Q1
mouse IgG antibody (Southern Biotech 0107-01): Quality testing for ELISA, FC, IHC done by Southern Biotech (https:// resources.southernbiotech.com/techbul/0107.pdf) and published in over 26 publications including: PMID: 9060677IF: 4.0 Q2 , PMID: 17548615IF: 3.6 Q2 , PMID: 23820392IF: 5.1 Q1 .
小鼠 IgG 抗体 (Southern Biotech 0107-01)：由 Southern Biotech（https:// resources.southernbiotech.com/techbul/0107.pdf）完成的 ELISA、FC、IHC 质量检测，并在超过 26 篇论文中发表，包括：pmid: 9060677, pmid: 17548615, pmid: 23820392：PMID: 9060677, PMID: 17548615, PMID: 23820392.

Eukaryotic cell lines 真核细胞系

Policy information about cell lines and Sex and Gender in Research
有关细胞系和研究中的性别的政策信息

Cell line source(s) 细胞系来源

Authentication 认证
Mycoplasma contamination 支原体污染

Commonly misidentified lines (See ICLAC register)
常见的错误识别线路（见 ICLAC 登记册）

State the source of each cell line used and the sex of all primary cell lines and cells derived from human participants or vertebrate models.
说明所使用的每种细胞系的来源，以及所有原代细胞系和来自人类参与者或脊椎动物模型的细胞的性别。

Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.
说明所使用的每种细胞系的验证程序，或声明所使用的细胞系均未经验证。
Confirm that all cell lines tested negative for mycoplasma contamination OR describe the results of the testing for mycoplasma contamination OR declare that the cell lines were not tested for mycoplasma contamination.
确认所有细胞系的支原体污染检测结果均为阴性，或描述支原体污染检测结果，或声明未对细胞系进行支原体污染检测。

Name any commonly misidentified cell lines used in the study and provide a rationale for their use.
列出研究中使用的任何常见的错误识别细胞系的名称，并说明使用这些细胞系的理由。

Animals and other research organisms
动物和其他研究生物

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research
关于涉及动物的研究的政策信息；建议报告动物研究的 ARRIVE 准则，以及研究中的性别和性别问题

Laboratory animals 实验动物

C57BL/6J Mus musculus Jackson Laboratory JAX:000664: 12 和 16 周大的 ROSA26::FLPe knock in Jackson Laboratory JAX:003946：10 到 18 周大的 C57BL/6-Agrptm1(cre)Lowl 杰克逊实验室 JAX:012899：10 至 18 周大的 B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J Jackson Laboratory JAX:007905：10-18 周大的 B6J.129(Cg)-RpI22tm1.1Psam/SjJ Mus musculus Jackson Laboratory JAX: 029977: 12 周大的 B6;129-Ptprdtm1Yiw/YiwRbrc Mus musculus RIKEN BioResource RBRC04925：12周龄和34周龄的C57BL/6N-Atm1Brd Ptprdtm2a(KOMP)Wtsi/Mmucd Wellcome Trust Sanger Institute MMRRC_065397-UCD：8至16周龄的B6.129-Tg(Pcp2cre)2Mpin/J Jackson Laboratory JAX#004146: 8 至 16 周龄 C57BL/6-Fbn1em1Chop/J Jackson Laboratory JAX# 033548: 14 和 16 周龄 Rosa26-LSL-tdTOMATO 小鼠 Jackson Laboratory JAX# 007914：8至16周龄

C57BL/6J Mus musculus Jackson Laboratory JAX:000664: 12- and 16-week-old

ROSA26::FLPe knock in Jackson Laboratory JAX:003946: 10- to 18-week old

C57BL/6-Agrptm1(cre)Lowl Jackson Laboratory JAX:012899: 10- to 18-week-old

B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J Jackson Laboratory JAX:007905: 10-18-week-old

B6J.129(Cg)-RpI22tm1.1Psam/SjJ Mus musculus Jackson Laboratory JAX: 029977: 12-week-old

B6;129-Ptprdtm1Yiw/YiwRbrc Mus musculus RIKEN BioResource RBRC04925: 12-week old and 34-week -old

C57BL/6N-Atm1Brd Ptprdtm2a(KOMP)Wtsi/Mmucd Wellcome Trust Sanger Institute MMRRC_065397-UCD: 8-to 16-week-old

B6.129-Tg(Pcp2cre)2Mpin/J Jackson Laboratory JAX#004146: 8- to 16-week-old

C57BL/6-Fbn1em1Chop/J Jackson Laboratory JAX# 033548: 14 and 16-week old

Rosa26-LSL-tdTOMATO mice Jackson Laboratory JAX# 007914: 8- to 16-week-old

Note that full information on the approval of the study protocol must also be provided in the manuscript.
请注意，手稿中还必须提供有关研究方案批准情况的完整信息。

$^{1}$ Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, OH, USA. ²Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, College of Medicine, University of Kentucky, Lexington, KY, USA. $^{3}$ Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA. $^{4}$ Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA. $^{5}$ Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA. $^{6}$ Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, TX, USA. $^{7}$ Department of Biology, College of Arts & Sciences, University of Dayton, Dayton, OH, USA. $^{8}$ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA. $^{9}$ Department of Electrical & Computer Engineering, School of Engineering, University of Dayton, Dayton, OH, USA. $^{10}$ Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA. $^{11}$ Development, Disease Models & Therapeutics Graduate Program, Baylor College of Medicine, Houston, TX, USA. $^{12}$ Department of Medicine, University Hospitals Cleveland Medical Center, Cleveland, OH, USA. $^{13}$ These authors contributed equally: Ila Mishra, Bing Feng. $⊠$ e-mail: yanlin.he@pbrc.edu; atul.chopra@case.edu
$^{1}$ 美国俄亥俄州克利夫兰市大学医院克利夫兰医学中心哈灵顿发现研究所。² 美国肯塔基州列克星敦肯塔基大学医学院内科学系内分泌、糖尿病和新陈代谢科。 $^{3}$ 美国洛杉矶巴吞鲁日路易斯安那州立大学彭宁顿生物医学研究中心。 $^{4}$ 美国俄亥俄州克利夫兰凯斯西储大学遗传学和基因组科学系。 $^{5}$ 美国德克萨斯州休斯顿贝勒医学院病理学与免疫学系。 $^{6}$ 美国德克萨斯州休斯顿市德克萨斯儿童医院扬和丹-邓肯神经研究所（Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital）。 $^{7}$ 美国俄亥俄州代顿市代顿大学文理学院生物系。 $^{8}$ 美国德克萨斯州休斯顿贝勒医学院分子与细胞生物学系。 $^{9}$ 美国俄亥俄州代顿市代顿大学工程学院电气与计算机工程系。 $^{10}$ 美国德克萨斯州休斯顿贝勒医学院儿科系。 $^{11}$ 美国德克萨斯州休斯顿贝勒医学院开发、疾病模型与治疗研究生课程。 $^{12}$ 美国俄亥俄州克利夫兰市克利夫兰大学医院医学中心医学系。 $^{13}$ 这些作者的贡献相同：Ila Mishra、Bing Feng。 $⊠$ 电子邮件：yanlin.he@pbrc.edu; atul.chopra@case.edu
All data used and generated in the study is available in source data files.
研究中使用和生成的所有数据均可从源数据文件中获取。

Thecerebellummodulates thirst 小脑调节口渴

Results 成果

AgRP neurons do not mediate the dipsogenic effect of asprosinAgRP神经元不介导天冬氨酸的致溺作用

Asprosin activates cerebellar Purkinje neurons阿司匹林激活小脑浦肯野神经元

Purkinje neuron activity modulation alters water intake浦肯野神经元活动调节改变了水的摄入量

Purkinje neuron-specific Ptprd deletion results in hypodipsia浦肯野神经元特异性 Ptprd 缺失会导致嗜睡症

Purkinje-specific deletion of Ptprd does not result in motor deficitsPurkinje特异性缺失Ptprd不会导致运动障碍

Purkinje-specific deletion of Ptprd renders mice unresponsive to dipsogenic effect of asprosinPurkinje特异性缺失Ptprd会导致小鼠对阿司匹林的致浸剂效应反应迟钝

Discussion 讨论

Online content 在线内容

References 参考资料

Methods 方法

Mice 小鼠

Validation of Ptprd deletion in Purkinje neurons在浦肯野神经元中进行 Ptprd 缺失验证

Assessment of Purkinje neuron morphology评估浦肯野神经元形态

AP staining AP 染色

Metabolic caging, water and food intake assessment代谢笼、水和食物摄入量评估

Overexpression of asprosin and recombinant asprosin treatment天冬氨酸的过表达和重组天冬氨酸的处理

DREADD manipulation of Purkinje neuronsDREADD 操纵浦肯野神经元

Optogenetic manipulation of Purkinje neurons浦肯野神经元的光遗传学操作

qPCR

In vivo anti-asprosin mAb treatment体内抗天冬氨酸 mAb 治疗

Plasma and urine parameters血浆和尿液参数

Motor function assays 运动功能检测

Ex vivo electrophysiology体外电生理学

Surgery for in vivo awake electrophysiology体内清醒电生理学手术

In vivo awake electrophysiology体内清醒电生理学

Fiber photometry 纤维光度测量

Quantification and statistical analysis量化和统计分析

Editing 编辑

Reporting summary 报告摘要

Data availability 数据可用性

References 参考资料

Acknowledgements 致谢

Author contributions 作者供稿

Competing interests 竞争利益

Additional information 其他信息

CNO+ AAV-hSyn-DIO-hM3Dq-mCherry TreatmentCNO+ AAV-hSyn-DIO-hM3Dq-mCherry 处理

CNO+ AAV-hSyn-DIO-mCherry TreatmentCNO+ AAV-hSyn-DIO-mCherry 处理

우

Reporting Summary 报告摘要

Statistics 统计资料

n/a ∣ ∣ ∣\mid Confirmed 不适用 ∣ ∣ ∣\mid 已确认

Software and code 软件和代码

Policy information about availability of computer code关于计算机代码可用性的政策信息

Data 数据

Field-specific reporting 针对具体领域的报告

Life sciences study design生命科学研究设计

Reporting for specific materials, systems and methods特定材料、系统和方法的报告

Eukaryotic cell lines 真核细胞系

Animals and other research organisms动物和其他研究生物

Thecerebellummodulates thirst
小脑调节口渴

AgRP neurons do not mediate the dipsogenic effect of asprosin
AgRP神经元不介导天冬氨酸的致溺作用

Asprosin activates cerebellar Purkinje neurons
阿司匹林激活小脑浦肯野神经元

Purkinje neuron activity modulation alters water intake
浦肯野神经元活动调节改变了水的摄入量

Purkinje neuron-specific Ptprd deletion results in hypodipsia
浦肯野神经元特异性 Ptprd 缺失会导致嗜睡症

Purkinje-specific deletion of Ptprd does not result in motor deficits
Purkinje特异性缺失Ptprd不会导致运动障碍

Purkinje-specific deletion of Ptprd renders mice unresponsive to dipsogenic effect of asprosin
Purkinje特异性缺失Ptprd会导致小鼠对阿司匹林的致浸剂效应反应迟钝

Validation of Ptprd deletion in Purkinje neurons
在浦肯野神经元中进行 Ptprd 缺失验证

Assessment of Purkinje neuron morphology
评估浦肯野神经元形态

Metabolic caging, water and food intake assessment
代谢笼、水和食物摄入量评估

Overexpression of asprosin and recombinant asprosin treatment
天冬氨酸的过表达和重组天冬氨酸的处理

DREADD manipulation of Purkinje neurons
DREADD 操纵浦肯野神经元

Optogenetic manipulation of Purkinje neurons
浦肯野神经元的光遗传学操作

In vivo anti-asprosin mAb treatment
体内抗天冬氨酸 mAb 治疗

Plasma and urine parameters
血浆和尿液参数

Ex vivo electrophysiology
体外电生理学

Surgery for in vivo awake electrophysiology
体内清醒电生理学手术

In vivo awake electrophysiology
体内清醒电生理学

Quantification and statistical analysis
量化和统计分析

CNO+ AAV-hSyn-DIO-hM3Dq-mCherry Treatment
CNO+ AAV-hSyn-DIO-hM3Dq-mCherry 处理

CNO+ AAV-hSyn-DIO-mCherry Treatment
CNO+ AAV-hSyn-DIO-mCherry 处理

n/a $∣$ Confirmed 不适用 $∣$ 已确认

Policy information about availability of computer code
关于计算机代码可用性的政策信息

Life sciences study design
生命科学研究设计

Reporting for specific materials, systems and methods
特定材料、系统和方法的报告

Animals and other research organisms
动物和其他研究生物