Understanding interactions between brain reward and sleep systems in driving maladaptive behaviors

了解大脑奖励和睡眠系统之间的相互作用在驱动适应不良行为中的作用

• 批准号: 10702275
• 负责人: Andrew Kesner
• 金额: $ 30.08万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702275
• 关键词: Abstinence; Acetylcholine; Action Potentials; Affect; Appetitive Behavior; Area; Arousal; Automobile Driving; Back; Behavior; Behavioral Model; Binding; Brain; Brain region; CNR1 gene; Calcium; Cannabis; Cell Nucleus; Chronic; Clozapine; Complex; Consummatory Behavior; Consumption; Data; Desire for food; Dopamine; Drug usage; Endocannabinoids; Enterobacteria phage P1 Cre recombinase; Exposure to; Face; Fiber; Fiber Optics; Fluorescence; Genes; Genetic Recombination; Glutamates; Goals; Homing; Human; Implant; Individual; Injections; Knock-out; Laboratories; Ligands; Light; Measures; Medial; Mediating; Messenger RNA; Modeling; Motivation; Mus; Nature; Neurons; Neurosciences; Neurotransmitters; Oxides; Pharmaceutical Preparations; Phase; Photometry; Play; Procedures; Process; Property; Proteins; Publications; Publishing; REM Sleep; Relapse; Reporting; Research; Research Project Grants; Rewards; Rodent; Role; Series; Sex Differences; Signal Transduction; Site; Sleep; Sleep Stages; Sleep disturbances; Substance Use Disorder; Sucrose; Sum; System; Techniques; Time; Training; Transgenic Organisms; Ventral Striatum; Ventral Tegmental Area; Viral; Withdrawal; Withdrawal Symptom; Work; behavior influence; brain circuitry; cannabis cessation; cannabis withdrawal; cognitive enhancement; designer receptors exclusively activated by designer drugs; dopaminergic neuron; endogenous cannabinoid system; experience; flexibility; gamma-Aminobutyric Acid; in vivo; interest; maladaptive behavior; marijuana use; motivated behavior; neural; neuromechanism; neurotransmitter release; non rapid eye movement; pre-clinical; presynaptic; presynaptic neurons; receptor; reduce symptoms; response; sensor; sleep behavior; sleep physiology; substance misuse; substance use; synergism; time use; vigilance

FY2022 was the first year of this project. Firstly, we published a series of studies from prior work performed within the Laboratory for Integrative Neuroscience (See: Kesner et. al., 2022. PMID: 35478010). This publication forms the basis of the work within the newly established Unit on Motivation and Arousal, which is dedicated to advancing this research project. This publication describes our work developing pre-clinical sleep and behavioral models of cannabis withdrawal symptoms. Prior to this work, there was controversy in our field as to whether rodents experience cannabis withdrawal symptoms purely upon cessation of this drug. It had previously been reported that withdrawal symptoms could be precipitated in rodents by administration of drugs that block the action of delta-9-tetrahydroannabinol (THC; the main driver of cannabis use and misuse), but this does not appropriately mimic the human condition where withdrawal symptoms occur after abrupt cessation (i.e spontaneous withdrawal) of cannabis/THC use. We found that, indeed, mice do experience withdrawal symptoms that have face validity to those experienced by humans: sleep disruption, irritability, and alterations in reward-seeking behaviors. In addition, we found sex differences in the strength of several of these mouse THC-withdrawal symptoms, which is consistent with the existence of sex-differences in the endocannabinoid (eCB) system (which THC directly acts on) and cannabis withdrawal symptoms in humans. The research summarized above suggests that brain regions that express cellular machinery related to eCB activity and are associated with both sleep and motivated behaviors may be important loci to assess the neural mechanisms governing withdrawal phenomena associated with cessation of cannabis, and potentially other misused substances. One such brain area that we feel is understudied is the medial septum (MS). The MS expresses cannabinoid receptor type 1 (CB1), which predominantly occupies presynaptic neuron terminals, and is the main target for the psychoactive properties and misuse liability of THC. Release of eCBs from neurons in the MS would act in a retrograde manner to reduce the release of neurotransmitters from neuron terminals in the MS. To assess whether eCB release within the MS correlates to vigilance state (e.g. wake, NREM sleep, or REM sleep), we used the newly developed genetically encoded eCB sensor, GRABeCB2.0. This is essentially a CB1 receptor genetically modified to contain a fluorescent protein whose fluorescence will increase when eCBs bind to the GRABeCB2.0 protein. Using viral strategies, we expressed this sensor in the MS of mice and implanted an optic fiber into the MS to record the changes in fluorescence from this sensor while simultaneously recording sleep physiology. We then aligned the fluorescence signals to changes in vigilance state (i.e. sleep stage) and found that MS eCB activity is reduced during NREM to REM transitions and comes back to baseline levels just before the transition from REM to wake. There is no change in eCB activity during Wake to NREM or NREM to wake transitions. These data are the first to show real time correlation of eCB activity with changes in vigilance state. We then reasoned that presynaptic CB1 on terminals within the MS would be influenced by this vigilance-state specific change in eCB tone. There are several brain regions that have neurons that make CB1 and project to the MS. One region, the supramammillary nucleus (SuM), is known to be involved in sleep-wake processes, heavily expresses mRNA for CB1, and densely projects to the septal complex. We used an intersectional viral strategy to express Cre-recombinase in SuM neurons projecting to the MS in mice that have the gene encoding for CB1 flanked by loxp sites. This results in a Cre-mediated recombination-mediated deletion of CB1 on SuM neurons that project to MS. We compared sleep in these mice to wildtype littermates that received the same intracranial viral injections and found that SuM to MS CB1 knockout increases NREM sleep during the active phase of the light cycle, while decreasing REM sleep in the inactive phase. These data confirm that both eCB release and its action on CB1 receptors in the MS is important for sleep-wake processes. Beyond its role in sleep, the MS has also been implicated in reward seeking behaviors, making it an interesting region to study synergy of sleep and reward related brain circuitry. The MS is comprised of neurons that primarily make and release the inhibitory neurotransmitter gamma-Aminobutyric acid, the excitatory neurotransmitter glutamate (GLU), or the modulatory neurotransmitter acetylcholine. Of these distinct subpopulations of neurons within the MS, the GLU neurons (MS-GLU) are particularly understudied and have recently been implicated in reward processes. We know that selective stimulation of these neurons using optogentics is reinforcing in mice, and they will actively press a lever to earn stimulation specifically of MS-GLU neurons. These neurons in turn project to the ventral tegmental area, a brain region highly implicated in motivated behavior, and influence dopamine neuron activity here and dopamine release in the ventral striatum. Beyond these recent findings, little is known about how MS-GLU neurons respond during natural reward seeking behaviors or how modulation of their activity can influence these behaviors. Our group has performed a set of studies to elucidate the role of these neurons in these processes. We expressed either the excitatory designer receptor exclusively activated by designer drug (DREADD) hM3D-Gq, the inhibitory DREADD hM4D-Gi, or control mCherry protein exclusively on MS-GLU neurons. We trained mice to perform a basic reward seeking procedure where one lever press on an active lever (of two levers) resulted in delivery of a sucrose reward. We found that administration of the DREADD ligand, clozapine-n-oxide (CNO) resulted in reduced lever pressing in hM4D-Gi-expressing mice, but not in hM3D-Gq or control mice. Additionally, consummatory behaviors were increased in the hM4D-Gi group but not in other groups. These data suggest that taking MS-GLU neurons offline alters the appetitive and consummatory processes associated with goal directed behaviors in a converse fashion. When we reversed the lever assignments, where the previously active lever did nothing, but pressing the other lever now delivers reward, the hM3D-Gq group adapted their behavior to this new contingency much faster than the control mice, and the hM4D-Gi mice never appropriately adapted. These findings suggest augmenting MS-GLU activity plays a role in enhancing cognitive flexibility. Finally, we used fiber photometry techniques to measure calcium activity in MS-GLU neurons. Calcium activity is a correlate of neuron activity, so increased calcium levels indicate action potentials and general neuronal activity. In mice performing the same reward-seeking behaviors we found profoundly reduced activity in MS-GLU neurons during reward consumption. MS-GLU neuron activity also appears to encode some valence of different components of the appetitive behaviors; as activity dynamics are different depending on which lever the mouse presses and whether reward was present during consummatory behaviors. These data are being prepared for article submission. Overall, we have begun homing in on brain circuitry that is involved in both motivation and arousal processes to assess the interaction between these processes in sleep and goal directed behavior.

2022财年是该项目的第一年。首先，我们发表了一系列从综合神经科学实验室进行的先前工作的研究（请参阅：Kesner等，2022。PMID：35478010）。该出版物构成了新成立的动机和唤醒部门中工作的基础，该单位致力于推进这项研究项目。该出版物描述了我们开发临床前睡眠和大麻戒断症状的行为模型的工作。在这项工作之前，我们领域存在争议，即啮齿动物是否纯粹是在停止这种药物的情况下会出现大麻戒断症状。以前据报道，施用戒断症状可以通过施用阻止Delta-9-四氢甲酸的作用的药物（THC；大麻使用和滥用大麻的主要驱动力）的作用，但这并不能适当地模拟cesseation cesseation（即戒断）的戒断症状（即戒断）的戒断（即戒断）。我们发现，实际上，老鼠确实会出现戒断症状，​​这些症状对人类所经历的人面临有效性：睡眠破坏，易怒和寻求奖励行为的改变。此外，我们发现了这些小鼠的强度Thc-WithDrawal症状的性别差异，这与内源性大麻素（ECB）系统中的性别差异（THC直接作用）和人类中大麻戒断症状是一致的。 上面总结的研究表明，表达与欧洲央行活动相关的细胞机制并与睡眠和动机行为相关的大脑区域可能是评估有关戒断与大麻停止的戒断现象的神经机制的重要基因座，以及潜在的其他滥用药物。我们觉得正在研究的大脑区域是内侧隔膜（MS）。 MS表达1型大麻素受体（CB1），主要占据突触前神经元末端，并且是THC的精神活性特性和滥用责任的主要目标。 MS中神经元的ECB释放将以逆行方式起作用，以减少MS中神经元末端的神经递质的释放。为了评估MS内的欧洲央行释放是否与警惕状态相关（例如唤醒，NREM睡眠或REM睡眠），我们使用了新开发的遗传编码的欧洲央行传感器GrabeCB2.0。这本质上是一种经过遗传修饰的CB1受体，它包含荧光蛋白，当ECB与grabecb2.0蛋白结合时，荧光将增加。使用病毒策略，我们在小鼠的MS中表达了该传感器，并将光纤植入MS中，以记录该传感器的荧光变化，同时记录睡眠生理学。然后，我们将荧光信号与警惕性状态的变化（即睡眠阶段）保持一致，并发现MS ECB活性在NREM期间降低了REM转变，并在从REM到Wake Wake的过渡之前恢复到基线水平。在NREM或NREM唤醒过渡期间，欧洲央行活动没有变化。这些数据是第一个显示欧洲央行活动与警惕状态变化的实时相关性的数据。然后，我们认为MS内终端上的突触前CB1将受到这种警惕状态特定的欧洲央行音调变化的影响。有几个大脑区域的神经元可以使CB1和向MS投影。已知一个区域，即超核核（SUM），与睡眠效益过程有关，大量表达CB1的mRNA，并密集地向中间复合体投射。我们使用截面病毒策略来表达在小鼠中投射到MS的总和神经元中的CRE聚合酶，这些神经元的基因编码为LOXP位点的CB1编码。这会导致CRE介导的重组介导的CB1对MS的总和神经元的缺失。我们将这些小鼠的睡眠与接受相同颅内病毒注射的野生型同窝仔进行了比较，并发现在光周期的活动阶段，与MS CB1敲除的总和增加了NREM睡眠，同时减少了不活跃阶段的REM睡眠。这些数据证实，欧洲央行释放及其对MS中CB1受体的作用对于睡眠觉醒过程很重要。 除了其在睡眠中的作用外，MS还与寻求奖励的行为有关，使其成为研究睡眠和奖励相关的脑电路协同作用的有趣区域。 MS由主要制造和释放抑制性神经递质γ-氨基丁酸，兴奋性神经递质谷氨酸（GLU）或调节性神经递质的神经递质的神经递质的神经元组成。在MS内神经元的这些独特的亚群中，GLU神经元（MS-GLU）特别研究了，最近与奖励过程有关。我们知道，使用副本对这些神经元的选择性刺激正在加强小鼠，它们将积极按下杠杆以赢得MS-GLU神经元的刺激。这些神经元反过来依次向腹侧侧侧侧面区域，这是一个高度涉及动机行为的大脑区域，在这里影响多巴胺神经元活动，并在腹侧纹状体中释放多巴胺。除了这些最新发现之外，对MS-GLU神经元如何在自然奖励寻求行为或其活动的调制如何影响这些行为的情况下响应的知之甚少。 我们的小组已经进行了一系列研究，以阐明这些神经元在这些过程中的作用。我们表达了由Designer Dress（Dreadd）HM3D-GQ，抑制性DREADD HM4D-GI（Dreadd）专门激活的兴奋性设计器受体，或者仅在MS-GLU神经元上控制MCHERRY蛋白。我们训练了小鼠执行基本的奖励寻求程序，其中一个杠杆按下（两个杠杆）的杠杆导致提供了蔗糖奖励。我们发现，施用Dreadd配体，氯氮平-N-氧化物（CNO）导致HM4D-GI表达小鼠的杠杆按压降低，但在HM3D-GQ或对照小鼠中却没有减少。此外，HM4D-GI组的完整行为增加，但在其他组中则没有增加。这些数据表明，使MS-GLU神经元离线改变了与目标定向行为相关的食欲和完整的过程。当我们逆转杠杆任务时，先前活跃的杠杆什么也没做，但是按下另一个杠杆现在会提供奖励时，HM3D-GQ组将其行为适应了这种新的偶然性，而HM4D-GI小鼠则从未适当适应过。这些发现表明，增强MS-GLU活性在增强认知灵活性中起作用。最后，我们使用纤维光度法技术来测量MS-GLU神经元中的钙活性。钙活性是神经元活性的相关性，因此钙水平提高表明作用电位和一般神经元活性。在执行相同奖励行为的小鼠中，我们发现在奖励消费期间，MS-GLU神经元的活动大大减少了。 MS-GLU神经元活性似乎也编码了食欲行为的不同组成部分的某些价。由于活动动力学不同，取决于小鼠按下的杠杆以及在完成行为期间是否存在奖励。这些数据正在为文章提交准备。 总体而言，我们已经开始在脑电路上进行归宿，这些脑电路既参与动机和唤醒过程，以评估这些过程在睡眠和目标定向行为中之间的相互作用。