神经科学

Despite substantial progress in preclinical cannabinoid research, translational studies on cannabis use disorders (CUD) are still insufficient due to the absence of robust, validated animal models that fully recapitulate the multifactorial clinical phenotype of human CUD. The complex nature of CUD and the incomplete understanding of its underlying neurobiological mechanisms contribute to the limited availability of effective treatments. To address this gap, we developed an operant conditioning–based mouse model that enables the identification of individual vulnerability or resilience to CUD development. This highly translational model is based on the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) criteria for substance use disorders. The model allows the assessment of addiction-like behaviors by evaluating three behavioral domains: 1) persistence of responding during periods of cannabinoid unavailability, 2) motivation for cannabinoid seeking measured using a progressive ratio schedule, and 3) compulsivity, assessed when cannabinoid reward is paired with an aversive consequence such as a mild electric foot shock. A major strength of this paradigm is its ability to quantify two phenotypic traits proposed as predisposing factors for addiction vulnerability and two parameters related to craving. In addition, the model is specifically designed to evaluate genetic and circuit-level manipulations using chemogenetic approaches, with minor modifications required by surgical viral-vector delivery. Using this protocol, we can determine whether altering the excitability of specific neural networks promotes resilience or vulnerability to developing cannabinoid addiction. Elucidating these mechanisms is expected to facilitate the identification of novel and more effective therapeutic interventions for CUD.

Tail vein catheterization in mice is a standard technique for precise drug delivery in pharmacological research, offering high accuracy and reproducibility. However, existing techniques face significant limitations in maintaining long-term stable catheter patency in awake, freely moving mice, and there is currently no standardized, detailed protocol for tail vein catheterization. Current methods suffer from high rates of catheter dislodgement, increased animal stress from repeated injections, and movement restrictions, all of which introduce confounding variables in behavioral and pharmacological studies. We have developed a simple and efficient fixation method that maintains stable tail vein catheter patency for more than 60 min while allowing complete freedom of movement. This protocol employs a strain relief loop design and multi-point fixation strategy, effectively preventing catheter dislodgement during extended periods while minimizing animal stress. This protocol has been successfully applied across multiple research areas, including metabolic studies, behavioral assessments, and neuropharmacological research in awake mice, achieving >95% catheter retention with normal animal behavior, providing a reliable technical platform for long-term awake-state research applications.

Autonomic regulation of heart and respiratory rates is essential for understanding brain–body interactions in health and disease. Preclinical cardiovascular recordings are often performed under anesthesia or via telemetry, both of which introduce physiological confounds such as stress or impaired recovery due to the need for acute or chronic implantation of sensors. Here, we present a minimally invasive protocol for simultaneous acquisition of high-quality electrocardiography and respiratory signals in awake mice. Using an in-house-modified physiological monitor in awake, head-fixed mice that were briefly habituated to experimental conditions, we ultimately enable stable, long-term physiological recordings alongside in vivo microscopy. This protocol provides a robust, low-stress method for acquiring physiological signals, enabling the simultaneous study of cardiovascular–cerebral dynamics in awake head-fixed mice, thereby enhancing the translational relevance of preclinical measurements.

Hair cells are the sensory receptors of the auditory and vestibular systems in the inner ears of all vertebrates. Hair cells also serve to detect water flow in the lateral line system in amphibians and fish. The zebrafish lateral line serves as a well-established model for investigating hair cell development and function, including research on genetic mutations associated with deafness and environmental factors that cause hair cell damage. Rheotaxis, the ability to orient and swim in response to water flow, is a behavior mediated by multiple sensory modalities, including the lateral line organ. In this protocol, we describe a rheotaxis assay in which station holding behavior, which employs positive rheotaxis to maintain position in oncoming water flow, serves as a sensitive measure of lateral line function in larval zebrafish. This assay provides a valuable tool for researchers assessing the functional consequences of genetic or environmental disruptions of the lateral line system.

Vestibulo-ocular reflexes (VORs) are compensatory ocular reflexes that maintain stable vision during head movements. In research, VORs encompass angular VOR (aVOR) and off-vertical axis rotation (OVAR) tests, which various groups have employed to assess vestibular function in mice. This protocol outlines the process for measuring VORs in mice, including eye rotation calibration, immobilizing the mouse with a noninvasive setup, configuring the aVOR and OVAR stimulus modes, and interpreting the obtained waveforms to derive VOR values. As technology advances, VORs are expected to yield more qualitative and quantitative insights into the function of the horizontal semicircular canal cristae (HSCC) and the otolith organs. This methodology can serve as a standard for evaluating common vestibular deficits in mice.

Changes in learning and memory are important behavioral readouts of brain function across multiple species. In mice, a multitude of behavioral tasks exist to study learning and memory, including those influenced by extrinsic and intrinsic forces such as stress (e.g., escape from danger, hunger, or thirst) or natural curiosity and exploratory drive. The novel object recognition (NOR) test is a widely used behavioral paradigm to study memory and learning under various conditions, including age, sex, motivational state, and neural circuit dynamics. Although mice are nocturnal, many behavioral tests are performed during their inactive period (light phase, subjective night) for the convenience of the diurnal experimenters. However, learning and memory are strongly associated with the animal’s sleep-wake and circadian cycles, stressing the need to test these behaviors during the animals’ active period (dark phase, subjective day). Here, we develop a protocol to perform the NOR task during both light (subjective night) and dark (subjective day) phases in adult mice (4 months old) and provide a flexible framework to test the learning and memory components of this task at distinct times of day and associated activity periods. We also highlight methodological details critical for obtaining the expected behavioral responses.

Cognitive flexibility is a process that involves dynamically adapting behavior to obtain a desired series of outcomes during continuously changing stimulus-response-reward associations. Attentional set-shifting is a multi-modal decision-making paradigm that tests cognitive flexibility, which can be useful for investigating neural circuitry that is disrupted in multiple neuropsychiatric disorders, including schizophrenia, Alzheimer’s disease, depression, and addiction. The canonical human attentional set-shifting paradigm for measuring cognitive flexibility is the Wisconsin Card Sorting Test, in which subjects sort cards based on rules that change periodically and adapt their behavior by utilizing feedback in the form of correct and incorrect outcomes. To transfer these tests to rodent models, previous techniques involved attentional set-shifting paradigms in free-roaming test chambers, in which animals associated cues with reward locations. The protocol presented here involves an analogous attentional set-shifting paradigm, in which water-restricted mice are head-restrained on a platform and must keep track of periodically switching stimulus-response-outcome associations. The mice must learn to associate odor or whisker vibration cues with a binary directional lick response that triggers water delivery. The mice are trained to respond by licking one of two spouts, in which the correct decision is dependent on the current stimulus rule. This protocol allows for behaviorally measuring cognitive flexibility alongside neural activity by pairing the head-restrained paradigm with 2-photon calcium imaging, optogenetics, and extracellular and intracellular physiology.

The Morris water maze (MWM) is one of the most widely used procedures to assess hippocampus-dependent spatial learning and memory in rodents. By varying test protocols, researchers can test several different domains of learning and memory. Over multiple testing days, animals learn to swim to a platform hidden just under the water surface by using the spatial relationship between distal cues and the platform. Probe trials, where the platform is rendered unavailable, measure rodents’ spatial bias for the area where the platform was previously located. The ability of researchers to control the availability of the platform “on-demand” offers both practical and methodological advantages. Despite MWM’s prominence in the field of behavioral neuroscience, the high cost of purchasing a commercial MWM package is often prohibitively expensive for many research labs, especially on-demand platforms. Here, we describe a low-cost strategy for a build-your-own MWM that includes a remote-controlled on-demand platform (~530 USD) and tank (~550 USD). It is our hope that disseminating low-cost strategies aimed at expanding access to high-quality research tools at underfunded research institutions will accelerate biomedical discovery and foster further innovation.

The global burden of stroke has increased in the past several decades, and post-stroke epilepsy (PSE) is a common complication. Contrasted with the advancement in knowledge of stroke pathophysiology, the exact pathogenesis of PSE is unclear. Various animal stroke models have been utilized to investigate the underlying mechanisms of PSE, but the success rate of PSE induction is low. To address this limitation, a novel PSE model was established in the rat by inducing status epilepticus using lithium-pilocarpine one week after photothrombotic stroke. Successful indication of status epilepticus and mortality rate at three days after status epilepticus were the main measurements. Potential usefulness of this model was also illustrated by preliminary results on locomotor activity, exploratory behavior, and anxiety level evaluated using the open-field test, as well as mossy fiber sprouting (MFS) in the hippocampal dentate granule cells using Zinc transporter 3 immunofluorescence staining at 8 weeks after PSE induction. This novel composite method of PSE induction may facilitate future studies on the pathogenesis and treatment of PSE.

Active sampling, such as respiration, is known to play a major role in modulating how sensory information is perceived and encoded in the field of olfaction. Hence, monitoring respiration is crucial for understanding olfactory-guided behavior and physiology. Several methods used to measure respiration, such as infrared cameras, piezoelectric sensors, video monitoring, temperature probes, intubation, and intranasal cannula, require the animal or at least its head to be fixed. However, telemetry-based sensors can be used wirelessly, allowing animals to move freely. Here, we describe the surgical protocol to implant a telemetry pressure sensor in the internal jugular vein to detect changes in thoracic pressure. The sensor can thus help in monitoring respiration by transmitting the signal wirelessly. We describe a way of inserting the probe into the right jugular vein aseptically while housing the transmitter underneath the skin on the back of the animal. Next, based on the optimal spot for the best signal, we secure the position of the probe and suture the skin. The animal then undergoes regular post-operative care with painkillers and soft diets for up to a week. The method offers two main advantages; first, it uses a strategy similar to the jugular vein catheterization, which is widely established in rodents. Second, it minimizes the need for extensive post-operative care, including not having to shift to a liquid diet post-recovery. This makes the animals fit for most behavioral experiments requiring water or food restrictions.