神经科学

Nociception is critically shaped by descending modulation of spinal circuits, yet its cellular and synaptic mechanisms remain poorly defined. Elucidating these mechanisms is technically challenging, as it requires simultaneous activation of primary afferents and descending fibers while monitoring the functioning of individual spinal neurons. Here, we present a method to investigate the influence of the rostral ventromedial medulla (RVM), a principal supraspinal structure mediating descending modulation, on the activity of spinal lamina I neurons. Our approach combines electrophysiological recordings in ex vivo intact spinal cord preparation with optogenetics, granting several advantages. First, ex vivo preparation spares rostrocaudal and mediolateral spinal architecture, preserving lamina I as well as primary afferent and descending inputs. Second, virally mediated channelrhodopsin-2 (ChR2) expression enables selective photostimulation of RVM-originating fibers. When coupled with patch-clamp recordings, this photostimulation allows identifying postsynaptic inputs from RVM to spinal neurons and revealing RVM-dependent presynaptic inhibition of primary afferent inputs. Overall, our approach is well-suited for investigating both pre- and postsynaptic mechanisms of descending modulation in physiological and pathological pain conditions.

Telomere length maintenance is strongly linked to cellular aging, as telomeres progressively shorten with each cell division. This phenomenon is well-documented in mitotic, or dividing, cells. However, neurons are post-mitotic and do not undergo mitosis, meaning they lack the classical mechanisms through which telomere shortening occurs. Despite this, neurons retain telomeres that protect chromosomal ends. The role of telomeres in neurons has gained interest, particularly in the context of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), where aging is a major risk factor. This has sparked interest in investigating telomere maintenance mechanisms in post-mitotic neurons. Nevertheless, most existing telomere analysis techniques were developed for and optimized using mitotic cells, posing challenges for studying telomeres in non-dividing neuronal cells. Thus, this protocol adapts an already established technique, the combined immunofluorescence and telomere fluorescent in situ hybridization (IF-FISH) on mitotic cells to study the processes occurring at telomeres in cortical neurons of the mouse ALS transgenic model, TDP-43 rNLS. Specifically, it determines the occurrence of DNA damage and the alternative lengthening of telomeres (ALT) mechanism through simultaneous labeling of the DNA damage marker, γH2AX, or the ALT marker, promyelocytic leukemia (PML) protein, together with telomeres. Therefore, the protocol enables the visualization of DNA damage (γH2AX) or the ALT marker (PML) concurrently with telomeres. This technique can be successfully applied to brain tissue and enables the investigation of telomeres specifically in cortical neurons, rather than in bulk tissue, offering a significant advantage over Southern blot or qPCR-based techniques.

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes.

Accurate labeling of excitatory postsynaptic sites remains a major challenge for high-resolution imaging due to the dense and sterically restricted environment of the postsynaptic density (PSD). Here, we present a protocol utilizing Sylites, 3 kDa synthetic peptide probes that bind with nanomolar affinity to key postsynaptic markers, PSD-95 and Gephyrin. eSylites (excitatory Sylites) specifically target the PDZ1 and PDZ2 domains of PSD-95, enabling precise and efficient labeling of excitatory postsynaptic density (ePSD). In contrast, iSylites (inhibitory Sylites) bind to the dimerizing E-domain of the Gephyrin C-terminus, allowing selective visualization of inhibitory postsynaptic density (iPSD). Their small size reduces linkage error and enhances accessibility compared to conventional antibodies, enabling clear separation of PSD-95 nanodomains in super-resolution microscopy. The protocol is compatible with co-labeling using standard antibodies and integrates seamlessly into multichannel immunocytochemistry workflows for primary neurons and brain tissue. This method enables robust, reproducible labeling of excitatory synapses with enhanced spatial resolution and can be readily adapted for expansion microscopy or live-cell applications.

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.

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.

Understanding the nanoscale organization and molecular rearrangement of synaptic components is critical for elucidating the mechanisms of synaptic transmission and plasticity. Traditional synaptosome isolation protocols involve multiple centrifugation and resuspension steps, which may cause structural damage or alter the synaptosomal fraction, compromising their suitability for cryo-electron tomography (cryo-ET). Here, we present an ultrafast isolation method optimized for cryo-ET that yields two types of synaptosomal fractions: synaptosomes and synaptoneurosomes. This streamlined protocol preserves intact postsynaptic membranes apposed to presynaptic active zones and produces thin, high-quality samples suitable for in situ structural studies. The entire procedure, from tissue homogenization to vitrification, takes less than 15 min, offering a significant advantage for high-resolution cryo-ET analysis of synaptic architecture.

The phototransduction cascade allows photoreceptors to detect light across a wide range of intensities without saturation, with cGMP serving as the second messenger and calcium feedback as the key regulatory mechanism. While experimental evidence suggests that cAMP may also play a role in modulating this cascade, such regulation would necessitate rapid changes in cAMP levels on a timescale of seconds. However, data on the dynamics of intracellular cAMP changes in photoreceptors remain scarce, primarily due to the limitations of conventional fluorescence-based methods in this specialized sensory system. To address this gap, we developed a methodology combining rapid cryofixation of retinal samples following light stimulation with the isolation of outer segment preparations. The rapid cryofixation setup comprises six computer-controlled sections, each with a high-speed stepper motor-driven lever that rapidly moves the specimen in a 180° arc within ~80 ms to press it against a liquid nitrogen-cooled copper cylinder for fixation. Using highly sensitive metabolomics techniques, we measured cAMP levels in these samples. This approach enables the investigation of rapid cAMP dynamics and its potential regulatory role in phototransduction, providing a foundation for understanding the interplay between cAMP and PKA signaling in photoreceptor function.

High-throughput sequencing has created a tremendous amount of information about the genes expressed in various cell types and tissues throughout the body. As such, there is a need for a quick and effective method to knock down genes of interest in order to investigate their roles. While there are many approaches for this in mammalian models, there are limited ways to knock down genes of interest in adult zebrafish. Unlike mammals, zebrafish have the natural ability to regenerate their neurons after injury or disease is detected, making them a staple in regenerative studies. Unfortunately, current approaches for gene knockdown in the retina of adult zebrafish are costly and provide a barrier for many scientists. We provide two cost-effective approaches for targeted gene knockdowns in adult zebrafish retinas. We describe this approach through the use of Vivo-morpholinos and lipid-encapsulated siRNAs that target the expression of the proliferating cell nuclear antigen (PCNA) gene in adult zebrafish. We also describe how to collect and process retina samples for downstream immunohistochemistry, imaging, and quantification. Overall, this protocol will provide researchers with a straightforward, cheap, and effective method to perform targeted gene knockdowns in adult zebrafish retinas.

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.