神经科学

Calcium-permeable AMPA receptors (CP-AMPARs) and kainate receptors (CP-KARs) play crucial roles in synaptic plasticity and are implicated in various neurological processes. Current methods for identifying neurons expressing these receptors, such as electrophysiological recordings and immunostaining, have limitations in throughput or inability to distinguish functional receptors. This protocol describes a novel approach for the vital identification of neurons containing CP-AMPARs and CP-KARs using calcium imaging. The method involves loading neurons with Fura-2 AM, a calcium-sensitive fluorescent probe, KCl application to identify all neurons, and further addition of specific AMPAR agonists (e.g., 5-fluorowillardiine) in the presence of voltage-gated calcium channel blockers and NMDAR/KAR antagonists to identify CP-AMPAR-containing neurons. CP-KAR-containing neurons are identified using domoic acid applications in the presence and absence of NASPM (a CP-AMPAR antagonist). This technique offers several advantages over existing methods, including the ability to assess large neuronal populations simultaneously, distinguish between different receptor types, and provide functional information about CP-AMPAR and CP-KAR expression in living neurons, making it a valuable tool for studying synaptic plasticity and neurological disorders.

Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology.

During neuronal synaptic transmission, the exocytotic release of neurotransmitters from synaptic vesicles in the presynaptic neuron evokes a change in conductance for one or more types of ligand-gated ion channels in the postsynaptic neuron. The standard method of investigation uses electrophysiological recordings of the postsynaptic response. However, electrophysiological recordings can directly quantify the presynaptic release of neurotransmitters with high temporal resolution by measuring the membrane capacitance before and after exocytosis, as fusion of the membrane of presynaptic vesicles with the plasma membrane increases the total capacitance. While the standard technique for capacitance measurement assumes that the presynaptic cell is unbranched and can be represented as a simple resistance-capacitance (RC) circuit, neuronal exocytosis typically occurs at a distance from the soma. Even in such cases, however, it can be possible to detect a depolarization-evoked increase in capacitance. Here, we provide a detailed, step-by-step protocol that describes how "Sine + DC" (direct current) capacitance measurements can quantify the exocytotic release of neurotransmitters from AII amacrine cells in rat retinal slices. The AII is an important inhibitory interneuron of the mammalian retina that plays an important role in integrating rod and cone pathway signals. AII amacrines release glycine from their presynaptic dendrites, and capacitance measurements have been important for understanding the release properties of these dendrites. When the goal is to directly quantify the presynaptic release, there is currently no other competing method available. This protocol includes procedures for measuring depolarization-evoked exocytosis, using both standard square-wave pulses, arbitrary stimulus waveforms, and synaptic input.

Because of its genetic tractability and amenability for live imaging, larval zebrafish (Danio rerio) have emerged as a model to study the cellular and synaptic properties underlying behavior. The accessibility of Mauthner cells, a pair of escape-organizing neurons located in the brainstem of teleost fish, along with their associated sensory inputs, enables exploration of the correlation between structural and functional synaptic features. This is the case of the endings of auditory afferents on the lateral dendrite of this cell, known as large myelinated club endings, which provide the excitatory drive for the initiation of auditory-evoked escape responses mediated by the Mauthner cell and its spinal network. Here, we describe the procedures that make it possible to expose the molecular composition of these synapses using protein-retention expansion microscopy (proExM). This method allowed us to generate a map of the distribution of synaptic proteins at these identifiable synapses, which could also be applied to examine the organization of other synaptic contacts in this cell.

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes—expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons.

Comparative cell biology relies on methods that disrupt protein function. Traditional approaches target the gene that encodes the protein of interest via conventional knockout (KO) methodology, conditional Cre-lox system, or recently, flexible protocols based on CRISPR/Cas9. However, these technologies lack precise temporal control (hours), whereby the slow half-lives of proteins may confound measurements, possibly resulting in misleading phenotypes. Targeting the protein itself bypasses issues pertaining to protein half-life, resulting in more acute disruption. An ideal system would enable controllable protein disruption, dependent on the presence or absence of a small molecule, with high temporal control achieved through washout/addition of the small molecule. Here, we outline the use of knockoff, a general method to disrupt membrane proteins based on the NS3/4A protease of the hepatitis C virus. This technique has been used in post-mitotic cells to study the function of long-lived integral membrane proteins and is suitable for the study of other membrane-bound proteins.

Graphic abstract:

Removal of the protease inhibitor induces cleavage from the membrane.

General model of knockoff method. Inh, Inhibitor; POI, Protein of Interest; NS3/4A, Hepatitis C viral protease.

Synaptic vesicles (SVs) are clustered in the presynaptic terminals and consistently trafficking along axons. Based on their release features, SVs are classified into different “pools”. Imaging of SVs that are traveling among multiple presynaptic terminals has helped define a new pool named “SV super-pool”. Here we describe a Fluorescent Recovery After Photobleaching (FRAP) approach to elucidate the relationship between SVs from the super-pool with SV clusters at presynaptic terminals. This method is powerful to investigate SV mobility regulation mechanisms.

Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses through immune-electrophysiology in Drosophila melanogaster. This technique allows the quantification of the molecular behavior of the neuromuscular synapses by correlating the structural configuration of the synaptic boutons with their electrical activity.

The synapse is a complex structure where the transmission of information takes place. Synaptic dysfunction is one of the earliest pathophysiological events in several diseases, such as traumatic brain injury, cerebral ischemia, and neurodegenerative diseases. Thus, a methodology to study synaptic structure and function is crucial for the development of potential strategies for the treatment of many neurological diseases. Synaptoneurosomes (SNs) are structures assembled by the sealed presynaptic bouton and the attached post-synaptic density. Despite the fact that for a long time it has been recognized that SNs are a powerful tool to study synaptic function, composition, and structure, its use has been limited by the requirement of relatively large amounts of material to successfully isolate them. Here we describe a three-step centrifugation procedure performed under hypotonic conditions to isolate SNs from small volumes of the cerebral cortex.

Graphic abstract

Schematic flowchart for the preparation of synaptoneurosomes.

In the pilocarpine model of temporal lobe epilepsy (TLE) in rodents, systemic injections of pilocarpine induce continuous, prolonged limbic seizures, a condition termed “Status Epilepticus” (SE). With appropriate doses, many inbred strains of mice show behavioral seizures within an hour after pilocarpine is injected. With the behavioral scoring system based on a modification of the original Racine scale, one can monitor the seizures behaviorally, as they develop into more prolonged seizures and SE. SE is typically associated with damage to subsets of hippocampal neurons and other structural changes in the hippocampus and generally subsides on its own. However, more precise control of the duration of SE is commonly achieved by injecting a benzodiazepine into the mouse 1 to 3 h after the onset of SE to suppress the seizures. Several days following pilocarpine-induced SE, electrographic and behavioral seizures begin to occur spontaneously. The goal of this protocol is to reliably generate mice that develop spontaneous recurrent seizures (SRS) and show the typical neuropathological changes in the brain characteristic of severe human mesial temporal lobe epilepsy (mTLE), without high mortality. To reduce mortality, multiple subthreshold injections of pilocarpine are administered, which increases the percentage of mice developing SE without concomitant mortality. Precise control of the duration of SE (1 or 3 h) is achieved by suppressing SE with the benzodiazepine Midazolam (Versed). We have found that this protocol is an efficient means for generating mice that subsequently develop characteristics of human mTLE including high-frequency interictal spike and wave activity and SRS. In addition, we and others have shown that this protocol produces mice that show excitotoxic cell death of subsets of hippocampal GABAergic interneurons, particularly in the dentate gyrus and compensatory sprouting of excitatory projections from dentate granule cells (mossy fiber sprouting). Aspects of this protocol have been described in several of our previous publications.