Neuroscience

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.

The process of autophagy is an essential cellular mechanism, required to maintain general cell health through the removal of dysfunctional organelles, such as the ER, peroxisomes and mitochondria, as well as protein aggregates, and bacteria. Autophagy is an extremely dynamic process, and tools are constantly being developed to study the various steps of this process. This protocol details a method to study the end steps of autophagy-lysosomal fusion and the formation of the autolysosome. Many techniques have been used to study the various steps of the autophagy process. Here we describe the RedGreen-assay (RG-assay), an immunofluorescence-based technique used to visualize the targeting of substrates to the autolysosome in live cells. This technique takes advantage of the low lysosomal pH and over-expression of a tandem GFP-mCherry tagged protein targeted to an organelle of interest. While in the neutral cytosol or autophagosome, both GFP and RFP will fluoresce. However, within the autolysosome, the GFP signal is quenched due to the low pH environment and the RFP emission signal will predominate. This technique is readily quantifiable and amenable to high throughput experiments. Additionally, by tagging the GFP-RFP tandem fluorescent protein with organelle specific targeting sequences, it can be used to measure a wide range of substrates of autophagy.

The elucidation of the molecular mechanisms of long-term synaptic plasticity has been hindered by both the compensation that can occur after chronic loss of the core plasticity molecules and by ex vivo conditions that may not reproduce in vivo plasticity. Here we describe a novel method to rapidly suppress gene expression by antisense oligodeoxynucleotides (ODNs) applied to rodent brain slices in an “Oslo-type” interface chamber. The method has three advantageous features: 1) rapid blockade of new synthesis of the targeted proteins that avoids genetic compensation, 2) efficient oxygenation of the brain slice, which is critical for reproducing in vivo conditions of long-term synaptic plasticity, and 3) a recirculation system that uses only small volumes of bath solution (< 5 ml), reducing the amount of reagents required for long-term experiments lasting many hours. The method employs a custom-made recirculation system involving piezoelectric micropumps and was first used for the acute translational blockade of protein kinase Mζ (PKMζ) synthesis during long-term potentiation (LTP) by Tsokas et al., 2016. In that study, applying antisense-ODN rapidly prevents the synthesis of PKMζ and blocks late-LTP without inducing the compensation by other protein kinase C (PKC) isoforms that occurs in PKCζ/PKMζ knockout mice. In addition, we show that in a low-oxygenation submersion-type chamber, applications of the atypical PKC inhibitor, zeta inhibitory peptide (ZIP) can result in unstable baseline synaptic transmission, but in the high-oxygenation, "Oslo-type" interface electrophysiology chamber, the drug reverses late-LTP without affecting baseline synaptic transmission. This comparison reveals that the interface chamber, but not the submersion chamber, reproduces the effects of ZIP in vivo. Therefore, the protocol combines the ability to acutely block new synthesis of specific proteins for the study of long-term synaptic plasticity, while maintaining properties of synaptic transmission that reproduce in vivo conditions relevant for long-term memory.

Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. The size of the readily releasable pool is an important property of synapses, which influences release probability and can dynamically change during various forms of plasticity. Here we describe a detailed protocol for estimating the readily releasable pool at a model glutamatergic synapse, the Drosophila neuromuscular junction. This synapse is experimentally robust and amenable to sophisticated genetic, imaging, electrophysiological, and pharmacological approaches. We detail the experimental design, electrophysiological recording procedure, and quantitative analysis necessary to determine the readily releasable pool size. This technique requires the use of a two-electrode voltage-clamp recording configuration in elevated external Ca²⁺ with high frequency stimulation. We have used this assay to measure the readily releasable pool size and reveal that a form of homeostatic plasticity modulates this pool with synapse-specific and compartmentalized precision. This powerful approach can be utilized to illuminate the dynamics of synaptic vesicle trafficking and plasticity and determine how synaptic function adapts and deteriorates during states of altered development, stress and neuromuscular disease.

Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that allows sub-second detection of oxidizable chemical species, including monoamine neurotransmitters such as dopamine, norepinephrine, and serotonin. This technique has been used to record the physiological dynamics of these neurotransmitters in brain tissue, including their rates of release and reuptake as well as the activity of neuromodulators that regulate such processes. This protocol will focus on the use of ex vivo FSCV for the detection of dopamine within the nucleus accumbens in slices obtained from rodents. We have included all necessary materials, reagents, recipes, procedures, and analyses in order to successfully perform this technique in the laboratory setting. Additionally, we have also included cautionary points that we believe will be helpful for those who are novices in the field.

Studies over several decades on the organization of the CA1 hippocampus–a particularly favorable model for learning, memory and certain forms of cognition–have shown that the synaptic network in this brain region is plastic (Fortin et al., 2012). Recent evidence suggests that a number of environmental and endogenous stimuli may have a substantial effect on hippocampus-dependent cognitive function, implying enhanced synaptic plasticity in this brain region. Stimuli (e.g., food restriction, enriched environment, social interaction, gene-loss [knock-out animals], etc.) can trigger structural and functional plasticity (e.g., spine formation, increased expression of neurotrophic factors, synaptic function and neurogenesis) in the hippocampus (Stewart et al., 1989; Andrade et al., 2002; Babits et al., 2016). Using quantitative electron microscopy, we can study the synaptic neuropil of CA1 hippocampus in rodents during short- or long-term treatments and/or stimuli. Within the scope of this electron microscopic methodological construct, the density of various synaptic connections, the morphology and internal structure of excitatory spine synapses (e.g., the mean length and width of postsynaptic densities) can be quantified. Such quantitative ultrastructural measurement using high-resolution electron-microscopy may be applied to observe structural manifestations of synaptic plasticity in rodent brain tissue. The presented ultrastructural protocol may empower researchers to reveal details and synaptic changes which may not be obvious using only light microscopy. Ultrastructural data may provide substantial advances in our understanding of the changes in hippocampal synaptic architecture under different conditions.