Current Protocols in Neuroscience

Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+ ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders...

Attentional set shifting is a measure of cognitive flexibility and executive functions widely assessed in humans by the Wisconsin Card Sorting Test (WCST) and the CANTAB Intra-/Extra-Dimensional set-shifting task (ID/ED). The recently established automated two-chamber "Operon ID/ED" task for mice has proved to be an effective preclinical tool for drug testing and genetic screening, with direct translational valence in healthy human subjects and patients with schizophrenia. Here, we describe an upgraded version of the Operon ID/ED task that is now commercially available...

Fluorescence lifetime microscopy (FLIM) and Förster's resonance energy transfer (FRET) are advanced optical tools that neuroscientists can employ to interrogate the structure and function of complex biological systems in vitro and in vivo using light. In neurobiology they are primarily used to study protein-protein interactions, to study conformational changes in protein complexes, and to monitor genetically encoded FRET-based biosensors. These methods are ideally suited to optically monitor changes in neurons that are triggered optogenetically...

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution...

Basic neuroscience research employs antibodies as key reagents to label, capture, and modulate the function of proteins of interest. Antibodies are immunoglobulin proteins. Recombinant antibodies are immunoglobulin proteins whose nucleic acid coding regions, or fragments thereof, have been cloned into expression plasmids that allow for unlimited production. Recombinant antibodies offer many advantages over conventional antibodies including their unambiguous identification and digital archiving via DNA sequencing, reliable expression, ease and reliable distribution as DNA sequences and as plasmids, and the opportunity for numerous forms of engineering to enhance their utility...

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results...

MagellanMapper is a software suite designed for visual inspection and end-to-end automated processing of large-volume, 3D brain imaging datasets in a memory-efficient manner. The rapidly growing number of large-volume, high-resolution datasets necessitates visualization of raw data at both macro- and microscopic levels to assess the quality of data, as well as automated processing to quantify data in an unbiased manner for comparison across a large number of samples. To facilitate these analyses, MagellanMapper provides both a graphical user interface for manual inspection and a command-line interface for automated image processing...

Elucidating the complex neural mechanisms that underlie cognition is contingent upon our ability to measure behavioral outputs reliably in animal models. While the development of open-source software has made behavioral science more accessible, behavioral research remains underappreciated and underutilized. One reason is the large real estate necessitated by traditional behavioral setups. Space must be specifically allocated for a controlled testing environment, accommodate the large footprint of mazes used in behavioral research, and allow a contiguous computerized area for data acquisition...

Recombinant adeno-associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last decades, rAAVs have become the gold standard for studying in vivo gene function and are especially favorable for gene transfer in the central nervous system. AAVs have been proven safe and provide stable gene expression over a long period of time...

Deficits in decision making are at the heart of many psychiatric diseases, such as substance abuse disorders and attention deficit hyperactivity disorder. Consequently, rodent models of decision making are germane to understanding the neural mechanisms underlying adaptive choice behavior and how such mechanisms can become compromised in pathological conditions. A critical factor that must be integrated with reward value to ensure optimal decision making is the occurrence of consequences, which can differ based on probability (risk of punishment) and temporal contiguity (delayed punishment)...

Ex vivo rodent whole nerves provide a model for assessing the effects of interventions on nerve impulse transmission and consequent sensory and/or motor function. Nerve impulse transmission can be measured through sciatic nerve compound action potential (CAP) recordings. However, de novo development and implementation of an ex vivo whole nerve resection protocol and an electrophysiology setup that retains nerve viability, that produces low noise CAP signals, and that allows for data analysis is challenging...

Utilization of functional ultrasound (fUS) in cerebral vascular imaging is gaining popularity among neuroscientists. In this article, we describe a chronic surgical preparation method that allows longitudinal studies and therefore is applicable to a wide range of studies, especially on aging, stroke, and neurodegenerative diseases. This method can also be used with awake mice; hence, the deleterious effects of anesthesia on neurovascular responses can be avoided. In addition to fUS imaging, this surgical preparation allows researchers to take advantage of common optical imaging methods to acquire complementary datasets to help increase the technical rigor of studies...

GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity...

Expansion microscopy (ExM) is a technique that physically expands preserved cells and tissues before microscope imaging, so that conventional diffraction-limited microscopes can perform nanoscale-resolution imaging. In ExM, biomolecules or their markers are linked to a dense, swellable gel network synthesized throughout a specimen. Mechanical homogenization of the sample (e.g., by protease digestion) and the addition of water enable isotropic swelling of the gel, so that the relative positions of biomolecules are preserved...

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAARs), exhibit highly dynamic trafficking and cell surface mobility. Regulated trafficking to and from the surface is a critical determinant of GABAAR neurotransmission. Receptors delivered by exocytosis diffuse laterally in the plasma membrane, with tethering and reduced movement at synapses occurring through receptor interactions with the subsynaptic scaffold. After diffusion away from synapses, receptors are internalized by clathrin-dependent endocytosis at extrasynaptic sites and can be either recycled back to the cell membrane or degraded in lysosomes...

We describe a set of protocols for doing visual psychophysical experiments in head-fixed mice. The goal of this approach was to conduct in mice the same type of precise and well-controlled tests of visual perception and decision making as is commonly done in primates. For example, these experimental protocols were the basis for our demonstration that mice are capable of visual selective attention in paradigms adapted from classic attention cueing paradigms in primates. Basic Protocol 1 describes how to construct the experimental apparatus, including the removable wheel assembly on which the mice run during the visual tasks, the lick spout used to deliver rewards and detect licks, and the behavioral box that places these components together with the visual displays...

Mitochondria are essential organelles involved in energy supply and calcium homeostasis. The regulated distribution of mitochondria in polarized cells, particularly neurons, is thought to be essential to these roles. Altered mitochondrial function and impairment of mitochondrial distribution and dynamics is implicated in a number of neurologic disorders. Several recent reports have described mechanisms regulating the activity-dependent distribution of mitochondria within astrocyte processes and the functional consequences of altered mitochondrial transport...

Astrocytes play numerous vital roles in the central nervous system. Accordingly, it is of merit to identify structural and functional properties of astrocytes in both health and disease. The majority of studies examining the morphology of astrocytes have employed immunoassays for markers such as glial fibrillary acidic protein, which are insufficient to encapsulate the considerable structural complexity of these cells. Herein, we describe a method utilizing a commercially available and validated, genetically encoded membrane-associated fluorescent marker of astrocytes, AAV5-GfaABC1D-Lck-GFP...

Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses...

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins...