Abstract: This unit presents two basic protocols that offer rapid assessments of anosmia (the absence of a sense of smell) in mice The buried food test is used to check for the ability to smell volatile odors The olfactory habituation/dishabituation test is used to test whether the animal can detect and differentiate different odors, including both nonsocial and social odors A non-contact method of odor presentation, along with a general method for collecting urine samples, is given as an alternate protocol The tests described in this unit only require simple equipment and can be adopted readily by most laboratories

Abstract: The technique of microdialysis enables sampling and collecting of small-molecular-weight substances from the interstitial space. It is a widely used method in neuroscience and is one of the few techniques available that permits quantification of neurotransmitters, peptides, and hormones in the behaving animal. More recently, it has been used in tissue preparations for quantification of neurotransmitter release. This unit provides a brief review of the history of microdialysis and its general application in the neurosciences. The authors review the theoretical principles underlying the microdialysis process, methods available for estimating extracellular concentration from dialysis samples (i.e., relative recovery), the various factors that affect the estimate of in vivo relative recovery, and the importance of determining in vivo relative recovery to data interpretation. Several areas of special note, including impact of tissue trauma on the interpretation of microdialysis results, are discussed. Step-by-step instructions for the planning and execution of conventional and quantitative microdialysis experiments are provided.

Abstract: Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.

Abstract: Alterations in multiple biological functions, such as transcription factor activity, are implicated in the neurobiology of depression, based primarily on the characterization of antidepressant efficacy in naive rodents rather than on models that capture the protracted feelings of anhedonia and helplessness that typify depression. This unit presents rat and mouse models of depression that involve chronic oral exposure to the stress-associated adrenal hormone, corticosterone (CORT), resulting in anhedonic- and helplessness-like behaviors that are persistent yet reversible by chronic antidepressant treatment. Prior CORT exposure also chronically influences molecular targets hypothesized to contribute to negative mood. One example is phosphorylation of cAMP response element binding protein in the hippocampus and nucleus accumbens. Prior chronic CORT exposure provides an alternative method to chronic mild stress models of depression that is easily replicable and persists well beyond the CORT exposure period, thereby modeling the persistent depressive-like state in humans.

Abstract: Amino acid analysis is used to determine the amino acid content of amino acid-, peptide- and protein-containing samples. With minor exceptions, proteins are long linear polymers of amino acids connected to each other via peptide bonds. The first step of amino acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are then separated, detected, and quantified. The method was first developed by Moore, Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable effort by many workers, the basic methodology remains relatively unchanged. This unit provides an overview and strategic planning for amino acid analysis, discussing a range of methodologies and issues. In addition, several common methods used for analysis of L-amino acids are described in detail, including: HCl acid hydrolysis, performic acid oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis, analysis of free amino acids, and analysis of reactive lysine.

Abstract: The electroencephalogram (EEG) is the most common tool used in sleep research. This unit describes the methods for recording and analyzing the EEG. Detailed protocols describe recorder calibration, electrode application, EEG recording, and computer EEG analysis with power spectral analysis. Computer digitization of an analog EEG signal is discussed, along with EEG filtering and the parameters of fast Fourier transform (FFT) power spectral analysis. Sample data are provided for a typical night's analysis of EEG during NREM (non-REM) and REM sleep.

Abstract: Parkinson disease (PD) is a progressive neurologic condition that causes motor and nonmotor manifestations. Treatment provides symptomatic benefit but no current treatment has been proven to slow disease progression. Research studies of PD require a means of rating the severity of disease by measurement of motor manifestations, assessment of ability to perform daily functional activities, and symptomatic response to medication. The most common rating scales are the Unified Parkinson Disease Rating Scale (UPDRS), Hoehn and Yahr staging, and the Schwab and England rating of activities of daily living. Each of these rating scales are described, including detailed instructions on how to implement these ratings. Although these are the most widely applied rating scales of PD, there are still substantial limitations to these scales that must be considered when using them for research. Finally, some common applications of these scales are described.

Abstract: Proteomics has evolved from genomic science due to the convergence of advances in protein chemistry, separations, mass spectroscopy, and peptide and protein databases. Where identifying protein-protein interactions was once limited to yeast two-hybrid analyses or empirical data, protein-protein interactions can now be examined in both cells and native tissues by precipitation of the protein complex of interest. Coupling this field to receptor pharmacology has recently allowed for the identification of proteins that differentially and selectively interact with receptors and are integral to their biological effects. It is becoming increasingly apparent that receptors in neurons do not exist as singular independent units, but rather are part of large macromolecular complexes of interacting proteins. It is a primary quest of neuroscience to piece together these interactions and to characterize the regulatory signalplexes of all proteins. This unit presents co-immunoprecipitation-coupled mass spectroscopy as one way of identifying signalplex partners.

Abstract: In recent years, several methods have been developed to analyze protein-protein interactions under native conditions. One of them, tandem affinity purification (TAP), combines two affinity-purification steps to allow isolation of high-purity protein complexes. This unit presents a methodological workflow based on an SF-TAP tag comprising a doublet Strep-tag II and a FLAG moiety optimized for rapid as well as efficient tandem affinity purification of native proteins and protein complexes in higher eukaryotic cells. Depending on the stringency of purification conditions, SF-TAP allows both the isolation of a single tagged-fusion protein of interest and purification of protein complexes under native conditions.

Abstract: By taking advantage of labeling methods such as hydrogen exchange (HX), many details about protein conformation, dynamics, and interactions can be revealed by mass spectrometry. In this unit, hydrogen exchange theory is discussed as it applies to HX-MS protocols, the practice of HX-MS including data analysis and interpretation is explained in detail, and recent advancements in technology which greatly increase the depth of information gained from the technique are highlighted.

Abstract: Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.

Abstract: The Fab molecule was the first generated antibody fragment and still dominates basic research and clinical applications. This unit describes the E. coli expression and purification of Fab antibody fragments with and without a His tag, and is designed to yield sufficient protein for the evaluation and characterization of a panel of Fab selected from a Fab library by phage display.

Abstract: Mouse embryonic stem (ES) cells are derived from mouse blastocyst and are able to generate all embryonic tissues in vitro. This propensity of ES cells has acquired considerable attention in recent years due to the promising potential for future cell replacement-based therapies. Therefore, it is of fundamental interest to establish protocols that allow the differentiation of ES cells into specific cell types. In recent years, several such differentiation procedures have been described for mouse and human embryonic stem cells. This unit describes a simple procedure that promotes the neuronal differentiation of mouse embryonic stem cells and yields a high proportion of midbrain dopaminergic neurons. Furthermore, this procedure permits the isolation of neural stem cell lines from mouse ES cells.

Abstract: The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution over a wide range of time scales from milliseconds to hours. In addition to providing topographical images of surfaces with nanometer- to angstrom-scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated from the nano-scale to the micro-scale. Importantly, the measurements are made in buffer solutions, allowing biological samples to “stay alive” within a physiological-like environment while temporal changes in structure are measured—e.g., before and after addition of chemical reagents. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, e.g., electron microscopy (EM). This unit provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins, cells, and tissues. The physical principles of the technique and methodological aspects of its practical use and applications are also described. © 2016 by John Wiley & Sons, Inc. Keywords: topography; force spectroscopy; manipulation; fluorescence microscopy; vibrational spectroscopy; high speed scanning

Abstract: In this overview, the Fab molecule is introduced and discussed as the first generated antibody fragment, which still dominates basic research and clinical applications. The unit contains a concepts section and an applications section. In the concepts section, the two principal methods for producing Fab, as well as the generation and directed evolution of Fab by phage display, are described. The applications section discusses Fab in clinical applications, as well as their increasingly important role in the determination of the three-dimensional structures of transmembrane proteins.

Abstract: Despite the development of various transfection methods, the transfection of post-mitotic cells, including neurons, poses a challenging task Nucleofection, a specialized form of electroporation described in this unit, achieves high transfection efficiencies in primary mammalian neurons, such as hippocampal neurons, while simultaneously maintaining high cell viability Therefore, it allows for biochemical analyses that rely on large numbers of transfected cells The recently developed 96-well shuttle system described in this unit further permits the transfection of up to 96 different constructs in a single experiment This opens up the possibility for large-scale experiments in primary neurons, such as shRNA-mediated knock-down of a wide range of target genes

Abstract: Methods for determining protein concentration that use progressively smaller amounts of material are continually being developed. A new way of minimizing the amount of sample used for spectroscopic analysis is providing more opportunities for greater quality control. Traditional spectrophotometric and fluorometric methods for determination of protein concentrations have long required placing samples into containment devices such as cuvettes or capillaries. A microsample retention system is changing that paradigm by using natural surface tension properties to capture and hold microvolume samples in place during measurement without traditional containment devices. The advantage of such a system is to dramatically reduce the amount of sample required (1 to 2 µl) while greatly increasing the dynamic range of protein concentrations that can be measured. Modifications to classic protein concentration determination protocols are presented to provide a microvolume alternative to traditional cuvette-based methods. Curr. Protoc. Protein Sci. 55:3.10.1-3.10.16. © 2009 by John Wiley & Sons, Inc. Keywords: microvolume; microvolume spectrophotometer; microvolume fluorospectrometer; microsample; microvolume spectroscopy; protein concentration; microsample analysis; microvolume analysis

Abstract: Tandem affinity purification (TAP) is a very efficient method to isolate proteins, protein complexes, or ribonucleoprotein particles from crude extracts. The method depends on the expression of one protein component fused N- or C-terminally to a TAP tag in the organism of interest. The TAP tag is a composite tag consisting of two different epitope domains and a protease cleavage site, and it facilitates the purification of the tagged protein in two consecutive, high-affinity chromatography steps. Combined, the two steps are typically so efficient that a protein complex can be purified virtually to homogeneity without the need for protein overexpression. If the tag does not interfere with protein function, TAP is likely to yield an intact protein complex because all steps of the procedure are carried out under nondenaturing conditions. In this unit, a TAP procedure is detailed which employs a novel epitope combination termed PTP.

Abstract: Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5‐aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site‐specific fashion. This reaction is applicable to proteins in solution and on the living cell surface. The method described in this unit only requires incubation of the target protein, which has been engineered to contain a sortase recognition site either at the C terminus or within solvent‐accessible loops, with a purified sortase enzyme and a suitably functionalized oligoglycine peptide. Curr. Protoc. Protein Sci. 56:15.3.1‐15.3.9. © 2009 by John Wiley & Sons, Inc.

Abstract: Alzheimer's disease (AD) is the most common form of dementia in older adults. It represents a significant public health concern because of its associated personal, social, and economic burden. As such, AD is the focus of considerable research worldwide. This unit reviews the major cognitive and behavioral impairments associated with AD, and the practical application of current neuropsychological procedures used to assess these deficits.

Abstract: This unit describes an acceptor-sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end-point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live-cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided.

Abstract: This unit describes the materials, methods, and analytical techniques available for the study of electrical activity of neural tissue in rodents in both homeostatic and disease states, with emphasis on epileptogenesis A table containing a list of suppliers of relevant materials and equipment is also provided

Abstract: This unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Although these techniques provide less information than complete sequencing of the sugar chains, the partial structural information derived is sufficient for many purposes. In the basic procedure presented in this unit, actively growing cell cultures are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady-state level of incorporation. In alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse-chase labeling procedure can be used to analyze precursor-product relationships. With sequential pulse-labeling, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimal amount of labeled precursor by using the same medium to pulse-label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.

Abstract: Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [14C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-l-isoaspartyl methyltransferase that converts aberrant l-isoaspartyl residues in peptides and proteins to normal aspartyl residues. Curr. Protoc. Protein Sci. 55:14.4.1-14.4.27. © 2009 by John Wiley & Sons, Inc. Keywords: protein oxidative modification; carbonylation; cysteine; dityrosine; isoaspartate; nitrotyrosine

Abstract: This unit presents a rapid and simple method for the nonchromatographic purification of recombinant proteins expressed in E. coli. This method relies on a thermally responsive elastin-like polypeptide (ELP) tag, where the tagged protein is precipitated using a mild temperature shift. The tag is then induced to self-cleave by a mild pH shift and is subsequently removed by a final thermal precipitation. The result is a purified native protein target, without the requirement for affinity apparatus or protease removal of the tag. This protocol describes the required cloning methods to insert a given target into the expression vector, as well as the general method for purifying the resulting expressed protein.

Abstract: In the 1950s, the discovery of anti-nerve growth factor, an immunotoxin stunting sympathetic neural development, signaled the advent of neurotoxins as research modalities. Other selective neurotoxins were discovered in rapid succession. In the 1960s, 6-hydroxydopamine and 6-hydroxydopa were shown to destroy noradrenergic and dopaminergic nerves. Excitotoxins (glutamate, aspartate, and analogs) were discovered in the 1970s. DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] proved to be selective for noradrenergic destruction, while 5,6- and 5,7-dihydroxytryptamines were relatively selective for serotonin neurons. Additional neurotoxins were discovered, but it was MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) that predominated neurotoxicity research in the 1980s. Eventually, Clostridium botulinum neurotoxin (BoNT), discovered as a "poisonous" principle in the late 1800s, resurfaced in purified and standardized forms as a clinically useful drug. Neurotoxins represent chemical tools, useful not only for discerning neuronal mechanisms and animal modeling of neurological disorders, but also for their use in medicine and potential as treatments for medical disorders. This unit reviews the early discovery of neurotoxins, describes categories of neurotoxins, and finally characterizes their usefulness--first as research tools, and eventually as clinical therapeutic agents.

Abstract: Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background-free immunostaining on the chip-based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high-density peptide arrays. Curr. Protoc. Protein Sci. 57:18.2.1-18.2.13. © 2009 by John Wiley & Sons, Inc. Keywords: solid phase peptide synthesis; peptide array; combinatorial synthesis; atom transfer radical polymerization (ATRP); amino acid particles

Abstract: Protein sequence comparison and search has become commonplace not only for bioinformatics researchers but also for experimentalists in many cases. Because of the exponential growth in sequence data, sequence comparison in particular has become an increasingly important tool. Relating a new gene sequence to other known sequences often reveals its function, structure, and evolution. Many sequence comparison and search tools are available through public Web servers, and biologists can use them easily with little knowledge of computers or bioinformatics. This unit provides some theoretical background and describes popular tools for dot plot, sequence search against a database, multiple sequence alignments, protein tree construction, and protein family and motif search. Step-by-step examples are provided to illustrate how to use some of the most well-known tools. Finally, some general advice is given on combining different sequence analysis tools for biological inference. Curr. Protoc. Protein Sci. 56:2.1.1-2.1.27. © 2009 by John Wiley & Sons, Inc. Keywords: protein sequence comparison; dot plot; multiple sequence alignment; protein tree; protein family; motif search

Abstract: This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included.