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The Y-maze can be used to assess short term memory in mice. Spontaneous alternation, a measure of spatial working memory, can be assessed by allowing mice to explore all three arms of the maze and is driven by an innate curiosity of rodents to explore previously unvisited areas. A mouse with intact working memory, and hence intact prefrontal cortical functions, will remember the arms previously visited and show a tendency to enter a less recently visited arm. Spatial reference memory, which is underlined by the hippocampus, can also be tested by placing the test mice into the Y-maze with one arm closed off during training. After an intertrial interval of for example 1 h, the mouse should remember which arm it has not explored previously and should visit this arm more often. This chapter describes the pre-test conditions, the materials required and the protocol for conducting and interpreting the results of these two related tests.

This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.

The open field test is used in studies of the neurobiological basis of anxiety and screening for novel drug targets and anxiolytic compounds. This test uses a camera to measure movement of the test animal in the peripheral and central zones of a 42 x 42 x 42 cm polyvinyl chloride box. This chapter describes a protocol for carrying out the open-field test for assessment of locomotion and anxiety-like behavior in mice.

The identification of biomarkers for toxicity is becoming increasingly important for drug discovery and development. This chapter describes the preparation and utilization of primary rat hepatocytes as a cellular model of steatosis. A protocol is presented for dosing the cells with the steatosis-inducing compound amiodarone, along with the conduction of assays for measuring lipid accumulation and mitochondrial function. A differential solubility extraction procedure is also presented, which can be used for proteomic profiling analysis.

Psychiatric disorders affect approximately one quarter of people worldwide at some point in their lifetime. This chapter provides a step-by-step guide to conduct behavioral tests in adult mice for investigations of social behavior, without the need for specific equipment. This test should allow the identification of key abnormalities in social interactions that can be followed up by targeted, more complex, behavioral analysis aimed at identification of new biomarkers and potential drug targets.

This chapter presents characterization of the db/db mouse model of type 2 diabetes. The protocol describes generation of the mice along with measurements of physical characteristics such as body weight and body composition, and behaviours including food and water consumption. Such a procedure can also be used for omics-based analyses of brain tissues to investigate the potential use of db/db mouse model in neuropsychiatric research. In addition, we present measurement of blood glucose and plasma insulin levels which tend to be elevated in this model and can modulated post treatment with anti-diabetic agents. All of these characteristics can be used for studying the pathophysiology and for monitoring response to potential therapeutics.

The islets of Langerhans release vital hormones involved in the regulation of blood sugar and other aspects of metabolism. The islets are housed in diffuse clusters of cells within the exocrine pancreas and, therefore, purification of these cells for research or transplant purposes is a difficult undertaking. Here, a detailed protocol is presented for purification of islets from rat pancreas using limited collagenase digestion and step gradient centrifugation techniques. In addition, a method for assessing islet viability is presented using perifusion under both basal and stimulatory glucose conditions, with measurement of the hormone released using an immunoassay for insulin.

Obsessive-compulsive disorder (OCD) can occur in several psychiatric illnesses such as autism spectrum disorders (ASD) and it is more prevalent in children. This condition is characterized by repeated and apparently meaningless behaviors such as frequent hand washing, counting, tapping, and rocking. This can disrupt normal socialization and in some cases lead to self-harm. Therefore there is interest in developing more effective therapies for individuals suffering from these conditions. This chapter describes how to conduct the mouse marble burying test as a sensitive measure of compulsive behaviors.

The Goto-Kakizaki (GK) rat is a model of type 2 diabetes mellitus, produced originally by selective inbreeding for a hyperglycemic trait. These rats are characterized as having insulin resistance and an insulin secretory defect. Pancreatic islets from these mice show abnormal morphology in the distribution of glucagon-secreting α cells and insulin-secreting β cells, which may affect paracrine functions involved in the secretory response. This chapter describes the characterization of GK rat islets using immunofluorescence microscopy and Western blot analyses to demonstrate the effects on islet architecture and how this translates to changes in expression of predominant islet proteins.

The elevated plus maze test is used to measure anxiety-like behavior in rodents. It can be used to gain insight into conditions such as posttraumatic stress disorder (PTSD) and other conditions marked by anxious behavior. It can also be used as a component in screening of novel compounds for anxiolytic properties. This model is based on aversion to open spaces, which is seen as the animal spending more time in the enclosed arms of the maze. This chapter describes the steps necessary for setting up and conducting the test, along with interpretation of the results.

This chapter describes the propagation and characterization of transplantable insulinoma cells as model of insulin-producing pancreatic islet cells in the rat. Here, the cells are propagated by transplantation into rats followed by harvesting after growth for approximately 1 month. The cells are then purified by Percoll density gradient centrifugation and characterized by pulse-chase radiolabelling and immunoprecipitation of the insulin-related peptides. The results show that the transplantable insulinoma cells produce insulin in a manner similar to that found in normal pancreatic islets.

This chapter presents a method for assessing general behavior, well-being, and sensorimotor gating. A detailed protocol is given for assessment of nest building performance in female mice using a strict scoring system. The test can be used for assessing moods and behaviors associated with psychiatric disorders such as depression and schizophrenia. It can also be useful for testing movement disorders such as Parkinson disease.

The forced swim test assesses learned helplessness, which is a feature of depression-like behavior in rodents. This test has also been used in testing the efficacy of existing and novel antidepressant drugs. It is based on the natural tendency of rodents to escape from water. Rodents are placed in a cylinder filled with water and the presumption is that those with a depression-like phenotype give up swimming earlier than those that are not depressed. Furthermore, antidepressant drugs reverse this effect. This chapter describes the basic setup and conduction of the test, along with interpretation of the results. It should be emphasized that this test should be conducted as part of a series of behavioral assessments in order to increase the accuracy of the results.

Poor nutrition during pregnancy leads to an increased risk of metabolic disorders and other diseases in the offspring. This can be modelled in animals through manipulation of the maternal diet. One such model is the maternal low-protein rat which gives rise to offspring characterized by insulin resistance. This chapter gives a detailed protocol for generation of the maternal low-protein rat, which has been used in the study of several disorders including diabetes and psychiatric disorders.

Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

There is a strong association between a suboptimal maternal environment and increased risk of developing age-associated diseases such as type 2 diabetes, obesity, and cardiovascular disease in the offspring. Blood clotting time may be altered in all of these conditions, and it is also an important factor that requires monitoring in postoperative and cardiovascular disorder patients who are on coagulant medications. This chapter describes patient self-management of blood coagulation activity using a test strip device and the Coagu app. The app can also be used as a reminder of treatment times and for monitoring the effects of treatment over time.

Many diseases result from programming effects in utero. This chapter describes recent advances in proteomic studies which have improved our understanding of the underlying pathophysiological pathways in the major psychiatric disorders, resulting in the development of potential novel biomarker tests. Such tests should be based on measurement of blood-based proteins given the ease of accessibility of this medium and the known connections between the periphery and the central nervous system. Most importantly, emerging biomarker tests should be developed on lab-on-a-chip and other handheld devices to enable point-of-care use. This should help to identify individuals with psychiatric disorders much sooner than ever before, which will allow more rapid treatment options for the best possible patient outcomes.

Over the last few decades, evidence has emerged that the pathogenesis of psychiatric disorders such as schizophrenia can involve perturbations of the hypothalamic-pituitary-adrenal (HPA) axis and other neuroendocrine systems. Variations in the manifestation of these effects could be related to differences in clinical symptoms between affected individuals and to differences in treatment response. Such effects can also arise from the complex interaction between genes and environmental factors. Here, we review the effects of maternal stress on abnormalities in HPA axis regulation and the development of psychiatric disorders such as schizophrenia. Studies in this area may prove critical for increasing our understanding of the multidimensional nature of mental disorders and could lead to the development of improved diagnostics and novel therapeutic approaches for treating individuals who suffer from these conditions.

Lab-on-a-chip assays allow rapid analysis of one or more molecular analytes on an automated user-friendly platform. Here we describe a fully automated assay and readout for measurement of vitamin D levels in less than 15 min using the Fraunhofer in vitro diagnostics platform. Vitamin D (25-hydroxyvitamin D3 [25(OH)D3]) dilution series in buffer were successfully tested down to 2 ng/mL. This could be applied in the future as an inexpensive point-of-care analysis for patients suffering from a variety of conditions marked by vitamin D deficiencies.

Many chronic diseases are associated with hypothalamic-pituitary-adrenal axis dysfunction. Therefore, proteomic profiling of the pituitary gland has potential to uncover new information on the underlying pathways affected in these conditions. This could lead to identification of new biomarkers or drug targets for development of novel therapeutics. Here we present a protocol for preparation of pituitary protein extracts and analysis of the major hormones and accessory proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS). The same methods can be applied in the study of other tissues of the diffuse neuroendocrine system.

Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of immune function in studies of multiple medical areas, such as metabolic diseases and immune dysfunction. This chapter describes a standardized technique for blood draw and preparation of PBMCs from whole blood using density gradient centrifugation, followed by cell culture. The main focus is on collection of the PBMC culture media and extraction of cellular proteins in order to provide the materials for biomarker studies.

This chapter describes innovations in biomarker testing that can facilitate earlier and better treatment of patients who suffer from metabolic disorders. The use of new microfluidic devices along with miniaturized biosensors and transducers enables analysis of a single drop of a blood within the time frame of a typical visit to a doctor's office. Steps are underway so that these approaches will incorporate both biochemical and clinical data, resulting in unique bioprofiles for each patient. This will allow earlier, personalized, and more effective therapeutic options. In addition, smartphone apps for self-monitoring will be used increasingly for the best possible patient outcomes.

Early parental nutritional interventions during prenatal development have been shown to result in neuropsychiatric sequelae in the adult offspring. In order to understand the impact of such nutritional interventions, the behavior of the animal has to be carefully analyzed. This chapter provides a step-by-step guide to conduct behavioral tests in adult mice for investigators without specific expertise or those without the equipment to carry out behavioral studies. We focus on tests tapping into the main behavioral abnormalities that correspond to mental illnesses. We describe the materials required and the detailed methods to conduct global assessment of parameters such as behavioral integrity and general well-being, psychomotor activity, social behavior, repetitive behavior, anxiety-like behavior, depression-like behavior, short-term spatial working memory, and spatial reference memory.

Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labeled proteins can then be immunoprecipitated and analyzed by electrophoresis and gel imaging techniques. This chapter presents a protocol for the biosynthetic labeling and immunoprecipitation of pancreatic islet proteins which are known to be affected in disorders such as diabetes, obesity, and metabolic syndrome.

Under- or overfeeding during pregnancy can lead to behavioral deficits in the offspring in later life. Here, we present a protocol for setting up and carrying out the hyperlocomotion test for assessing behavioral symptoms such as psychosis or mania. As an example, we use the acute rat phencyclidine-injection model which exhibits hyperlocomotion and stereotypic behaviors, resembling the positive symptoms of schizophrenia.

Developmental programming resulting from maternal malnutrition can lead to an increased risk of metabolic disorders such as obesity, insulin resistance, type 2 diabetes and cardiovascular disorders in the offspring in later life. Furthermore, many conditions linked with developmental programming are also known to be associated with the aging process. This review summarizes the available evidence about the molecular mechanisms underlying these effects, with the potential to identify novel areas of therapeutic intervention. This could also lead to the discovery of new treatment options for improved patient outcomes.

In metabolic diseases such as obesity and type 2 diabetes mellitus, the conversion of proinsulin to mature insulin can be impaired. This could mean that insulin molecules with lower activity toward the insulin receptor can be released under conditions of high metabolic demand, resulting in an inadequate glucoregulatory response. The chapter describes a fluorescent monoclonal antibody-based protocol for measurement of human proinsulin and the proinsulin conversion intermediates (split proinsulins). An example assay is presented using serum from non-diabetic, normal body mass index individuals.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.

Proteomic tools can only be implemented in clinical settings if high-throughput, automated, sensitive, and accurate methods are developed. This has driven researchers to the edge of mass spectrometry (MS)-based proteomics capacity. Here we provide an overview of recent achievements in mass spectrometric technologies and instruments. This includes development of high and ultra definition-MSE (HDMSE and UDMSE) through implementation of ion mobility (IM) MS towards sensitive and accurate label-free proteomics using ultra performance liquid chromatography (UPLC). Label free UPLC-HDMSE is less expensive than labeled-based quantitative proteomics and has no limits regarding the number of samples that can be analyzed and compared, which is an important requirement for supporting clinical applications.

This chapter describes how current and future innovations driven by application of multiplex biomarker techniques can help in earlier and more efficacious treatment of patients, suffering from the world's most devastating and costly diseases. The application of new miniaturized biosensors and transducers will enable point-of-care testing by facilitating analysis of a single drop of a blood within the time span of a visit to the doctor's office. It is anticipated that the scoring algorithms used with future tests will incorporate both biochemical and clinical data, resulting in specific profiles for each patient or tested subject to enable personalized medicine approaches.

A major challenge in single or panel of biomarker discovery and validation is the inherent biological complexity underlying disease heterogeneity and inconsistent responses to treatment. Moreover, the lack of standardization in the sampling, processing, and storage of biological fluids such as plasma and serum disrupts the discovery and validation of blood-based biomarker tests in preclinical and clinical settings. This chapter presents a reproducible sample collection and handling procedure that aims to enhance analyte stability and ensure compatibility with the corresponding multiplex biomarker profiling platforms. The importance of defining bio-sample acquisition and processing, study design, and profiling platform guidelines for blood-based biomarker measurements is paramount for the success of personalized healthcare strategy and development of companion diagnostics.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well. The availability to multiplex therefore allows the measurement of the expression levels of several targets or genes of interest quickly. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.

This chapter describes the basics, applications, and limitations of two-dimensional gel electrophoresis (2DE) and two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of distinct proteomes. We also propose a basic protocol for 2D-DIGE, technique that allows the analysis of paired protein extracts, which are labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed with a Cy2-labeled standard extract on the same 2DE gels. Scanning the gels at wavelengths specific for each dye allows direct overlay the two different proteomes and the differences in abundance of specific protein spots can be determined.

Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with 35S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE. This provides a means of determining whether or not the biosynthetic rates of the entire granule constituents are coordinately regulated.

DNA microarrays contain microscopic DNA spots attached to a solid surface. Each spot contains picomolar levels of a specific DNA probe sequence and hybridization to the corresponding gene products can be detected and quantitated through the use of fluorescently labeled target DNA. In this format, DNA microarrays can be used to measure the expression level of thousands of genes in a single experiment. Here, we present a method to detect the mRNA transcriptional changes in neuronal precursor cells following differentiation using high density cDNA microarrays.

Multiplex biomarker tests are becoming an essential part of the drug development process. This chapter explores the role of biomarker-based tests as effective tools in improving preclinical research and clinical development, and the challenges that this presents. The potential of incorporating biomarkers in the clinical pipeline to improve decision making, accelerate drug development, improve translation, and reduce development costs is discussed. This chapter also discusses the latest biomarker technologies in use to make this possible and details the next steps that must undertaken to keep driving this process forwards.

Type 2 diabetes mellitus is a multifactorial condition resulting in high fasting blood glucose levels. Although its diagnosis is straightforward, there is not one set of biomarkers or drug targets that can be used for classification or personalized treatment of individuals who suffer from this condition. Instead, the application of multiplex methods incorporating a systems biology approach is essential in order to increase our understanding of this disease. This chapter reviews the state of the art in biomarker studies of human type 2 diabetes from a proteomic and metabolomic perspective. Our main focus was on biomarkers for disease prediction as these could lead to early intervention strategies for the best possible patient outcomes.

Psychiatric disorders are complex diseases involving exogenous and endogenous factors. Biomarkers for diagnosis or prediction of successful treatment are not existent. In addition, the molecular basis of these diseases is still poorly understood. Blood plasma represents the most complex proteome as it contains subproteomes from several body tissues. However, the high abundance of some little proteins can obscure the analysis of hundreds of low abundance proteins, which are potential biomarkers. Therefore, removal of these high abundance proteins is pivotal in any proteomic study of plasma. Here, we present a method of depleting these proteins using immunoaffinity liquid chromatography.