1Inserm, U1191, Institute of Functional Genomics, 2CNRS, UMR-5203, 3Université de Montpellier
1Department of Emergency Medicine, University of Cincinnati College of Medicine
1Department of Neurology, Johns Hopkins University
The light microscope is an instrument used by researchers in many different fields to magnify specimens to as much as a thousand times their original size. In its simplest form, it is composed of a clear lens that magnifies the sample and a light source to illuminate it. However, most light microscopes are much more complex and house numerous fine-tuned lenses with tightly controlled dimensions all within the body of the microscope itself and in components such as the objectives and eyepieces. In this video, the major components of the light microscope are described and their uses and functions are explained in detail. The basic principles of magnification, focus, and resolution are also introduced. Basic light microscope operation begins with bringing light to the sample and ensuring that the light source is of the correct intensity, directionality, and shape in order to produce the best quality image. Next, the sample must be magnified properly and brought into focus to view the region of interest. There are many practical applications for light microscopy including the viewing of stained or unstained cells and tissues, resolving small details of specimens, and even magnifying a region of interest during surgery to assist with complex procedures on the micron scale.…
General Laboratory Techniques
Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.
This video reviews proper experimental design when using these oligonucleotides. Following that, an explanation of morpholino microinjection techniques in zebrafish and the analysis of resulting phenotypes will be discussed. Finally, we showcase examples of specific applications where morpholino technology is used to model developmental disorders or to study tissue regeneration.…
Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are unfamiliar with or have difficulty adjusting to. Since it is hard to examine mechanisms of social stress in humans, modeling this condition in animals may help scientist in developing new therapies for treating this commonly encountered problem.
This science education video begins by discussing the known anatomy and physiology behind stress response. Then, we explain a well-established paradigm for modeling social stress in rodents, the Resident-Intruder task. In the applications section, we review some example studies in which response to stress is measured.…
Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One extremely useful technique for investigating the biophysical properties of these channels is called patch clamp recording. In this method, neuroscientists place a polished glass micropipette against a cell and apply suction to form a high resistance seal. This process isolates a small “patch” of membrane that contains one or more ion channels. Using an electrode within the micropipette, researchers can “clamp” or control the electrical properties of the membrane, which is important for analysis of channel activity. The electrode also allows for changes in the voltage across the membrane, or the flow of ions through the membrane, to be recorded. This video begins with an overview of the principles behind patch clamp electrophysiology, an introduction to the necessary equipment, and descriptions of the various patch configurations, including whole cell, cell-attached, perforated, inside-out, and outside-out patches. Next, the key steps of a typical whole-cell patch clamp experiment are outlined, in which a current-voltage (IV) curve is generated. Finally, applications of patch clamp recording are provided to demonstrate how the biophysical propert…
The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to their small size, large numbers of zebrafish can be housed in the laboratory at low cost. Although zebrafish are relatively easy to maintain, special consideration must be given to both diet and water quality to in order to optimize fish health and reproductive success.
This video will provide an overview of zebrafish husbandry and maintenance in the lab. After a brief review of the natural zebrafish habitat, techniques essential to recreating this environment in the lab will be discussed, including key elements of fish facility water recirculation systems and the preparation of brine shrimp as part of the zebrafish diet. Additionally, the presentation will include information on how specific zebrafish strains are tracked in a laboratory setting, with specific reference to the collection of tail fin samples for DNA extraction and genotyping. Finally, experimental modifications of the zebrafish environment will be discussed as a means to further our understanding of these fish, and in turn, ourselves.…
Researchers study learning of a behavior through the use of operant conditioning. This type of learning involves associating the behavior with a consequence, which is a reward or punishment. If the consequence is a reward, it leads to reinforcement of the desired behavior. One type of reinforcement approach is positive reinforcement, where the behavior is rewarded with an artificial, natural, or social reinforcer. Studies using positive reinforcement as a tool can help tease out important details about neurological functioning associated with different behaviors.
This video reviews the concepts behind reinforcement studies by using an example of a man training a dog to sit. Following this, we look at a generalized procedure of positive reinforcement commonly used by behavioral researchers. This involves, training rodents to perform a behavior (lever press) to get a reward (food). Lastly, specific applications demonstrate how scientists use positive reinforcement to understand behavior.…
Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
Here, the neurobiology behind spatial memory is briefly reviewed and some general principles behind maze tests are discussed. Then, the video explains generalized protocols on how to conduct trials with specific types of mazes. Lastly, it will explain how behavioral researchers are using these tools to conduct some specific experiments.…
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and changes in cell morphology. Studying these processes is essential to our understanding of nervous system function as well as our ability to diagnose and treat disorders that result from abnormal development. However, neural tissues are relatively inaccessible for experimental manipulations, especially in embryonic mammals. As a result, many scientists take advantage of explant culture in order to study neurodevelopmental processes in an “organotypic” environment, meaning that the tissue is removed from the organism but its complex cellular architecture is maintained. Generally, explant cultures are created by careful dissection of neural tissue that is then submerged in carefully designed growth media and cultured in vitro.
This video will first provide a brief overview of neural explant culture, including its advantages over other in vitro methods and important considerations for maintaining healthy tissue. Next, a general protocol will be provided for setting up an explant culture from embryonic mouse brain, outlining the isolation of embryos from the mother and dissection of the brain. The presentation also includes an overview of slice culture…
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production