This manuscript was supported by NIH/NIGMS K08 123321 (to N.L.) and by funds from the Department of Anesthesiology at the University of Virginia. The Authors wish to thank Alev Erisir (Department of Biology, University of Virginia, Charlottesville, VA) for excellent training and technical assistance with TEM, and for her invaluable manuscript criticism. The Authors also thank the Advanced Electron Microscopy facility at the University of Virginia for technical assistance with specimen sectioning and post-staining.

<ol>
	<li>Lunardi, N., Oklopcic, A., Prillaman, M., Erisir, A., Jevtovic-Todorovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+exposure+to+general+anesthesia+disrupts+spatial+organization+of+presynaptic+vesicles+in+nerve+terminals+of+the+developing+subiculum.">Early exposure to general anesthesia disrupts spatial organization of presynaptic vesicles in nerve terminals of the developing subiculum.</a> Molecular Neurobiology. 52 (2), 942-951 (2015).</li><li>Lunardi, N., Ori, C., Erisir, A., Jevtovic-Todorovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anesthesia+causes+long-lasting+disturbances+in+the+ultrastructural+properties+of+developing+synapses+in+young+rats.">General anesthesia causes long-lasting disturbances in the ultrastructural properties of developing synapses in young rats.</a> Neurotoxicity Research. 17 (2), 179-188 (2010).</li><li>Sanchez, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anesthesia+causes+long-term+impairment+of+mitochondrial+morphogenesis+and+synaptic+transmission+in+developing+rat+brain.">General anesthesia causes long-term impairment of mitochondrial morphogenesis and synaptic transmission in developing rat brain.</a> Anesthesiology. 115 (5), 992-1002 (2011).</li><li>Boscolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+abolishment+of+anesthesia-induced+cognitive+impairment+by+timely+protection+of+mitochondria+in+the+developing+rat+brain:+the+importance+of+free+oxygen+radicals+and+mitochondria+integrity.">The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: the importance of free oxygen radicals and mitochondria integrity.</a> Neurobiology of Disease. 45 (3), 1031-1041 (2012).</li><li>Aghajanian, G. 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The Authors have nothing to disclose.

Handling of tissue sections during specimen preparation for TEM requires a considerable degree of finesse, concentration and patience. When using a micropipette to add and remove solutions, specimens can be sucked into the pipette tip by surface tension, so great care should be taken to avoid tissue damage by the pipette. Also, certain steps of the dehydration sequence can be as quick as 1 min, hence the operator needs to work swiftly to ensure that the next dehydration step is started on time and the specimen does not d...

We describe methods for the preparation of newborn rat brain tissue to obtain high-quality electron micrographs for in-depth morphometric analysis of synaptic vesicle spatial distribution at nerve terminals1,2. The high-contrast micrographs that can be obtained by processing specimens following these methods can also be used to study the detailed morphology of a number of cellular components and their dimensional structural relations3,4.
The transmission electron microscope (TEM) is a powerful tool to study the morphology of organelles and other cellular structures quantitatively. As of this decade, there are no other methods of investigation that can provide the same degree of resolution of lipid membranes and organelles without immuno-tagging, with the exception of cryofixation by high pressure freezing. However, freeze substitution techniques are not widely used, and normally require expensive equipment and long preparation times.
In order to take advantage of TEM&#39;s high resolving power, optimal specimen preparation is of paramount importance. The main goals of specimen preparation are to preserve tissue structure with minimum alteration from the living state, enhance specimen contrast, and stabilize the tissue against extraction of cellular components during processing and exposure to the electron beam. Numerous protocols for TEM tissue preparation have been introduced and perfected by several laboratories over the years. Many of them have focused on methods for optimal visualization of synaptic vesicles5,6,7,8,9,10,11. Among a number of well-established, gold standard methods currently in use, we chose procedures for chemical fixation, post fixation, en bloc staining, sequential dehydration, resin embedding and post staining that aim to preserve optimal tissue structure and achieve excellent image contrast. Of note, preservation of fine ultrastructure can be particularly challenging when working with newborn rat brain tissue. In fact, the central nervous system of very young animals is characterized by a higher water content than the adult brain, more prominent enlargement of extracellular spaces, and looser connections between cells12. This makes newborn rat brain tissue profoundly sensitive to changes in osmolarity, and exquisitely prone to artifactual shrinkage and/or swelling when processed through sequential solutions of different tonicity12. Therefore, our methods employed solutions for specimen processing that are of osmolarity as close as possible to that of rat newborn brain. Our goal was to obtain high-quality, high-resolution electron microscopy images for quantitative assessment of synaptic vesicle spatial distribution at nerve terminals. Specifically, we sought to measure the number of vesicles within the nerve terminal, the distance of synaptic vesicles from the pre-synaptic plasma membrane, the number of vesicles docked at the pre-synaptic membrane, the size of synaptic vesicles and the inter-vesicle distances1.
Satisfactory chemical fixation is a prerequisite for obtaining high-quality electron micrographs that can provide the morphological detail necessary to study synaptic vesicle morphology and spatial organization. Although several modes of fixation exist, fixation of brain tissue by vascular perfusion is decidedly superior to other methods. Since fixation via vascular perfusion begins immediately after the arrest of systemic circulation, it shortens the interval between deoxygenation of the brain tissue and cross-linking of proteins with fixatives, resulting in minimum alterations in cell structure. Furthermore, it accomplishes fast and uniform penetration, because of the rapid flow of the fixative from the vascular bed to the extracellular and cellular compartments12,13,14. Primary fixation with glutaraldehyde, followed by secondary fixation (post-fixation) with osmium tetroxide, yields excellent preservation of the fine structure15,16,17. A mixture of glutaraldehyde and paraformaldehyde has the additional advantage of more rapid penetration into the tissue12.
Since biological tissues are not sufficiently rigid to be cut into thin sections without the support of a resin matrix, they need to be embedded in a medium before thin sectioning. Water-immiscible epoxy resins are commonly used as an embedding medium in TEM. When this type of matrix is used, all specimen&#39;s free water must be replaced with an organic solvent before resin infiltration. Water is removed by passing the specimen through a series of solutions of ascending concentrations of ethanol and/or acetone12. In this protocol, specimens are first flat embedded between flexible aclar sheets, then embedded in a capsule. The final result is tissue situated at the tip of a cylindrical resin block, which has the ideal geometry to be least affected by vibrations arising during microtome sectioning.
Staining with heavy metals to enhance endogenous tissue contrast is another important aspect of specimen preparation. Image contrast in TEM is due to electron scattering by the atoms in the tissue. However, biological materials consist largely of low atomic weight molecules (i.e., carbon, hydrogen, oxygen and nitrogen). Therefore, the generation of sufficient scattering contrast requires the incorporation of high atomic weight atoms into the cellular components of the tissue. This is achieved through staining of the specimen with heavy metals12,18,19. Osmium tetroxide, uranium and lead, which bind strongly to lipids, are the most common heavy metals used as electron stains.
Osmium (atomic number 76) is one of the densest metals in existence. It is both a fixative and a stain, although its primary role in TEM is as a reliable fixative12. Among various fixation protocols in use, the method of double fixation with glutaraldehyde followed by osmium is the most effective in reducing the extraction of cell constituents during specimen preparation. These two fixatives are used to stabilize the maximum number of different types of molecules, especially proteins and lipids, and result in superior preservation of tissue ultrastructure12,14,15,16,17.
Uranium (atomic number 92) is the heaviest metal used as electron stain, most typically in the form of uranyl acetate. Similarly to osmium, it acts as a stain and fixative, although its primary role in TEM is as a stain20,21. Nucleic acid-containing and membranous structures are strongly and preferentially stained with uranyl salts in aldehyde-fixed tissues22,23. Treatment of tissues with uranyl acetate after osmication and before dehydration results in stabilization of membranous and nucleic acid-containing structures, as well as enhanced contrast, and permits identification of some structural details that would not be easily detected in specimens stained with osmium alone12,24,25. It is thought that uranyl acetate may stabilize the fine structure by combining with reduced osmium that has been deposited on lipid membranes during osmication24. Maximum contrast is achieved when uranyl acetate is applied before embedding and as a post-stain in thin sections12.
Lead (atomic number 82) is the most common stain used for TEM and is mainly employed for post-staining of thin sections. Lead salts have high electron opacity and show affinity for a wide range of cellular structures, including membranes, nuclear and cytoplasmic proteins, nucleic acids and glycogen26,27. When the double staining method is employed (i.e., staining with uranyl acetate is followed by treatment with lead), the latter acts as a developer of uranyl acetate staining. For instance, lead post-staining of chromatin fixed with glutaraldehyde increases uranyl acetate uptake by a factor of three28,29,30,31,32. Lead also enhances the staining imparted by other metals such as osmium. It is thought that lead salts stain the membranes of osmium-fixed tissues by attaching to the polar group of phosphatides in the presence of reduced osmium33. A potential disadvantage of staining with both uranyl acetate and lead, especially for prolonged durations, is that many different structural elements are stained equally and non-specifically, and thus may not be easily distinguished from one another12.
The recent introduction of alternative light sources, such as in optical super-resolution photo-activated localization microscopy, has significantly improved light microscopy resolution34. However, because light microscopy relies on histochemical and immune-cytochemical methods to visualize individually-labelled proteins or enzymes, the power of TEM to display all structural elements at once remains unsurpassed for in-depth study of the morphology and dimensional relationships of tissue structures. In particular, no other technique can provide the morphological detail necessary to perform morphometric analysis of synaptic vesicle distribution at pre-synaptic nerve boutons. Nevertheless, it is important to note that electron micrographs capture the structure of the tissue after the organism dies, and therefore they cannot provide information regarding the dynamics of pre-synaptic vesicle trafficking and exocytosis. Hence, other tools, such as FM dye-live imaging and patch-clamp electrophysiology, should be considered when the main objective is to study dynamic and/or functional aspects of synaptic vesicle trafficking and exocytosis.

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preparation of newborn rat brain tissue for ultrastructural morphometric analysis of synaptic vesicle distribution at nerve terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

General criteria that are mostly accepted as indicative of satisfactory or defective preservation of specimen for TEM have been established. These criteria are exemplified in four selected electron micrographs (two examples of optimal preparation, two examples of defective preparation) that were obtained by treating young rat brain tissue following the methods described in this protocol.
In general, a good-quality electron micrograph appears as an orderly, distinct and overall grayish image. I...

Watch this Scientific Journal Video about Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals at JoVE.com

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

preparation-newborn-rat-brain-tissue-for-ultrastructural-morphometric

Research

JoVE Journal

Neuroscience

6.8K Views.  University of Virginia Health System. We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Video: Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Biology

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal resolution from shallow and deep brain regions. Their use has been hindered, however, by mechanical and structural mismatches between the probes and brain tissue that commonly lead to micromotion and gliosis with resulting signal instability in chronic recording experiments. In contrast, following the implantation of ultraflexible mesh electronics via syringe injection, the mesh probes form a seamless, gliosis-free interface with the surrounding brain tissue that enables stable tracking of individual neurons on at least a year timescale. This protocol details the key steps in a typical mouse neural recording experiment using syringe-injectable mesh electronics, including the fabrication of mesh electronics in a standard photolithography-based process possible at many universities, loading mesh electronics into standard capillary needles, stereotaxic injection in vivo, connection of the mesh input/output to standard instrumentation interfaces, restrained or freely moving recording sessions, and histological sectioning of brain tissue containing mesh electronics. Representative neural recordings and histology data are presented. Investigators familiar with this protocol will have the knowledge necessary to incorporate mesh electronics into their own experiments and take advantage of the unique opportunities afforded by long-term stable neural interfacing, such as studies of aging processes, brain development, and the pathogenesis of brain disease.

Mesh electronics probes seamlessly integrate and provide stable, long-term, single-neuron level recording within the brain. This protocol uses mesh electronics for in vivo experiments, involving the fabrication of mesh electronics, loading into needles, stereotaxic injection, input/output interfacing, recording experiments, and histology of tissue containing mesh probes.

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal ...

Bioengineering

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...