Neuroanatomical Methods Chapters

CHAPTER 1 Neuroanatomical Methods INTRODUCTION he functional complexity of the brain that is responsible for behavior is an emergent property of the diverse neuronal cell types that make up neural systems, the specificity of connections between these neurons, the neurochemical interactions that occur through these connections, and the functional response of neurons that result from such interactions. Neuroanatomical procedures provide for the visualization of the structural organization of neural systems. Over a century ago, the Golgi techniques revealed the incredible morphologic diversity of neurons. Neuroanatomical methods developed in the past 30 years have demonstrated an equal or even greater diversity in the neurochemicals expressed by particular neuronal types. Chapter 1 presents neuroanatomical procedures for studying the structural and functional organization of the central nervous system. The procedures described are focused on those that provide visualization of various neurochemical markers in the brain.

T

describes some of the basic procedures for preparation of the brain for histologic processing that are common to most of the methods described in subsequent units. These include methods for preparing the brain in either a fixed or unfixed state as well as for sectioning the brain for subsequent histologic processing. The choice among these basic procedures depends on the type of histologic processing that is to be performed.

UNIT 1.1

UNIT 1.2 presents methods for localization of messenger RNA (mRNA) by in situ hybridization histochemistry (ISHH). ISHH involves the specific binding of a labeled probe to a complementary portion or all of an RNA in brain cells. Methods are described for the localization of mRNA using oligonucleotide cDNA probes and using ribonucleotide probes which are detected using either radioactive or nonradioactive methods. Techniques described provide for both regional localization (localization to brain regions) and cellular-level resolution. In addition, combined methods provide for localization of multiple mRNAs in the same tissue section and for the use of ISHH localization in association with other techniques such as tract tracing or immunohistochemistry. UNIT 1.3 describes localization of biochemicals by immunohistochemistry. Immunohistochemistry involves the localization of specific proteins, peptides, or glycoproteins by the binding of antibodies to specific antigenic sites on these biochemicals, and the visualization of the bound antibodies using histochemical processes. Methods are described for the histochemical procedures used on brain tissue sections. The production and characterization of antibodies are described in UNITS 5.5-5.7. UNIT 1.4 describes localization of neurotransmitter receptor binding sites in brain sections. These methods involve the use of radioactively tagged ligands that are used to bind specifically to neurotransmitter receptor binding sites in brain tissue sections. In many cases, these ligands have been characterized by pharmacologic binding assays, and demonstrated to have receptor-specific binding. Obtaining similar receptor-specific binding in brain tissue sections requires that conditions be determined to block nonspecific binding while retaining receptor-selective binding.

describes the use of neurotropic viruses as transneuronal tracers of neuronal circuitry. Nearly 50 years ago, neurotropic viruses such as the polio virus were among the

UNIT 1.5

Contributed by Charles Gerfen Current Protocols in Neuroscience (2007) 1.0.1-1.0.3 C 2007 by John Wiley & Sons, Inc. Copyright


Neuroanatomical Methods

1.0.1 Supplement 39

first agents used to trace the central connections of axonal projections. In the intervening years, a wide range of axonal tracers have been used, including radiolabeled amino acids, horseradish peroxidase, plant lectins such as PHA-L, and fluorescent tracers that are transported in both anterograde and retrograde directions. These tracers have been employed to elucidate many of the complexities of neural circuit connections and have set the stage for the next step of analysis—that of second-order transneuronal circuits within neural systems. Recent characterization of a variety of neurotropic viruses provides a powerful approach for tracing transneuronal connections in neural circuits. Protocols in UNIT 1.5 describe the use of attenuated pseudorabies virus as a transneuronal tracer in rodents. UNIT 1.6 describes a method for determination of metabolic activity in single neurons using high-resolution [3 H]2-deoxyglucose (2DG) labeling. The 2DG method is commonly used for measurement of alterations in metabolic activity in distinct brain regions. This unit extends this methodology to the single-cell level, allowing metabolic activity to be measured in neurons identified by neurochemical phenotype, in conjunction with immunohistochemical staining. This technique, together with cytochrome oxidase histochemistry, provides measures of alterations in neuronal metabolic function that are likely to be closely correlated with altered physiologic activity. UNIT 1.7 provides methods utilizing targeted toxins to selectively lesion neuronal pathways.

Lesions of brain regions or neuronal pathways have provided an important tool for study of the functional organization of neural systems. Common lesion techniques that utilize mechanical, thermal, or excitotoxic mechanisms provide effective destruction of neurons or neuronal pathways, but are nonspecific. In some cases, neurotoxins that selectively target specific neuronal systems have been developed, such as 6-hydroxydopamine, which selectively lesions catecholamine neurons. In this unit, strategies and methods are provided for selectively lesioning other specific neuronal phenotypes or pathways. In general, the approach utilizes toxin constructs that are made of distinct components, one of which binds to proteins expressed by specific neuronal phenotypes, and another which disrupts the normal function of the neuron, thus leading to cell death. These methods allow for the design of experiments to understand the contribution of specific components to the function of neural systems. UNIT 1.8 describes a technique for demonstrating the activation of neuronal populations at two distinct time points, in vivo. The induction of immediate early genes (IEGs) is a useful measure of neuronal activation in response to a broad variety of physiological, pharmacological and behavioral stimuli. This unit describes a novel technique that exploits the fact that induced IEGs display a reproducible shift in their neuronal compartmental distribution with time. In the immediate post-stimulus time frame (2 to 15 min), induced IEGs are localized to the nuclear cellular compartment, from which they disappear and are translocated to the cytoplasmic compartment (between 30 and 45 min post-stimulus). Cellular compartmental analysis of temporal activity with fluorescent in situ hybridization histochemistry (catFISH) provides the ability to discriminate cells activated by two distinct stimuli separated by 30 min. This technique provides a powerful extension of the use of IEGs to analyze neuronal system function.

Introduction

UNIT 1.9 describes a method to detect DNA damage in tissue sections by in situ nick translation (ISNT). DNA strand breaks occur in cells undergoing degeneration, either as a result of cellular injury, in response to a variety of insults or due to programmed cell death. ISNT using radiolabeled nucleotides provides for the detection of DNA damage at the cellular level in brain tissue sections. The cellular resolution of INST in combination with other histochemical methods allows for characterization and identification of neurons undergoing degeneration. Thus, although this method does not specifically identify cells undergoing apoptosis, it provides a useful tool for analyzing cellular degeneration due to a wide variety of processes.

1.0.2 Supplement 39

Current Protocols in Neuroscience

UNIT 1.10 details quantitative methods to measure gene expression in brain sections using in situ hybridization histochemistry (ISHH). In many animal models, changes in the expression of a variety of genes have provided important insights into the mechanisms underlying certain neurologic and mental disorders. Localization of mRNAs with ISHH, particularly with radioactive probes, provides neuroanatomical material that lends itself to quantitative measurement. Two levels of analysis are described. Macroscopic analysis involves densitometric measurement of X-ray film which has been exposed to brain sections labeled by radioactive ISHH methods. Cellular analysis is accomplished by counting silver grains in labeled brain sections that have been dipped in film emulsion. UNIT 1.11 presents methods for obtaining estimates of cell density and cell number from brain sections at the light-microscopic level. Two methods are detailed. One uses the so-called “optical disector” or “3-D counting” method, which is a stereologic method providing a mathematically “unbiased” measure. A second method, Abercrombie’s twosection comparison, though less often used, is described and compared. Support protocols provide supplementary information on histologic techniques as well as correction and calibration tools.

provides details for labeling individual or small numbers of neurons with the marker biocytin, which labels all processes of the neuron, including both dendritic and axonal projections. Two methods are described, one in which neurons are injected intracellularly, and the other in which the marker is ejected extracellularly, to allow for uptake by nearby neuron cell bodies. These methods are most often used as part of neurophysiologic studies to enable identification of the neurons from which recording is taking place. As such labeling provides nearly complete filling of both dendrites and axonal projections, important information is obtained about the connections of the neuron from which recording is taking place.

UNIT 1.12

outlines a method for quantification of measures of axonal innervation within terminal areas. A process is described for determining the average arbor size of an axonal terminal tree from a given neuron by dividing the total number of terminals by the number of neurons contributing to the projection. This method nicely complements the cell counting methods described in UNIT 1.11. UNIT 1.13

UNIT 1.14 describes methods for tracing axonal connections in the anterograde direction in the brain, that is, from the neuron cell body out to the axonal terminals. These methods involve the injection of tracers, a plant lectin PHA-L or biotinylated dextran amine (BDA), into brain areas. These tracers are transported exclusively, in the case of PHA-L, in the anterograde direction from the cell body to axonal terminals, or bidirectionally, in the case of BDA, in both anterograde and retrograde directions. Following axonal transport, brains are processed to visualize the tracers using immunohistochemical or other techniques. By combining anterograde axonal tracing with other neuroanatomical methods, the connectional organization of neuroanatomical circuits can be revealed.

describes neuroanatomical methods for detecting apoptosis, a common form of programmed cell death that occurs in neurologic disease. Detection of apoptosis is based on immunohistochemical localization of caspaces or their cleavage products. Two protocols are described utilizing variations of immunohistochemical chromogen visualization, which may be combined with other techniques to identify, for example, chromatin clumping or the phenotype of the apoptotic cell type. An additional protocol for detection of non-apoptotic cell death is described. These protocols are useful for studies designed to detect neuron death at the light microscope level in tissue sections derived from animal models of neurologic disease. UNIT 1.15

Charles Gerfen

Neuroanatomical Methods

1.0.3 Current Protocols in Neuroscience

Supplement 39