AMER. ZOOL., 30:513-529 (1990) New Dimensions in Neuroanatomy: Visualizing the Morphology, Physiology and Chemistry of Neurons1 BARBARA S. BELTZ Department of Biological Sciences, Wellesley College, Wellesley, Massachusetts 02181 SYNOPSIS. Traditional neuroanatomical methods provide a means for understanding some aspects of cell and tissue organization in the nervous system. However, the boundaries of the field of neuroanatomy have been blurred in the past twenty-five years by interdis- ciplinary techniques, such as dye-injection methods that combine electrophysiological and Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 anatomical protocols and immunocytochemistry which combines immunological and his- tological methods. This review therefore takes a very broad view of neuroanatomy, includ- ing within this field a variety of "anatomically-based" methods that allow visualization of physiological and molecular features of neurons. Several representative methods are dis- cussed and examples of the data achieved are provided. Although this area of neurobiology has evolved rapidly in recent years, the future holds promise for an even more dramatic revolution as molecular, computer and confocal-microscopic methods are more widely applied to neuroanatomical problems. Neuroanatomy is therefore viewed in this paper as an interdisciplinary field, and this paper might just as appropriately be entitled, "How are neurons visualized?" The answer involves a wide range of methods that allows descrip- tion not only of static situations, but also dynamic phenomena in neurons. Our concept of the neuron is rapidly they make in the brain, and the fine struc- evolving, largely because of dramatic ture that is the basis of neuronal function. improvements in research technologies. Santiago Ramon y Cajal, a late nineteenth Neuroanatomical methods are no excep- century neuroanatomist, used the Golgi sil- tion. In addition to the purely topographic ver staining method (Golgi, 1883) to vis- histological techniques of 25 or 30 years ualize cell types in the mammalian brain ago, a host of anatomically-based methods and to understand the tissue organization are now available that allow examination of brain regions. Figure 1A illustrates that of the dynamic phenomena involved in Cajal drew neurons as discrete cellular neuronal function. Neuronal architecture units. One of CajaPs most important con- can be defined with histological, ultrastruc- tributions to neurobiology was to establish tural and dye-injection techniques. Fur- neurons as distinct cells rather than as part thermore, active processes in neurons can of a continuous network, as many at that be examined using optical recording meth- time believed (see Hodgson [1990] this ods that require no electrodes and using symposium, "Long-range Perspectives on ion-sensitive dyes such as the calcium sen- Neurobiology and Behavior" [Reticular- sor Fura-2. The molecular composition of ists vs. Anti-reticularists]). Such classical neurons also can be denned using antibod- histological methods are still popular today ies and immunocytochemical techniques, for solving modern neurobiological prob- or radioactively-labeled compounds and lems. The Golgi method, for instance, has autoradiographic methods. This paper been used to define the morphologies of briefly reviews a variety of these tech- Purkinje neurons in normal (Fig. IB) and niques. staggerer mutant (Fig. 1C) mice (Berry et ah, 1980). The primary lesion in the stag- DEFINING NEURONAL ARCHITECTURE gerer mutant appears to reside in the Pur- kinje cells, which have stunted dendrites Histological and dye-injection methods and very few spines. The Golgi technique reveal the shapes of neurons, the pathways is also frequently used with counterstains that enhance the contrast of the method 1 From the Symposium on Science as a Way of Know- (Color Plate 1A). ing—Neurobiology and Behavior organized by Edward S. Hodgson and presented at the Centennial Meeting However, with all of the virtues that the of the American Society of Zoologists, 27-30 Decem- Golgi method affords, it suffers from the ber 1989, at Boston, Massachusetts. limitation that the particular neurons that 513 514 BARBARA S. BELTZ -\ Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 i FIG. 1. A. Drawing by Ramon y Cajal of a section through the hen cerebellum stained with the Golgi method showing cellular and fiber layers. Purkinje neurons (arrows) are shown at upper left in cellular layer. B. & C. Purkinje neurons from a normal (B) and staggerer (C) mouse cerebellum, stained with the Golgi method. The stunted neuron pictured in C is typical of this mutant, which has a characteristic staggered gait. (From Berry et ai, 1980.) stain are not predictable. The method stains which allow penetration of specific neu- only 5% or fewer of the neurons, appar- rons with electrodes and passage of dye ently at random, but these cells are stained into those cells using either pressure or in their entirety. Therefore, although it is electrical current (Kater and Nicholson, possible to learn what types of cells com- 1973). pose a neural tissue, it is not possible to There are two basic methods for filling select the individual cells that one would neurons with dye (Fig. 2). For anterograde like to visualize. This problem is best filling, dye is injected into the neuronal cell addressed using a combination of anatom- body and is then transported (or diffuses) ical and electrophysiological methods, via the axon to the terminals, thus filling NEW DIMENSIONS IN NEUROANATOMY 515 the entire cell. Alternatively, dyes can be Anterograde Retrograde injected into particular brain regions, where axon terminals take up the dye and transport it back to the cell bodies. In the peripheral nervous system, it is possible to simply dip a cut nerve end into the dye, and wait for the dye to travel into the cen- tral nervous system. By such retrograde filling methods, it is possible to label cell bodies that project to particular targets, Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 thereby suggesting a functional role for the labeled neurons. With all of these methods, the "ideal" label should stay confined within the cell(s) of interest and should pro- vide a complete morphological profile of the labeled neuron(s). Anterograde methods One of the most widely used dyes for examining neuronal anatomy is Lucifer yellow (Stewart, 1978), which can be passed into a neuron via an electrode by ionto- FIG. 2. Diagrammatic representation of two types of phoresis. The dye flows out of the elec- dye-filling procedures for neurons. For anterograde trode with the current and throughout the filling of a neuron, dye is usually pressure-injected or neuron, as shown in Color Plate IB. While iontophoresed into the cell body; the dye then diffuses yielding very powerful results, such tech- or is transported throughout the rest of the cell. In niques are relatively straightforward, espe- order to fill neurons retrogradely, dye is injected at the target site, or a cut end of a peripheral nerve is cially when working with large neurons dipped in dye. Dye then travels toward the cell bodies (such as the leech Retzius cell shown in projecting to that region. Color Plate IB) that are easy to penetrate and hold for long periods of time. In addi- tion to Lucifer yellow, several other com- scope of this paper, references are pro- pounds, such as biocytin (Color Plate 1C; vided for further details of the methods Horikawa and Armstrong, 1988), the plant described. lectin Phaseolus vulgaris leukoagglutinin HRP serves as a particularly versatile (PHA-L; Gerfen et al., 1989), the carbo- label. For example, in the lobster Homarus cyanine dyes Dil and DiO (see Color Plate americanus a cluster of neurons located at ID and Godement et al, 1987), and the the bifurcation of a peripheral nerve can enzyme horseradish peroxidase (HRP; see be visualized in whole mounts with neutral Fig. 3 and Bishop and King, 1982; Beltz red stain (Fig. 3 A, and Kravitz et al., 1980). and Kravitz, 1987) also can be injected into An electrode containing HRP can be placed neurons utilizing very similar methods. in the cell body of one of these neurons Many of these compounds are observable and the neuron filled with the enzyme by because they are pigmented or fluoresce; others, such as biocytin and PHA-L, must iontophoresis or pressure injection. The be used in concert with avidin-labeling preparation is then reacted with diamino- methods or immunocytochemistry, respec- benzidine, causing the HRP to form a tively, for the distribution of the com- brown-black precipitate (Fig. 3B). A neu- pound to be visually detectable. Each of ronal cell body, its processes, and a few the compounds mentioned has particular filled varicosities are visualized (Fig. 3B). advantages and disadvantages depending In contrast to fluorescent compounds that upon the desired application. While a full fade with time, HRP is a permanent discussion of these factors is beyond the marker. In addition, HRP is electron dense, and the ultrastructural features of labeled 516 BARBARA S. BELTZ Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 FIG. 3. A. A cluster of neurosecretory neurons in the second nerve root of the third thoracic segment in the lobster Homarus americanus is stained by the dye neutral red. Homologous clusters of neurons are found in all of the thoracic segments, as well as in nerve roots emanating from the subesophageal ganglion. Although the contents of these cells have not been identified, they have the ultrastructural characteristics of neurose- cretory neurons. See Evans et al., 1976, and Kravitzei al, 1980, for further details. B. Horseradish peroxidase (HRP) has been iontophoretically injected into the cell body of one of the thoracic second root neurons stained in A.