New Dimensions in Neuroanatomy: Visualizing the Morphology, Physiology and Chemistry of Neurons1

AMER. ZOOL., 30:513-529 (1990)

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 interdisciplinary 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 histological 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 discussed 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 description 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

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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 central 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 provide 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 addition 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 neurosecretory 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. Following injection the preparation was incubated for 12-15 hr to allow the HRP to fill the cell completely, and was then reacted with diaminobenzidine (DAB) which causes the HRP to form a black precipitate. The cell body (~40 jim diameter), its projections, and a few terminals (arrows) have been filled with HRP by this procedure. C. Following the DAB reaction, the preparation was fixed for electron microscopy by standard methods, thin sectioned, and viewed. HRP-filled terminals of the injected cells are consistently filled with large dense granules, a typical characteristic of neurosecretory cells. A reprinted from Evans et al., 1976, with permission; B and C unpublished photographs from B. S. Beltz and E. A. Kravitz. tissues therefore can be examined. The Dil (Color Plate ID and Godement et al., HRP-labeled neuron shown in Figure 3B 1987) are often injected into dendritic was fixed, thin sectioned, and examined regions in the brain or sensory epithelia, with the electron microscope revealing where they are taken up by the dendrites clusters of neurohormonal release sites (Fig. and subsequently are transported or dif- 3C). Three of the terminals contain the fuse throughout the cells. HRP label and therefore must belong to the injected cell. Some ultrastructural Retrograde dye-filling characteristics of the filled terminals, such Brain areas and ganglia are connected as large dense granules, can be seen (see via highly ordered pathways, and neurons also Carson and Mesulam, 1982). that share a target are often functionally Anterograde labeling of neurons also can related. Therefore, if the neurons that be used to mark projections of small groups project to particular brain areas or periph- of cells rather than an individual cell eral targets can be visualized, this infor- (Swanson and Sawchenko, 1985). For mation suggests possible functional iden- instance, PHA-L (Gerfen et al., 1989) and tities for the neurons. Numerous methods NEW DIMENSIONS IN NEUROANATOMY 517 are available for tracing fibers back to their VISUALIZING DYNAMIC PHENOMENA cell bodies of origin (Mesulam, 1982; IN NEURONS Swanson, 1983; Heimer and Zaborszky, 1989). A variety of compounds (many Optical imaging of physiological activity of them the same that are used for anter- A number of voltage-sensitive dyes that ograde-labeling methods), such as Lucifer change color when depolarized have been yellow (Color Plate IE; Mavder etal., 1987), discovered and utilized to record physio- carbocyanine dyes (Dil and DiO; Gode- logical activity in relatively simple neural ment et al., 1987), fluorescently-labeled networks (Cohen and Lesher, 1986; Cohen

latex microspheres (Color Plate IF; Katz and Salzberg, 1978; Salzberg, 1983). Most Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 et al., 1984), cobalt chloride (Pitman et al., recently, a combination of optical and elec- 1972; Bishop and King, 1982) and HRP trode recording methods was used to obtain (Mesulam, 1982; Freund and Somogyi, an overview of neuronal activity in the 1983) can be used for these types of studies. abdominal ganglion of Aplysia in response As illustrated in Color Plate IE, Lucifer to light touch to the siphon skin (Zecevic, yellow is used to retrogradely label (via the 1989). This ganglion has been studied inferior ventricular nerve) neurons in the intensively using conventional recording esophageal ganglion of the crab Cancer methods to analyze habituation and sen- borealis (Marder et al., 1987). In a second sitization of the gill-withdrawal reflex example rhodamine-labeled latex micro- (Kandel et al, 1967; Kupfermann et al., spheres mark neurons in the nucleus teg- 1971; Hawkins et al., 1981; Frost et al., menti peduncularis, pars candalis (TPc) of 1988). Over the course of the approxi- the canary midbrain. The microspheres mately 25 years spanning these studies, it were injected into the lobus parolfactorius has been estimated that more than a (LPO) of the forebrain and traveled retro- hundred neurons may be involved in this gradely to the labeled cell bodies, thereby reflex, and the complete circuit of neurons demonstrating a neuronal pathway between has still not been defined. Because the these areas (Burd et al., 1985). The purpose numbers of neurons involved in this reflex of these retrograde filling methods is, activity are so large, understanding the therefore, to begin to understand function roles of each of these neurons in an inte- by determining the projections of neurons. grated network is an awesome task, and By learning the target areas to which cells one that is not suited to conventional project, it is possible to discover related recording methods because of the num- groups of cells. bers of electrodes required. Therefore, optical recording methods, which theoret- The methods reviewed thus far make it ically can monitor simultaneously the activ- possible to visualize the varieties of neu- ity patterns of each of the neurons in an ronal types that compose tissues (Golgi entire ganglion, have been used to study method), examine the morphology of this reflex. Spike activity was detected in selected individual neurons (intracellular up to 150 neurons during reflex activity, injection of dyes), define the targets of and habituation and sensitization of the groups of neurons (dye injection and anter- reflex was accompanied by large changes ograde transport), and discover function- in the numbers of activated neurons ally related neurons (dye administration at (Zecevic, 1989). target sites and retrograde transport). A neuron's structure and projection pattern To accomplish these studies, a living are obviously of critical importance in its abdominal ganglion was incubated in the function. However, each cell's physiologi- dye oxonol until the neurons were visibly cal profile and ionic composition also define stained; the absorption of photons by this its functional abilities. The optical meth- dye is dependent upon the transmembrane ods described below allow "visualization" potential. Therefore, once stained with this of physiological changes in neurons and of dye, each neuron undergoes a spectral shift some of the ionic fluctuations underlying every time it fires an action potential. These physiological activity. minute color changes in individual neu- 518 BARBARA S. BELTZ

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rons are captured by placing the ganglion in a specialized recording apparatus that includes a photodiode detection system; the output of each diode is sent to amplifiers and computers for analysis (Fig. 4A). The output of the photodiodes can be recorded directly (Fig. 4B) or can be analyzed with the computer and displayed as neural activity (Fig. 4C). Optical recording methods,

although still not fully developed, there- Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 fore provide the possibility to obtain information about single-cell activity without the use of electrodes. One of the major advantages of this method is its ability to examine the individual activity patterns of Two-step large numbers of neurons simultaneously, a task not currently possible with conven- Method tional recording methods. FIG. 5. Diagrammatic representation of the two-step (indirect) immunocytochemical method. Antigen molecules are represented by the filled triangles, anti- Calcium measurement and the dye Fura-2 bodies by the "Y-shaped" forms, and the label molecule by the solid circle. The primary antibody binds It has long been known that there is a to the antigen, while the secondary antibody, carrying frequency-dependent enhancement of the the label, binds to the primary antibody. Two-step excitatory junction potential (EJP) at the methods are more efficient than directly labeling the crayfish claw opener muscle (Atwood and primary antibody because several labeled secondary Wojtowicz, 1986). Several minutes of stim- antibody molecules can bind to each molecule of primary antibody (not indicated in diagram), thereby ulation of the excitor axon to the opener resulting in more label bound to the tissue for each muscle at high frequencies results in a sus- antigen molecule. Reprinted from Beltz and Burd, tained enhancement of the EJP known as 1989, with permission from Blackwell Scientific Pub- long-term facilitation. It has been sug- lications. gested that an increase in resting calcium in excitatory presynaptic terminals may 1989). However, a more sensitive method account for some parts of this facilitation using the dye Fura-2 does allow measure- (Wojtowicz and Atwood, 1985). Direct ment of direct calcium levels within indi- measurements of intraterminal calcium vidual presynaptic terminals in prepara- concentrations following high-frequency tions that also provide for simultaneous stimulation have not been possible using recording of EJP amplitudes. measurements based upon absorbance Fura-2 is a fluorescent dye whose emis- changes of Arsenazo III because of the sion spectrum is altered by the presence of small size (2-10 fim) of terminals (Tsien, free calcium. The dye can be injected into

FIG. 4. A. Schematic diagram of the optical recording apparatus. Light from a tungsten halogen lamp was passed through a 720 ± 25 nm interference filter and focused on the preparation using a modification of Kohler illumination. A 12 x 12 photodiode array was placed at the plane where the objective forms the real, inverted image. With this optical arrangement, the image of a three-dimensional ganglion is formed on the two-dimensional array. Photodiode currents were individually amplified. Amplifier outputs were multiplexed, digitized, and stored in a computer. The structure of the oxonol dye used for these experiments is shown at the lower right. B. & C. Comparison of photodiode outputs (B) and the analyzed results from that data, neuron activity (C). The output traces of 7 photodiodes (indicated in the boxes at the left) are shown at the right. The numbers to the left of each trace indicate the photodetector number. Examination of the photodiode outputs indicated that activity in four neurons would account for all of the large optical signals. The activity of these four neurons is shown in the roster diagram in C. The boxes at the left indicate which photodiodes are detecting which neuronal signals. See Zecevic et ai, 1989, for further details. A and B reprinted from Zecevic et al., 1989, with permission. BARBARA S. BELTZ Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021

PLATE 1. A. This section of the dentate gyrus of the hippocampus from cat brain has been stained with two procedures: the Golgi stain, which causes some neurons to stand out in black silhouette with all their processes revealed, and the Nissl stain, which renders every neuronal cell body blue. Reprinted from Nauta and Fiertag, 1979. B. A Retzius cell in a ganglion of the leech Hirudo mediririalis was filled with Lucifer yellow by iontophoresis. NEW DIMENSIONS IN NEUROANATOMY 521 a neuron, and spectral changes monitored suggested that there is a dendritic segre- by fluorescence ratio imaging; computers gation of calcium conductances in Purkinje then generate "pseudocolor images" that neurons (see Milburn, 1990; Llinas and reflect calcium concentrations (Connor, Hess, 1976; Llinas and Sugimori, 1980). 1986). In an elegant example of this Fura-2 imaging has shown that the highest method, the excitor neuron to the crayfish levels of calcium in an active Purkinje neu- claw opener muscle was filled with Fura-2 ron are, indeed, segregated in the den- by penetrating the axon with an electrode drites (see Color Plate 2B and Tank et al, containing the dye, and passing hyperpo- 1988).

larizing current (Delaney et al., 1989). In Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 Color Plate 2 A, pseudocolor images of the MOLECULAR MAPPING IN THE axon and terminals are seen. At rest, free NERVOUS SYSTEM calcium is low in the axons and terminals The methods thus far discussed reveal (Fig. 2A, top panel, left frame). At various some of the anatomical and physiological times following stimulation, a rapid increase (and underlying ionic) properties of neu- in free calcium is seen in the terminals, rons. What now of the neuron's chemical followed by a slow decline that lasts and molecular architecture—for instance, approximately 35 min. These images illus- the transmitters, enzymes and receptors trate the long time course for the decay of that are so closely tied to a neuron's func- calcium back to rest following tetanic stim- tion? Several anatomically-based methods, ulation. This time course is linearly cor- such as autoradiography (Cowan and related with the post-tetanic enhancement Cuenod, 1975), immunocytochemistry of transmission at the synapse, thereby pro- (Beltz and Burd, 1989), and in situ hybrid- viding further evidence for the involve- ization techniques (Siegel, 1987) can be ment of calcium in this process. used to define the molecular elements that Fura-2 technology also has been utilized compose neural tissues. Because immuno- to examine the spatial distribution of cal- cytochemical methods are particularly ver- cium currents in Purkinje cells of the cer- satile and rapid to use, and are not tech- ebellum (Tank et al., 1988). The calcium nology intensive, these techniques are current distribution is likely to reflect the described below as one example of the distribution of calcium channels. It has been methods available for identifying and

The dye was passed from the electrode with hyperpolarizing pulses (5-10 nA) at a frequency of 0.5 Hz for 10-15 min. For further details of the method, see Stewart, 1978. Photo courtesy of Kristy Coyle and Katrina Russo, Biology 306 class, Wellesley College, 1989. C. Three cells (arrows) in a crayfish (Pacifaslacus leniusculus) abdominal ganglion were filled with biocytin by iontophoretic injection using hyperpolarizing current. The preparation was then fixed and labeled with streptavidin-Texas Red. For further methodological details, see Horikawa and Armstrong (1988). Photograph courtesy of Carolyn Sherffand Brian Mulloney, University of California at Davis. D. The carbocyanine dye Dil was injected into the nasal capsule (NC) of Xenopus laevis larvae. Dil was picked up by the tips of the dendrites of the olfactory receptor cells and filled the cells to their terminals in the glomerular layer of the olfactory bulb (OB). Although the labels appear on the right side, both sides of the bilaterally symmetrical system are filled. Dil also can be used retrogradely to fill the pathways from the axon terminals. Photo courtesy of Gail D. Burd, University of Arizona at Tucson. For further details see Perkins and Burd, 1989. E. Retrograde labeling methods were used to fill these esophageal ganglion neurons of Cancer borealis with Lucifer yellow via the inferior ventricular nerve (IVN). For further details see Marderefa/., 1987. Photograph courtesy of Eve Marder and Michael Nusbaum, Brandeis University. Reprinted from Beltz and Burd, 1989, with permission of Blackwell Scientific Publications. F. Rhodamine-labeled latex microspheres were injected into the lobus parolfactorius of the canary forebrain and traveled retrogradely to the cell bodies labeled in this photograph (arrows) that are found in the nucleus tegmenti peduncularis, pars caudalis of the midbrain. Photograph courtesy of Gail Burd, John Paton and Fernando Nottebohm, Rockefeller University. For further details see Burd et al., 1985. Reprinted from Beltz and Burd, 1989, with permission of Blackwell Scientific Publications. 522 BARBARA S. BELTZ Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 NEW DIMENSIONS IN NEUROANATOMY 523 localizing molecules of interest in the ner- way for dopamine. Using an antibody vous system. against tyrosine hydroxylase, and a sec- The term "antibody" often evokes ondary antibody conjugated to fluorescein, thoughts of the body's disease response. sites of tyrosine-hydoxylase-like immuno- However, just about any molecule that is reactivity have been detected in sections of found in a cell can serve as an antigen. This the canary midbrain (Color Plate 3 A). Both includes enzymes that are in synthetic neuronal cell bodies and processes are pathways for transmitters, or the trans- labeled. Whole mounts of tissues also can mitters themselves. One of the simplest be used with immunocytochemical meth- ways to obtain an antibody is to inject a ods (Color Plates 3B and C; see also Beltz Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 purified form of the compound into a rab- and Kravitz, 1983). This approach is par- bit repeatedly over a period of time (usu- ticularly useful in tissues where preserva- ally several months) and harvest the rab- tion of three-dimensional information is bit's serum, which will contain antibodies critical, and avoids the necessity of recon- against the injected compound. Once anti- structing tissues from mountains of labeled bodies have been obtained, they can be used sections. For example, serotonin-like with immunocytochemical methods to immunoreactivity has been localized in a detect the presence of the antigen in fixed thoracic ganglion of the crustacean Gam- tissues. marus lacustris, where two lateral cell bod- Using one of many available immuno- ies, several longitudinal fibers, and a neu- cytochemical methods, referred to as the ropil region are intensely labeled (Color indirect staining or two-step method, fixed Plate 3B). Likewise, serotonin-like immu- tissues are incubated in primary antibody noreactivity has been found in fibers and generated against a molecule of interest varicosities in a neurohormonal region (Fig. 5). The primary antibody binds to located on the thoracic second roots of antigen molecules in the tissue. The tissue Homarus americanus (Color Plate 3C). is then rinsed and incubated with the sec- Because of the excellent resolution of these ondary antibody; this secondary antibody methods, it is possible from immunocyto- is raised against immunoglobulin of the host chemical data to construct detailed chem- for the primary antibody, and therefore ical maps of the nervous system, as has been binds to the primary antibody. The sec- done for many transmitter substances in ondary antibody is conjugated to a visual both vertebrate and invertebrate nervous marker, such as a fluorophore or horse- systems (Beltz and Kravitz, 1983; Stein- radish peroxidase. Thus, the sites of label- busch, 1984; Siwicki and Bishop, 1986). ing in the tissue represent areas where the Similar immunocytochemical methods also antigen resides. Tissues illustrated in Color have been used to localize a variety of Plates 3A-D and 3F have all been pro- neural antigens, such as molecules that cessed by similar two-step techniques. serve developmentally significant func- tions (Slemmon et al., 1985; Bastiani et al., Immunocytochemical methods have 1987) and that compose the extracellular been used to localize dopamine-containing matrix (Sanes, 1989). In some cases, anti- neurons by labeling tyrosine hydroxylase, body technology has provided a means to one of the enzymes in the synthetic path-

PLATE 2. A. This multi-panel photograph shows pseudocolor images of calcium in presynaptic terminal boutons and pre-terminal axon of the excitor motor neuron of crayfish (Procambarus clarkii) claw opener muscle. These images were obtained immediately before and at the times indicated after tetanic stimulation of the excitor axon at 33 Hz for 7 min and illustrate the long time course for the decay of calcium back to rest, which correlates with post-tetanic enhancement of transmission at this synapse. For further details see Delaney et al., 1989. Photo courtesy of Kerry Delaney,* David Tank* and Robert S. Zucker,+ University of California at Berkeley* and AT&T Bell Labs.* B. Using Fura-2 imaging, calcium concentration was mapped in a Purkinje neuron during the peak of calcium accumulation during physiological activity. The quantitative color scale used was purple-blue = 100 nM; green-yellow = 200-300 nM; red = 500 nM. Photo reprinted with permission from Tank et al., 1988. 524 BARBARA S. BELTZ Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021

PLATE 3. A. Tyrosine-hydroxylase immunoreactivity is demonstrated in this section of the canary midbrain. Both cell bodies and processes are labeled with fluorescein using a two-step immunocytochemical method. Photograph courtesy of Gail D. Burd, University of Arizona at Tucson. Reprinted from Beltz and Burd, 1989, with permission of Blackwell Scientific Publications. B. A two-step method using a fluorescein-labeled secondary antibody was used to demonstrate serotonin immunoreactivity in a third thoracic ganglion from the crustacean Gammarus lacustris, which was processed as a whole mount. Photograph courtesy of Simone M. Helluy. C. Serotonin immunoreactivity is demonstrated in fibers and varicosities in a neurohormonal region in the lobster Homarus americanus. This region was processed as a whole mount using a two-step immunocytochemical method employing a fluorescein-labeled secondary antibody. Reprinted from Beltz and Kravitz, 1983, with permission from the Journal of Neuroscience. D. Double-exposure photograph of double immunocytochemical labeling for tyrosine hydroxylase-like (fluorescein label) and serotonin-like (Texas Red label) immunoreactivities. Two-step immunocytochemical NEW DIMENSIONS IN NEUROANATOMY 525 localize region-specific or cell-specific pro- specific for each of the hosts and labeled teins even before actually identifying the with different visual markers, the two protein. immunoreactivities can be readily distin- guished in the same section of tissue. Color Coexistence of transmitters: Analysis Plate 3D, for example, is a double-expo- with double immunocytochemical sure photograph of serotonin-like (Texas labeling methods Red label) and tyrosine-hydroxylase (TH; Immunocytochemical methods have fluorescein label) immunoreactivities in a contributed not only to our understanding section from the shark medulla. The pri- of where individual transmitters reside in mary antibodies were generated in rabbit Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 the nervous system, but also to studies of (anti-TH) and rat (anti-serotonin). The sec- coexistence of transmitters. The idea that ondary antibodies are therefore able to dis- individual neurons use a single transmitter tinguish between the primary antibodies is now outdated; in fact, such neurons would based upon their species-specific affinities. now be considered exceptions, rather than the rule (Milburn, 1990). By combined COMBINED METHODS autoradiographic and immunocytochemi- The varieties of methods described above cal methods (Chan-Palay et al, 1978) or become even more powerful when they are double-labeling immunocytochemistry combined. For instance, combining retro- (Callaway et al., 1987; Carroll et al., 1988), grade fluorescent tracers with immunocy- it is possible to localize two or more com- tochemistry provides simultaneous infor- pounds in the same tissue or cell. One of mation about the target projection and the the simplest approaches, which often is used transmitter specificity of the retrogradely- when the available antibodies were raised labeled neurons (Skirboll et al., 1989). The in the same host, is to stain alternate serial use of such combined methods dates back sections with one or the other of the anti- to the mid 1970s (Ljungdahl et al., 1975), bodies of interest. By examining the stain- and since that time many successful labeling patterns for each antibody in the serial ing combinations have been used for the sections, it is possible to demonstrate tracing of transmitter-specific pathways in coexistence of two or more immunoreac- the brain (see Skirboll et al, 1989, for tivities in the same neural tissue and often detailed descriptions of several of these within the same cells (see Fig. 6 and Siwicki methods). In an elegant example of such a etal, 1987). combined method, neurons in the stoma- Coexistance of two substances also can togastric ganglion of the crab Cancer borealis be demonstrated using simultaneous dou- have been retrogradely filled with Lucifer ble immunocytochemical labeling in the yellow via the stomatogastric nerve (Color same tissue section. By this method, anti- Plate 3E), while FMRFamide-like immu- bodies against each of the molecules of noreactivity has been revealed in the same interest must be generated in different whole mount of tissue using immunocyto- hosts. Then, using secondary antibodies chemical methods and a rhodamine-labeled

methods were used. The arrow in this figure points to a fluorescein-labeled neuron and an arrowhead illustrates a neuron labeled with Texas Red. Some neuronal processes appear to be co-labeled with the two antibodies. Photograph courtesy of Joel White, University of Florida at Miami. Reprinted from Beltz and Burd, 1989, with permission of Blackwell Scientific Publications. E and F. The stomatogastric ganglion of the crab Cancer borealis has been double-labeled by combining a Lucifer yellow backfill of the stomatogastric nerve (E) with anti-FMRFamide immunolabeling using a two- step method utilizing a rhodamine-labeled secondary antibody (F). This experiment demonstrates intense FMRFamide-like immunoreactivity in the neuropil of the stomatogastric ganglion (F), while cell bodies surrounding that neuropil are labeled with the retrograde transported dye. Photographs courtesy of Eve Marder and Michael Nusbaum, Brandeis University. For further details see Marder et al., 1987. Reprinted from Beltz and Burd, 1989, with permission of Blackwell Scientific Publications. 526 Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021

FIG. 6. Demonstration that serotonin and proctolin immunoreactivities coexist in paired neurons in the first abdominal ganglion of the lobster Homarus amerkanus. In ganglion whole mounts, the bilateral cell pair is indicated with arrows in preparations stained with (A) a serotonin antiserum and a two-step fluorescent method; and (B) a proctolin antiserum and a three-step, avidin-biotin-peroxidase method (for further details of the latter method, see Belu and Burd, 1989). In adjacent 10 jim cryostat sections of the first abdominal ganglion, the same large paired cells (arrows) stain with (C) serotonin antiserum and (D) proctolin antiserum. Both sections were processed with a three-step avidin-biotin-peroxidase method. Scale bars =100 nm. Reprinted in a revised format with permission from Siwicki et al., 1985. secondary antibody (Color Plate 3F). This with immunocytochemistry (Beltz and experiment demonstrates intense FMRF- Kravitz, 1987; Kitai et al., 1989); in situ amide-like immunoreactivity in the neu- hybridization combined with retrograde ropil of the stomatogastric ganglion, while fluorescent tract-tracing (Chronwall et al., cell bodies surrounding that neuropil are 1989); and double retrograde tract-tracing labeled with the retrogradely transported using fluorescent latex microspheres dye, but not by the anti-FMRFamide anti- (Cornwall and Phillipson, 1988). This list- body. ing of combined methods is by no means In addition to combining retrograde flu- complete, and it is constantly expanding orescent labels and immunocytochemistry, with new possibilities and novel applica- a variety of other labeling methods have tions. been used successfully together: HRP or PHA-L tract-tracing with transmitter CONCLUSION immunocytochemistry (Zaborszky and In summary, methods utilized for visu- Heimer, 1989); biocytin, HRP, or Lucifer alizing neurons today, unlike traditional yellow single-cell labeling in combination neuroanatomical techniques, are not con- NEW DIMENSIONS IN NEUROANATOMY 527 fined to looking at static processes in neu- Callaway, J. C, B. Masinovsky, and K. Graubard. rons. The various optical and visualization 1987. Colocalization of SCPB-!ike and FMRF- amide-like immunoreactivities in crustacean ner- methods illustrated in this paper demon- vous systems. Brain Res. 405:295-304. strate that modern neuroanatomy is a Carroll, S., S. DiNardo, P. O'Farrell, R. A. H. White, dynamic science—capable of describing not and M. P. Scott. 1988. Temporal and spatial only structural characteristics of neurons, relationship between segmentation and homeotic but also the active physiological and chem- gene expression in Drosophila embryos: Distri- bution of the fushi tarazu, engrailed, Sex combs ical processes upon which neuronal func- reduced, Antennapedia, and Ultrabithorax pro- tion is dependent. However, the methods teins. Genes Dev. 2:350-360. presented here have still only scratched the Carson, K. A. and M.-M. Mesulam. 1982. Electron surface in terms of future technical possi- microscopic tracing of neural connections with Downloaded from https://academic.oup.com/icb/article/30/3/513/224946 by guest on 24 September 2021 bilities. Additional methodologies not even horseradish peroxidase. In M.-M. Mesulam (ed.), Tracing neural connections with horseradish peroxi- discussed in this paper, such as in situ dase, pp. 153-184. 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