Glutamic-Acid-Decarboxylase-and Parvalbumin-Like-Immunoreactive Structures in the Olfactory Bulb of the Human Adult T.G. OHM, H. M ~ L E RN. , ULFIG, AND E. BRAAK Zentrum der Morphologie, J.W. Goethe-Universitat, D-6000 Frankfurt, Federal Republic of Germany

ABSTRACT This study examines the distribution and morphological characteristics of glutamic-acid-decarboxylase-like(GAD)- and parvalbumin-like (PA)immunoreactive structures in the olfactory bulb of the human adult. GAD-immunoreactivesomata occurred in the glomerular layer, the external granule cell layer, the more superficial portion of the external plexiform layer, and the internal granule cell layer. The cells were small- to mediumsized. Demonstration of lipofuscin pigment revealed the presence of unpigmented as well as pigmented neurons, thus suggesting the existence of two subpopulations of GAD-positive neurons. GAD-immunoreactive puncta and/ or fibers were mainly seen in the periglomerular region and the internal granule cell layer. All other layers of the bulb, as well as the intrabulbar portion of the anterior olfactory nucleus, displayed considerably less of these puncta and/or fibers. The olfactory nerve layer remained practically clear of immunoreact.ive material. PA-immunoreactive somata occurred in the glomerular layer and both the external and internal granule cell layer. Only a small number of immunoreactive nerve cells were encountered within the white matter or the olfactory tract. Most PA-positive neurons displayed characteristics of short axon cells whereas a few others resembled van Gehuchten cells. All of the PA-immunoreactive neurons were devoid of lipofuscin pigment. Immunoreactive puncta and fibers were present in all layers though predominating in the periglomerular region, the olfactory nerve layer, and the internal granule cell layer. The intrabulbar portions of the anterior olfactory nucleus did not show any immunoreactive structures. Key words: glutamic acid decarboxylase, parvalhumin, internenrons, short axon cells, man

Gamma-aminobutyric acid (GABA) is generally accepted as an inhibitory transmitter and is frequently found in local circuit neurons of the cerebral cortex. Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme in GABA synthesis. It can immunocytochemically be demonstrated (Oertel et al., '81),thus marking GABAergic neurons, fibers, and terminals (puncta). Parvalbumin (FA) is a calcium-binding protein exhibiting a distinct distribution pattern within the central nervous system (Celio and Heizmann, '81; Berchtold et al., '85). The functional properties of this neuronal marker have been associated with the regulation of calciumdependent activities (Heizmann, '84). It has been empha-


sized that the distribution of PA-positive neurons is similar to that of GABAergic neurons (Celio and Heizmann, '81). The partial colocalization of these substances has recently been demonstrated (Celio, '86; Kosaka et al., '87; Celio et al., '88).

In the main olfactory bulb of the rat. only small neurons within the periglomerular region were labeled by PA anti-

Accepted August 9,1989. Address reprint requesta to T.G. Ohm, Zentrum der Morphologie, J.W. Goethe-Universitat,D-6000 Frankfurt, Federal Republic of Germany.






f \

Fig. 1. a: Diagram demonstrating the consecutive steps of the pigment-Nissl technique. The chromogen 4C1N is used for the demonstration of the immunoreaction. Immunoreactive neurons (light circle) are visualized hy using 4C1N in the first step. T h e second step shows neighboring cells (dark circles) surrounding the still-visible immunoreactive neuron. Destaining of immunoreactive structures and consequent application of the pigment stain follow in the third step. Previously immunoreactive neurons are now relocated by using the distinct arrangement of

surrounding cells. Decolorized immunoreactive neurons may contain lipofuscin pigment (small dark spots, upper half) allowing for the analysis of the distribution pattern of these deposits or may be devoid of lip+ fuscin granules (lower part). b: Micrographs of a GAD-positive neuron demonstrating the three steps of the staining procedure. The arrow points to intraneuronal deposits of lipofuscin. c: Micrographs of a PAimmunoreactive nerve cell. PA-like neurons have consistently been found to be nonpigmented.

sera (Celio and Heizmann, ’81; Endo et al., ’86a). GAD- and GABA-immunoreactive neurons were demonstrated within the glomerular layer, the superficial part of the external plexiform layer, and the internal granule cell layer of the rat (Ribak et al., ’77; Halasz et al., ’79; Ottersen and StormMathisen, ’84; Kosaka et al., ’85; Gall et al., ’87). In the brain of the human adult, intraneuronal lipofuscin deposits can he utilized as a natural marker of different neuronal types (Braak, ’80, ’84). The distinct pattern and morphological characteristics of these deposits remain unchanged even though the amount of lipofuscin increases with advancing age. As an additional neuronal feature, the pigmentation pattern may therefore help differentiating

Fig. 2. Micrographs of GAD and PA neurons in the human olfactory bulb. a-d: Neurons immunoreactive for GAD. e-1: PA neurons. a-c: Immunoreactive neurons in the periglomerular region. d Nerve cell in the internal granule cell layer. e: Superficial short axon cell. f:Immunoreactive neuron characterized by twisted processes and a dendritic arborization remaining relatively confined to the vicinity of the nerve cell soma-due to its features, which are obviously different from short axon cells, it could possibly represent a van Gehuchten cell. g: Superficial short axon cell; note the process running perpendicularly to the pial surface. h Periglomerular cell-arrows indicate “wraps.” i: Neuron possibly corresponding to a van Gehuchten cell. j: “Wrap” of a deep short axon cell. k: PA neuron located in the white matter. 1: Deep short axon cell. Scale bars represent 40 rm.



Figure 2



Fig. 3. a,h:Micrographs of the superficial layers of the human olfactory bulb. Note that olfactory glomeruli (GLO) in sections stained for GAD material are occupied by intensely stained immunoreactive fibers and puncta. In contrast to this the glomeruli in sections stained with PA

antisera appear encircled by immunoreactive material. c,d: PA fiber bundles of different calibers in the white matter adjacent the internal granule cell layer of the human olfactory bulb.

cells sharing a similar morphological appearance while exhibiting divergent immunohistochemical properties. Furthermore, different aspects in pigmentation may render the distinction of neurons being immunoreactive for the same substance possible. The aim of the present study is to delineate the distribution of GAD- and PA-immunoreactive structures within the olfactory bulb of the human adult. Moreover, immunoreactive neurons are correlated with the characteristics of nerve cell types distinguished in pigment-Nissl preparations.

biotin-peroxidase complex method (Hsu et al., '81). Longitudinally cut frozen sections (40-60 gm) were treated with 1OC, normal rabbit serum (for GAD) or with 10% bovine serum albumin (for PA) for 2 hours a t room temperature prior to the incubation with the primary antibodies. Polyclonal anti-GAD serum (sheep, dilution 1:2,000, 1:4,000) or monoclonal anti-PA serum (15,000, l:lO,OOO) was allowed to react for 45 hours a t 4 O C . Sections were then incubated with biotinylated rabbit-antisheep immunoglobulin G (IgG) or biotinylated horse-antimouse IgG and finally immersed in a solution containing the avidin-biotin-peroxidase complex (Vectastain, Vector Laboratories). The peroxidase BCtivity was visualized by adding 3.3'-diaminobenzidine-tetrahydrochloride (DAB) or 4-chloro-1-naphthol (4ClN) and H202as second substrate. Control sections were incubated in a preimmune serum (GAD) or in P A antiserum adsorbed with PA antigen. For further specificity tests see Oertel et al. ('81) and Celio et al. ('88). When employing DAB as a chromogen it is generally possible to trace even quite delicate processes for considerable distances. Tissue sections treated with 4C1N, on the other hand, have the advantage of allowing further staining procedures since the chromogen can be bleached out (Fig. 1).This renders the identification of cytoarchitectonic units as well as of neuronal types in analyzed sections possible. The procedure includes the examination of immunoreactive neu-

MATERIALS AND METHODS Human olfactory bulbs from five adult individuals (one female, four males; age range 45-88 years) were obtained at autopsy. Postmortem delay varied between 3.5 and 11 hours. The cases showed no clinical signs of cerebral disorders nor did they display on histopathological examination any relevant alterations of the brain. The olfactory bulbs were fixed by immersion in a mixture of paraformaldehyde and picric acid (Somogyi and Takagi, '82-the glutaraldehyde was omitted) for 1to 2 days. They were then stored in 0.1 M phosphate buffer (ph 7.3) containing 0.9% NaCl and 0.01 Sb thimerosal. The immunohistochemical procedure was carried out on free-floating sections (Oertel et al., '82) by using the avidin-



GAD fibers and puncta

TABLE 1. Laminar Distribution of GAD and PA Structures in the Human Olfadorv Bulb' PARV


olfactory nerve layer (ONL) II glommlar layer (GLO) 111 external granulecell layer (EGL) IV external p l e x l l m layer (EPL)


V mitral cell layer (MCL) VI internal pluiform layer (IPL) VII internal granulecell layer (IGL) V l l l white matter (WM) anterior olfactory nuc!eus (AON)








++ ++

+ ++ f





+t -

+ +t


++ ti+

+++ + -



+ t b t

++ -


'Plus and minus indicatequalitativefrequeneyofimmnnoreactiveatyucLure. t rareuccmence of immunoreactive structures, + t medium ocnurence, + + + fmuent occurrence, - no unmunoreactivem a t e d .

GAD-immunoreactive fibers and/or puncta are located in all layers of the olfactory bulb with the exception of the olfactory nerve layer (Table 1). The internal granule cell layer and, in particular, the superficial portion of the external plexiform layer, the external granule cell layer, and the glomerular layer present themselves as zones being densely packed with puncta. Only on rare occasions a few dispersed immunoreactive puncta, probably belonging to the glomerular layer, are seen in the inner half of the olfactory nerve layer. In general, fibers are less frequent than the abundant puncta; the ghmeruli, however, are clearly marked by a dense network of these fibers (Fig. 3a).

PA somata rons and fibers and their consequent documentation with both micrographs and camera lucida drawings. The sections are then stained with gallocyanin chrome alum in order to visualize the pattern of surrounding nonimmunoreactive cells. The immunoreactive structures as well as their nowvisible cellular environment are again documented. Subsequently, the sections are decolorized with ethanol and rest,ained with aldehydefuchsin and Darrow-red, rendering the demonstration of lipofuscin granules and Nissl substance within the previously immunoreactive neurons possible (Fig. 1). The position of immunoreactive neurons demonstrated in sections treated with DAB was determined by correlating these sections to consecutive sections stained with aldehydefuchsin and Darrow-red. The nomenclature proposed by Humphrey and Crosby ('38) was adopted.

RESULTS GAD somata GAD-immunoreactive somata are located in two main zones of the olfactory bulb. The inner zone is represented by the internal granule cell layer. The outer one comprises the glomerular layer, the external granule cell layer, and the external plexiform layer. All other layers including white matter and anterior olfactory nucleus (AON) remain devoid of immunoreactive nerve cell bodies (Table 1,Fig. 4). The immunoreactive somata are small in size and often ovoid in shape (Figs. 2a-d, 4). Only occasionally, mediumsized bipolar or multipolar neurons are recognized. The nucleus appears paler than the encompassing cytoplasm. The immunoreaction continues only rarely far enough into the cell processes to allow a reliable description of the dendritic branching pattern. Frequently, the proximal trunk of one faintly stained process of the small neurons within the internal granule cell layer rises towards the outer layers of the olfactory bulb (Figs. 2d, 4). Immunoreactive neurons of the outer zone are more intensely stained than those of the inner granule cell layer. Their dendrites are oriented either parallel to the respective layer or tangential to the shape of a glomerulum (Fig. 4). The demonstration of lipofuscin pigment reveals the presence of pigmented (Fig. 1h) as well as nonpigmented GADimmunoreactive neurons.

PA-immunoreactive structures stand out against a virtually unstained background. PA-positive somata are predominantly located in the periglomerular region and in the internal granule cell layer (Table 1).In addition to that the rare occurrence of immunoreactive nerve cells in the white matter as well as the adjoining proximal portion of the olfactory tract is also observed (Fig. 2k). Most of the immunoreactive somata display an ovoid to polygonal outline (Figs. 2e-1). Spindle-shaped somata are only rarely seen. Within the periglomerular region the largest diameter of P A somata ranges from 8 to 22 pm. The majority, however, have diameters between 15 and 20 pm, while a small group of neurons exhibits diameters of around 11pm. The processes of the immunoreactive neurons are mostly arranged in a multipolar manner, giving many of these cells a stellate-like appearance (Fig. 4). Bipolar cells are only infrequently encountered. Dendritic trees of PA-immunoreactive nerve cells can be followed for considerable distances (300 p m ) .

Neuronal processes generally arborize in the same layer in which their respective somata are located (Fig. 2e). Only a few neurons send their processes in deeper layers (Fig. 2g). Most of the processes display a ragged outline and generally dichotomize close to their parent soma. In some cases thin and varicose fibers are visible close by the soma, most probably representing an axonal arborization. Occasionally fairly thick processes are seen to terminate by dividing into two thin and varicose processes. A peculiar feature of some processes of PA neurons located in the periglomerular region is their clasp-like arrangement around olfactory glomeruli. In other cases processes wrap themselves for two or three times around an invisible structure (Fig. 2h arrows, and 2j). A small number of neurons exhibit an elaborate arborization pat,tern, which remains, when compared to a short axon cell, more confined to the area around the respective soma. Moreover, these cells are characterized by an intensive branching of their heavily twisted processes (Fig. 2f,i). As their counterparts in the periglomerular region, immunoreactive neurons of the internal granule cell layer frequently display a stellate-like appearance (Fig. 4).Neuronal processes coiled up around an invisible structure are equally observed. PA-irnmunoreactive neurons situated in the lower portion of the internal granule cell layer send some of their processes into the white matter. The latter in turn is characterized by the presence of a very limited number of fusiform neurons which are oriented parallel to the long axis of the bulb. In aldehydefuchsin/Darrow-red preparations the nuclei of the PA-immunoreactive nerve cells appear t o be rather













. AON 4

4 Figure 4




pale when compared to those of neighboring cells. The nuclei are surrounded by only a small rim of cytoplasm. All of the analyzed PA-immunoreactive neurons remain devoid of lipofuscin deposits (Fig. lc).

PA fibers and puncta PA-immunoreactive fibers and puncta occur in all layers yet show their highest density in the periglomerular region (glomerular layer and external granule cell layer). Olfactory glomeruli tend to be outlined by a rim of diffusely scattered immunoreactive puncta, thus frequently simplifying their recognition in DAB preparations (Fig. 3b). Around, and occasionally within, the glomeruli thin fibers and puncta are discernable being arrayed in a pearl-string-like manner. Bundles of thin fibers exhibiting periodically repeated spindle-shaped varicosities are present in the internal granule cell layer as well as in the white matter (Fig. 3c). There are also thick fibers in the same region which run-like the former-parallel to the pial surface extending centrally towards the olfactory tract (Fig. 3d). Some of these fibers also show spindle-shaped puffs and side branches arising at right angles. In the outer layers of the bulb immunoreactive puncta are more frequent than immunoreactive fibers. The latter predominate in the inner layers as well as white matter. The analyzed portion of the anterior olfactory nucleus remains devoid of immunoreactive fibers.


GAD-like structures Demonstration of GAD-immunoreactive neurons in human postmortem material is incomplete and difficult. We, therefore. cannot conclusively correlate the observed cells to one of the neuronal types described in the olfactory bulb. In animals, GAD-immunoreactive neurons in the main olfactory bulb were classified as granule cells and periglomerular cells (Ribak et al., '77), or as short axon cells, granule cells, and periglomerular cells (Halasz et al., '79; Mugnaini et al., '84; Gall et al., '87). Some authors even reported the presence of GAD-like cells (Kosaka et al., '85) or GABA-immunoreactive neurons (Gall et al., '87) exhibiting features which apparently resembled those of superficial tufted cells. Furthermore, there is evidence for the existence of two populations of GABAergic periglomerular cells (Gall e t al., '87), one associated with tyrosine-hydroxylase-like immunoreactivity, the other with a calcium-binding protein though not PA (Baimbridge and Miller, '82; Garcia-Segura e t al., '84; Halasz et al., '85). Interestingly, our results point to the presence of two populations of GAD-immunoreactive neurons in the periglomerular region of the human olfactory bulb, one containing a few scattered lipofuscin granules, the other devoid of any pigment. When focusing on the fact that all PA-immunoreactive neurons were nonpigmented, it becomes obvious that a t least the pigmented GAD-immunoreactive neurons cannot be colocalized with PA.

Fig. 4. Schematic distribution pattern of GAD and PA neurons in the human olfactory bulb. Camera lucida drawings are representative in their laminar arrangement,size, and orientation. In order to simplify the recognition of the various layers several symbols were inserted. Small circles indicate the periglomerular region as well as internal granule cell layer. Triangles signify the relative position of the mitral cell layer whereas rhombi represent the anterior olfactory nucleus.

Nevertheless, GAD- and PA-immunoreactive neurons exhibit a considerable overlap in their respective cytoarchitectonical distribution (Table 1, Fig. 4). This finding is in accordance with previous results established in the rat (Celio and Heizmann, '81; Celio, '86; Gall et al., '87). The distribution of GAD-immunoreactive fibers and puncta in the human is similar to that found in the olfactory hrllh of the rat. However, some (Halasz et al., '79; Ottersen and Storm-Mathisen, '84) but not all authors (Ribak et al., '77) note, in addition, the occurrence of GABAergic fibers and puncta within the olfactory nerve layer. Except for a few dispersed puncta close to the outer borders of the glomeruli, there are no GAD-positive structures in the olfactory nerve layer of adult man. It may be possible that the amount of GAD present in the olfactory nerve layer is too low to be detected (as has previously been discussed by Ribak et al., '77).

PA-like structures Anti-PA sera demonstrate even distal portions of the processes of a subpopulation of neurons in the olfactory bulb of adult man. Due to this fact, it is possible to match their features to neuronal identities established in Golgi preparations. Classification of PA-immunoreactive nerve cells in the human olfactory bulb is impeded by the fact that the morphological criteria for the differentiation of neuronal types are almost exclusively based on data derived from Golgi studies in animals (Ram6n y Cajal, '11; Price and Powell, '70a,b; Pinching and Powell, '71; Schneider and Macrides, '78). The PA-positive nerve cells of the internal granule cell layer closely match the description given for deep short axon cells (Ram6n y Cajal, '11;Price and Powell, '70a). In Golgi-impregnated material these cells, which probably represent inhibitory interneurons (Price and Powell, '70a), can be subdivided in four different types (RamBn y Cajal, '11; Stephan, '75). In our material the large majority of PAimmunoreactive deep short axon cells closely resemble Golgi cells. However, some neurons may belong to the class of horizontal cells of Cajal (Price and Powell, '70a; Schneider and Macrides, "B),even though they do not occur in their typical position, e.g., the internal plexiform layer. A very limited number of similarly fusiform and horizontally oriented cells are found in the white matter and proximal portions of the olfactory tract. For the time being i t cannot be decided whether these cells should be regarded as displaced horizontal cells or interstitial cells of Cajal (Ram6n y Cajal, '11).In a recent study similarly shaped somatostatinlike immunoreactive cells have been described to occur in a comparable location (Ohm et al., '88). Most of the PA-immunoreactive neurons in the periglomerular region correspond to superficial short axon cells. However, a small number of these neurons exhibit distinctly different morphological details. Because of the elaborate arborization pattern of their twisted dendrites, which remain restricted to a relatively small area around the parent soma, these cells possibly correspond to van Gehuchten cells (Schneider and Macrides, '78). It is, nevertheless, necessary to note that they are located in the deep portion of the periglomerular region and not in the external plexiform layer as described in the hamster (Schneider and Macrides, '78). An even more limited number of neurons could not successfully be classified as superficial short axon cells or van Gehuchten cells. Their small soma1 size and their shape resemble those



of periglomerular cells described in Golgi impregnations (Pinching and Powell, '71). In the rat, all PA-positive neurons in comparable locations are classified as periglomerular neurons (Celio and Heizmann, '81). This evaluation has chiefly been based on topological criterea (Celio, personal communication). It remains an open question whether this observation points to interspecies differences or rather represents an artificial discrepancy caused by neuronal identification on topographical grounds. P A neurons in the human olfactory bulb remain entirely devoid of lipofuscin deposits. It is thus possible to postulate the existence of a subpopulation of nonpigmented short axon cells in the human olfactory bulb. All suggested neuronal types for PA-immunoreactive cells are classified as local circuit neurons (interneurons). This is consistent with findings that PA is generally located in neurons displaying interneuronal properties (Celio, personal communication; Endo et al., '86b;Heizmann, '84).

ACKNOWLEDGMENTS We are indepted to Ms. B. Ganger and to Ms. A. Biczysko for technical assistance as well as to Ms. I. Szasz for the illus-

trations. We also thank Profs. Hubner and Stutte (Frankfurt) and Dr. Probst (Basle) for their contribution of brain tissue. Prof. Celio kindly donated the antibodies directed against parvalbumin. The anti-GAD sera were generously contributed by Prof. Oertel. This study was supported by the Scheidel-Stiftung and the Deutsche Forschungsgemeinschaft.

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Glutamic-acid-decarboxylase-and parvalbumin-like-immunoreactive structures in the olfactory bulb of the human adult.

This study examines the distribution and morphological characteristics of glutamic-acid-decarboxylase-like (GAD)- and parvalbumin-like (PA)-immunoreac...
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