Neuroscience Research, 5 ( 1987) 126-I 39

Elsevier Scientific Publishers Ireland Ltd.

126

NSR 00202

A Golgi study on the olfactory bulb Lampetra japonica

in the lamprey,

Nobuharu Iwahori, Etsuko Kiyota and Kaori Nakamura

Department of Anatomy, Faculty of Medicine, Nagasaki University, Nagasaki (Japan)

(Received 18 April 1987; Revised version received 1 June 1987; Accepted 10 June 1987)

Key words: Olfactory bulb; Lamprey; Golgi method

SUMMARY

The intrinsic organization ofthe olfactory bulb in the lamprey was studied using the rapid Golgi method. Although not as discrete as in many vertebrates, a lam&u organization was recognized. From the periphery inward, the following layers were discernible: the layer of the olfactory fibers, the olfactory glomeruli with the mitral cells, the granule cells, and the ependymal cells. Just beneath the surface of the olfactory bulb, the olfactory fibers extended over the entire bulb forming a dense fiber plexus terminating in the olfactory glomeruli which were arranged in one to two layers internally to the layer of the olfactory fibers. The mitral cells formed no discrete layer and were located mainly around the olfactory glomeruli. The mitral cells in the lamprey were lacking in secondary dendrites, but had two or more primary dendrites which terminated in the olfactory glomeruli. The axons of the mitral cells proceeded inwardly and accumulated diffusely in the granule cell layer which occupied a wide area internally to the layer of the olfactory glomeruli with the mitral cells. The granule cell layer was composed of densely packed small spindle or fusiform axonless cells, the processes of which extended superficially to be distributed in the olfactory glomeruli. At the deepest region of the bulb was a layer of the ependymal cells lining the surface of the olfactory ventricle. The external and internal plexiform layers were not evident. Thus, while the major constituents of the olfactory bulb of the vertebrate could be identified in that ofthe lamprey, the general laminar organization seemed indiscrete.

INTRODUCTION

Cyclostomes are considered to be the most primitive among vertebrates. The nervous system of the cyclostome represents in many ways a more primitive state than that of other vertebrates and is important for tracing the phylogeny of various brain structures.

The olfactory bulb in the lamprey is a fairly large structure protruding rostrodorsally and separated only incompletely from the caudally located olfactory lobe. Other investi-

Correspondence: N. Iwahori, Department of Anatomy, Faculty of Medicine, Nagasaki University, Nagasaki 852, Japan.

0168-0102/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd.

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gators have studied the olfactory bulb in the cyclostome, using various me- thods1,3*5,6,8,1 1*12*‘4*17-22. However, there are discrepancies among the reported data, particularly concerning the laminar patterns and the morphology of the neurons consti- tuting each lamina. In the present study, we examined the intrinsic organization of the olfactory bulb in the lamprey, using the rapid Golgi method, and compared the structure of the olfactory bulb of the lamprey with that of other vertebrates.

MATERIALS AND METHODS

Approximately 300 adult lampreys, 40-60 cm in body length and weighing 450-600 g, were studied. The animals were decapitated, the brains quickly removed, sliced into 5 mm thick slices with a razor blade and fmed in an osmium tetroxide-potas- sium dichromate mixture for 2-7 days at room temperature. The fvted materials were washed briefly in a freshly prepared 1 y0 silver nitrate solution and were kept in the same solution for 2-3 days at room temperature. The impregnated materials were then dehydrated in absolute ethanol, embedded in 14% celloidin and cut into loo-120 pm serial sections. The sections were dehydrated in absolute alcohol, cleared in xylol and mounted in dammar-gum.

The thickness of the sections and the complete lack of each neural array in a single focal plane made for poor photos of the Golgi sections; therefore photographs were taken only when instructive data were obtained. All other results are shown in the form of free-hand drawings, made using a monocular microscope.

Several brain series stained by the Nissl or Weil methods were utilized for orientation purposes.

RESULTS

The olfactory bulb in the lamprey is a rostrodorsal protrusion of the rostral half of the lateral lobe and is separated from the caudal portion of the lateral lobe, the olfactory lobe, by a shallow groove considered to represent the olfactory peduncle. Although not as discrete as in many vertebrates, a laminar structure was observed in the olfactory bulb of the lamprey. From the periphery inward, the olfactory bulb of the lamprey consisted of the following layers: a layer of olfactory fibers, olfactory glomeruli with mitral cells, granule cells, and ependymal cells.

Layer of the olfactory fibers This layer was composed of the olfactory fibers arranged in numerous bundles. As

seen in a sag&al section, the olfactory fibers ran ventrocaudahy and spread over the bulb. In a frontal section cut at the middle of the bulb, the olfactory fibers extended over almost the entire surface of the bulb, especially over the dorsal and lateral aspect, and

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formed a dense fiber plexus around the bulb (Fig. 1). As seen in the Golgi sections, the olfactory fibers ran in bundles along the free surface of the bulb (Fig. 2). Each bundle travelled in various directions, and crossed in a complex fashion. Finally, the fiber bundles swung inward and entered the olfactory glomeruli to terminate therein, forming

Fig. 1. The olfactory bulb (OB) of the lamprey as seen in a frontal section cut through approximately the rostral one-third of the OB. The left side of the figure is medial, and the upper, dorsal. The olfactory fibers (OF) extend over the OB, especially over its dorsal and lateral aspect. Cell-sparse ovoid areas are the olfactory glomeruli (OG), around which are seen large cells, the mitral cells (MC). Internally to the OG is a wide area composed of densely packed small cells, granule cells (GC). Drawing from a thionine-stained

section. The scale line is 200 pm.

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/ OF

Fig. 2. The olfactory bulb (OB) of the lamprey as seen in the frontal sections cut near the caudal end of the OB. The left side ofthe figure is medial, and the upper, dorsal. The olfactory fibers (OF) run superficially over the OB forming a dense fiber plexus and terminated in the olfactory glomeruli (OG), arranged in one or two layers internally to the OF layer. Around the OG are numerous mitral cells (MC), the dendrites of which end in the OG. Internally to the OG are numerous granule cells (GC) with long processes which terminate in the OG. The ependymal cells (EC) are located in the deepest layer and line the olfactory

ventricle (OV). Drawing from the rapid Golgi sections. The scale line is 200 pm.

a dense fiber plexus. Each fiber did not ramify on the way to the glomeruli. Once inside the glomeruli, they became coarse through the entire width of a glomerulus and ended by means of a free varicosity (Fig. 3).

Layer of the olfactory glomeruli with the mitral cells In this layer were seen numerous olfactory glomeruli, around which were distributed

a number of mitral cells. In the Nissl preparations, the olfactory glomeruli were identified as spherical cell sparse areas of 140 pm in average diameter (Fig. 1, OG). In the frontal sections, they were closely packed in single, and sometimes double layers internally to the layer of the olfactory fibers. In the Golgi preparations, the olfactory glomeruli were seen as ovoid areas filled with dense terminal arborizations of the olfactory fibers, dendrites of the mitral cells and processes of the granule and ependymal cells (Fig. 2, OG).

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Fig. 3. Terminal portion of olfactory fiber (A-E) and olfactory fiber bundle (F) as seen in the frontal sections. The upper side of the figure is the surface of the olfactory bulb. Drawing from the rapid Golgi

sections. The scale line is 50 pm.

The mitral cells were conspicuous cellular components of the bulb and were located mainly around the olfactory glomeruli (MC in Figs. 1 and 2). The mitral cells in the lamprey had two or more primary dendrites, but were lacking in secondary ones (Fig. 4). The primary dendrites were coarse, sparsely spinous and extended toward the olfactory glomeruli. They usually branched into several collaterals on the way to the olfactory glomeruli. On arriving at the glomeruli, the dendrites arborized repeatedly to break up into a large and dense end-bunch, and terminated totally within the glomeruli. The end tuft had an extention of loo-150 pm and fiued much of the glomeruli. Based on the size of the somata and the thickness of the primary dendrites, the mitral cells in the lamprey were divided into two types. Type I cells had spindle or fusiform cell bodies which measured, on average, 21 x 35 pm (Fig. 5D, F, G). The primary dendrites were 2-4 in number, 4-5 pm in thickness and extended toward the glomeruli. Type II cells were more frequently impregnated than type I. The somata of the type II cells were spindle- like, triangular or polygonal in shape and had an average diameter of 13 x 25 pm (Fig. 5A-C, E). From the somata extended 2-4 primary dendrites with a width of l-2 pm. Type I and II cells intermingled and had no distinct areas of distribution. Usually the primary dendrites of 3-5 mitral cells converged on a single glomerulus (Figs. 2, OG; 5H). The majority of the mitral cells sent dendrites to one glomerulus. In rare cases, however, dendrites of a mitral cell distributed to two or more glomeruli (Fig. 5E). The axons of the mitral cells emerged from the proximal portion of a dendrite (Fig. 5C, D, F) or from a deep pole of the somata (Fig. 5B, E, G). They proceeded to

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Fig. 4. A photomicrograph of the mitral cell in the lamprey. The upper side of the figure is the surface of the olfactory bulb. Rapid Golgi method. The scale line is 100 pm.

the depth of the bulb and could be traced to the granule cell layer (Fig. 9, open triangle). In the olfactory bulb of the lamprey, the axons of the mitral cells did not gather in a definite layer. Although their further courses could not be followed in a single fiber, the axons, as a whole, ran medially or caudally. In the frontal sections, they travelled medially toward the dorsal commissure. In the sag&al or horizontal sections, they were followed caudally into the olfactory lobe. Their exact terminal regions could not be determined.

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Fig. 5. Mitral cells of the lamprey, as seen in the frontal sections. Each cell was depicted from regions indicated in the guide figure at the upper right corner. Cells A-C, and E are type II, while D, F and G are type I mitral cells. Note that the mitral cells have two or more primary dendrites but are lacking in secondary ones. The primary dendrites of cell E distribute to two glomeruli, while in H, three mitral cells converge into a single glomerulus. Open triangles indicate axons. Drawing from the rapid Golgi sections. The scale

line is 100 pm.

Layer of the granule cells As seen in the Nissl preparations, the granule cell layer occupied approximately

two-thirds of the total depth of the bulb and was composed of densely packed small cells (Fig. 1, GC). In the Golgi preparations, the granule cells were small cells with thin long processes (Figs. 2,6,7). The cell bodies were minute in size and the processes were so long that the granule cells appeared to be made up almost exclusively of the processes, and the somata seemed to be a mere regional swelling of the process. The cell bodies of the granule cells were spindle-like, fusiform or triangular and measured on average 14 x 24 pm (Fig. 6). Each cell had 2-4 processes which extended toward the surface of the bulb. The processes were varicose, sparsely spinous, and branched with several collaterals. The tips of the processes entered into the olfactory glomeruli to end totally therein, branching out into several branchlets (Figs. 6C; 7A-E). The processes of a single cell distributed at least 6-9 glomeruli. On the other hand, the processes of several cells converged into a’ single glomerulus. As more caudal levels of the bulb were approached, the granule cell layer was gradually replaced by the anterior olfactory nucleus.

Fig. 6. Photomicrographs of the granule cells (CC). Two processes of the CC in C (indicated by

arrowheads) can be traced to the terminal in the olfactory glomeruli. Rapid Go& method. The

scale line is 100 pm.

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Fig. 7. The granule cells (A-E) of the lamprey, as seen in the frontal sections. The left side of the figure is medial, and the upper, dorsal. The granule cells have small cell bodies (indicated by arrows) which extend 2-3 thin long processes terminating in the olfactory glomeruli (OG). OV, olfactory ventricle. Drawing from

the rapid Golgi sections. The scale line is 100 pm.

Layer of the ependymal ceils At the deepest portion of the bulb, a layer of the ependymal cells was seen lining the

olfactory ventricle (Fig. 2, EC). The ependymal cells were small spindle cells, the long axis of which oriented perpendicular to the surface of the olfactory ventricle. The internal pole of the cell body extended deeply to protrude into the olfactory ventricle. From the external pole of the cell body, extended a long process which branched into numerous collaterals covered with thin thorns. The processes proceeded toward the surface of the bulb and the tips of some processes could be followed until they reached the vicinity of the free surface of the bulb. Many processes passed through the confines of the olfactory glomeruh, and at least some of the collaterals seemed to terminate therein.

Organization of the olfactory glomeruli The olfactory glomeruli in the lamprey were mainly composed of a dense

agglomeration of the following 4 components: the terminal portion of the olfactory fibers, the primary dendrites of the mitral cells, the terminal arborization of the granule

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Fig. 8. The ependymal cells (A, B) of the lamprey, as seen in the frontal sections. Each cell was depicted from points indicated in the guide figure at the lower right comer. Cell bodies of the ependymal cells are

indicated by open triangles. Drawing from the rapid Golgi sections. The scale line is 100 pm.

cell processes and the terminal portion of the processes of the ependymal cells. The olfactory fibers were derived from peripheral olfactory cells and constituted principal afferents to the glomeruli (Figs. 1,2). The main efferents of the glomeruli, on the other hand, were the primary dendrites of the mitral cells (Fig. 9A). The primary dendrites of the mitral cells ended in the glomeruli branching dense terminal collaterals with numerous spines. Usually primary dendrites of several mitral cells converged into a single glomerulus, and occasionally a single mitral cell extended dendrites to two or more glomeruli. Thus, the glomeruli usually had several efferents; sometimes, two or more different glomeruli had common efferents. Besides the mitral cell dendrites, processes of the granule cells terminated in the glomeruli (Fig. 9B-D). The processes of a single granule cell terminated in at least 6-9 glomeruli, while terminals from several

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Fig. 9. Convergence of the mitral cell (A) dendrites and granule cell (B-D) processes in the olfactory glomeruli (OG) as seen in the frontal sections. The figure was depicted from the squared area indicated in the guide figure at the lower left corner. The left side of the figure is medial, and the upper, dorsal. Open

triangle indicates the axon of the mitral cell. Drawing from the rapid Golgi sections. The scale line is 100 pm.

granule cells converged into one glomerulus. Finally, the processes of the ependymal cells passed through the confines of the glomeruli and some collaterals seemed to end therein, emitting numerous thorns.

DISCUSSION

The olfactory bulb of the lamprey proved to be a sessile protrusion of the rostral half of the lateral lobe, separated incompletely from the caudal half of the lateral lobe, the olfactory lobe, by a shallow sulcus considered to represent the olfactory peduncle. These features exhibited a striking contrast to many vertebrates with long olfactory peduncles.

Cytoarchitectonically, the following 4 layers were recognized in the lamprey bulb: the layer of the olfactory fibers, the olfactory gfomeruli with the mitral cells, the granule cells, and the ependymal cells. The laminar structure of the lamprey bulb has been repo~ed6.‘4.‘8-20.22. It was generally agreed that the first and second layers were

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composed of the olfactory fibers and olfactory glomeruli with mitral cells, respectively. Internal to the layer of the olfactory glomeruli with the mitral cells, a definite mitral cell layer has been noted by other investigators 6*18*2022. Moreover, Heier6 divided the granule cell layer into a superficial and deep sublayer. In the lamprey which we studied, the mitral cells were seen around and between the glomeruli. Only a few mitral cells were distributed internally to the glomeruli, mainly in the lateral portion of the bulb. Thus, a distinct mitral cell layer could not be distinguished in our materials.

The mitral cells of the lamprey have been observed in ammocoetes22 as well as in mature specimens . 6,1 l The morphology of the mitral cells of the lamprey is different from that found in mammals. Johnston” described that “there are no well developed mitral cells to be found” in the lamprey bulb. In the mitral cells of the lamprey, usually two or more primary dendrites were seen, whereas no secondary dendrites were observed. The absence .of secondary dendrites in the mitral cells seemed to be one of the main causes for the lack of a clear laminar organization in the lamprey bulb. The external plexiform layer, the main constituents of which were the secondary dendrites of the mitral cells, was absent in the lamprey bulb. This type of mitral cell did not seem to be specific to the lamprey, as it was noted in the myxine’, fishlo, frogi and salamander7. This type of mitral cell might be common in cyclostome, fish and amphibia, however.

In the present study, the mitral cells were divided into two types, based primarily on the size of the somata and the thickness of the primary dendrites. Two types of mitral cells were also recognized in the ammocoetes 22 It has been well documented that in .

the mammalian bulb, two types of projection neurons can be distinguished: the mitral and the tufted cells2. It was not certain, however, whether the two types of mitral cells in the lamprey corresponded to the mitral and the tufted cells in mammals.

The axons of the mitral cells proceeded inward and accumulated diffusely in a wide area of the granule cell layer to travel further medially or caudally. The axonal courses in the lamprey have apparently not been heretofore followed experimentally, but were traced only in normal preparations 4,6~1 1J4*17,22. According to these studies, the axons of the mitral cells accumulated in diffuse fiber bundles, termed as a whole the olfactory tract. The olfactory tract was roughly divided into a dorsal, lateral and medial com- ponent which projected to the dorsal portion of the hemispherium and hippocampal primordium, the lateral portions of the hemispherium, and the medial portions of the hemispherium, respectively. Part of the medial and dorsal components proceeded to the contralateral olfactory lobe by way of the dorsal and partly by the supraoptic commis- sure. On the way, the olfactory tract travelled through the anterior olfactory nucleus to branch out terminal collaterals therein. In the present study, the axons of the mitral cells as a whole were seen to run medially or caudally. Fibers travelling medially were considered to project to the medial portion of the hemispherium and contralateral forebrain. On the other hand, the axons running in a caudal direction seemed to correspond to fibers which terminated in the dorsal and lateral portions of the hemispherium.

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The granule cell layer was composed of densely packed granule cells which projected processes to the olfactory glomeruli. It is well known that processes of the granule cells in mammals distribute to the external plexiform layer to make synaptic contact with the secondary dendrites of the mitral celIs2*‘3*15,‘6. I n the lamprey, the mitral cells lacked the secondary dendrites and mere synapse sites were a distal portion of the primary dendrites which distributed in the glomernli. As a result, in the lamprey, the primary dendrites of the mitral cells in the glomeruli should make contact with the granule cell processes as well as with the olfactory fibers. Thus, the synaptic organizations of the glomeruli in the lamprey might differ from those in mammals in which granule cell processes do not terminate in the glomeruli. These events were not elucidated in the present light microscopic study.

In the present study, only one type of cell, the granule cell, was observed in the granule cell layer. Besides the granule cell, however, various cells have been detected in the granule cell layer ‘J ‘,22 In ammocoetes, bipolar cells similar to the mitral cells . were present in the superficial portion of the granule cell layer and termed as the third type of mitral cell 22 Cells with dendritic arbors similar to those of the granule cells and .

with a fine axon were also described and were termed horizontal or vertical neurons, based on axonal courses6. Finally, neurons with dendrites distributed mainly in the granule cell layer were seen in ammocoetes”.

Many tips of the processes of the ependymal cells in the lamprey were seen to enter into the confines of the olfactory glomeruli and at least some processes seemed to end therein. In mammals, the majority of the ependymal cell processes were described to terminate in the external plexiform layer, and only a few processes ended in the glomerular layer’6*23. Although the meaning of the termination of ependymal cell pro- cesses in the glomeruli is not clear, it is important to elucidate the structural organization of the glomeruli.

In mammals, the olfactory bulb has been reported to receive afferent fibers from other brain regions, especially from the basal forebrain areas2*13. In ammocoetes, Mayer14 found that fibers from the contralateral thalamus and hypothalamus projected to the olfactory glomeruli and their vicinity by way of the dorsal commissure. In mature specimens, however, Heier6 observed no tierent fibers from other brain regions, except for the interbulbar fibers. In the present study, no afferent fibers from other brain regions were observed. As the Golgi method is capricious, it is still uncertain whether the lamprey bulb receives tierent fibers from other brain regions.

In conclusion, the fundamental organization of the lamprey bulb is similar to that of the mammalian bulb2,13. The main differences between the lamprey bulb and the mammalian one are as follows: (1) the mitral cells are located mainly in the periglomeru- lar areas, especially in areas between the olfactory glomeruli; (2) the mitral cells lack secondary dendrites; (3) the external plexiform layer is absent; and (4) the granule cell processes terminate in the olfactory glomeruli. These features of the lamprey bulb resemble those of the bulb in the myxineg, fish” and amphibia7.

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ACKNOWLEDGEMENTS

We thank M. Ohara of Kyushu University for commenting on the manuscript, M. Saito at Ebetsu, Hokkaido for collecting the materials and T. Okubo, M. Kaneko and M. Hashiguchi for their excellent technical and photographic assistance. This work was supported in part by a Grant-in-Aid for Scientific Research (61570029) from the Japanese Ministry of Education, Science and Culture.

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