VETERINARSKI ARHIV 69 (3), 167-177, 1999
ISSN 1331-8055 Published in Croatia
Development of accessory lobes in the spinal cord
of the turkey (Meleagris
gallopavo)
Snjezana Vukovic*, Hrvoje Lucic, and Snjezana Curkovic
Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
* Contact address:
Dr. Snjezana Vukovic,
Department of Anatomy, Histology and Embryology,
Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000
Zagreb, Croatia,
Phone: 385 1 23 90 249
VUKOVIC, S., H. LUCIC, S. CURKOVIC: Development of accessory lobes in the spinal cord of the turkey (Meleagris gallopavo). Vet. arhiv 69, 167-177, 1999.
ABSTRACT
Accessory lobes are unusual structures of the lumbosacral segment of the avian spinal cord, which are characterized by glycogen accumulation. They consist of neuroglial cells and neurons, which are similar to the multipolar neurons of the ventral horns of the grey matter. Development of the accessory lobes is different from that of other glycogen-rich regions of the avian spinal cord. At the 8th day of embryonic development in turkey, neuroblasts are visible at the ventrolateral margins of the spinal cord, and they will differentiate in multipolar neurons. At the 15th day of embryonic development glioblasts appear in accessory lobes. At the 16th day, accessory lobes achieve their final form and obtain first PAS-positive granules of glycogen. Glycogen accumulates in neuroglial cells, and the appearance of a lesser amount of glycogen in multipolar neurons is very interesting.
Key words: turkey, Meleagris gallopavo, spinal cord, accessory lobes, multipolar neurons, neuroglial cells, glycogen, embryo
Introduction
The avian spinal cord is unique among vertebrates because of its specific structures, which can accumulate glycogen. These structures are glycogen body and accessory lobes. Glycogen body is a gelatinous structure that rises from the dorsomedian portion of the spinal cord at the lumbosacral plexus level. Accessory lobes are less visible than the glycogen body, consisting of segmental bulges arranged bilaterally along the lumbosacral segment of the avian spinal cord. Accessory lobes were first so described by Lachi at 1889, and are frequently referred to as Lachies lobes (DeGENNARO and BENZO, 1976). Also, Hofmanns nuclei (von KOLLIKER, 1902), Hofmann-Kollikers nuclei (BERLINGER, 1902), and the main marginal nuclei (STREETER, 1904) are very often mentioned in literature.
Glycogen body consists of oval or circular cells with little (or without) connective tissue. A small amount of connective tissue is found between the net of blood vessels and nerves which pass between glycogen cells. Glycogen body cells consist of a thin layer of cytoplasm, located at the edge of the cell, and glycogen accumulation is located in the central section. The nucleus is located at the edge of the cell (DeGENNARO, 1959). Accessory lobe tissue is similar to that of the glycogen body and consists of oval cells filled with glycogen. There is a thin layer of cytoplasm and nucleus located peripherally. It is supposed that specific glycogen cells belong to the neuroglial cells of the astrocyte type, which are extremely differentiated for the glycogen accumulation (LYSER, 1973; DeGENNARO and BENZO, 1976). The only one difference between these two types of cells is the interlacing of neuroglial cells of accessory lobes with neurons, which is visible by light microscope. Neurons are similar to the multipolar neurons of grey matter, but are much smaller.
The morphological similarity between glycogen body and accessory lobes can indicate their common function, or embryonic origin, or both (DeGENNARO and BENZO, 1978).
The embryonic base of the glycogen body is visible after the formation of neural epithelium zones in the spinal cord. According to Langman (SADLER, 1995), the ventricular zone, composed of a few layers of neuroepithelial cells, is located around the canal of the neural tube. These cells first differentiate in primitive neurons or neuroblasts, and later in primitive supporting cells or glioblasts. During neuroblast differentiation, two neighbouring zones are created. After differentiation of the neuroblasts from the neuroepithelial cells of the ventricular zone, they migrate and form an intermedial ("mantle") zone around the ventricular zone. The cells form a base for the grey matter of the spinal cord. Their nuclei are large, circular, and pale coloured, with a dark nucleolus.
The marginal zone is situated around the mantle zone and consists of the nerve fibres of neuroblasts from the intermedial zone. In the intermedial (or mantle) zone, the circular neuroblast is subject to two processes on opposite sides of the cell. One becomes a branch-like primitive dendrite, and the other grows out to the periphery of the spinal cord, enters the marginal zone, and becomes primitive axon. This zone is a base for the white matter of the nerve cord, after myelinization. When the differentiation of neuroblasts is over, neuroepithelial cells develop to the glioblasts, which then migrate to both neighbouring zones. Glioblasts of the intermedial zone develop to the astrocytes, and glioblasts of the marginal zone develop to the glial cells.
Glycogen body cells originate from the cells of the intermedial zone and form the paired primordia, the latter joining to one organ (WATTERSON, 1952).
Development of the glycogen body was investigated in the chicken (WATTERSON, 1952; MATULIONIS, 1972; UEHARA and UESHIMA, 1982), Japanese quail (DeGENNARO and BENZO, 1987; DeGENNARO and BENZO, 1991), and turkey (VUKOVIC, 1993), but there is no information on development of the accessory lobes. The purpose of this investigation is to describe the development of accessory lobes in the turkey. We hope that similarities with, or differences between, accessory lobes and glycogen body will result in new data about their connection with the avian central nervous system. Both of these structures are placed in the lumbosacral segment of the spinal cord of birds. However, cells which can accumulate glycogen can be visible along the whole length of the spinal cord and are located mainly dorsally from the central canal (SANSONE and LEBEDA, 1976; SANSONE, 1977). These cells originate from astrocytes also, but are different from the cells of the glycogen body and accessory lobes because they have a lighter cytoplasm, with a lesser amount of glycogen granules (SANSONE, 1980).
Materials and methods
In this investigation, eggs of the turkey (Meleagris gallopavo) of the Nicholas line, supplied from "Puris" (Pazin, Croatia), were used. Eggs were incubated in a laboratory incubator at 37.5 °C and at a relative humidity of 60%. Eggs were turned, and temperature and humidity were controlled, every day. During the experiment 230 eggs were placed in an incubator, from which 156 embryos were developed. From the 8th day of incubation, embryos were sacrificed at 24-hour intervals. Eggs were frosted at a temperature of -60 °C for four hours, and than decapitated. The lumbosacral segment of the spinal cord was separated, bones were extracted, and the spinal cord was taken out and fixed in 10% buffered formalin ("Kemika", Zagreb, Croatia). The tissue was processed by a standard histological procedure. It was embedded in paraffin and cut by microtome into serial sections 8 micrometers thick. Serial sections were then stained with hematoxylin and eosin, the PAS method for the presentation of polysaccharides by McManus (PEARSE, 1968), and control reaction with diastase enzyme. This was done at the earlier established order, and first staining was PAS, second PAS-diastase, and then hematoxylin and eosin (H/E). Histological analyses were performed by light microscopy ("Nikon-Microphot FXA", Japan).
Results
At the 8th day of the embryonic development of turkey, division in three zones was very apparent in the spinal cord.
The ventricular zone is situated around the central canal of the embryonic spinal cord, and consists of a few ranges of elongated neuroepithelial cells. These cells have a well defined oval nuclei concentrated in the lateral part of the cell.
The mantle zone is visible when it is fulfilled by neuroblasts whose dorsal and ventral groups can also be seen very clearly. The nuclei of neuroblasts are large, circular, and pale coloured, with dark nucleolus. Especially distinguishable are ventral groups of neuroblasts, known as the basal plate.
The marginal zone lies outside and is composed of fibres of neuroblasts from the mantle zone. Inside the marginal zone there are no other cells, except for small groups of nuclei which are visible on both sides of the spinal cord in a ventrolateral position. These are the first cellular elements of the accessory lobes (Fig. 1). Nuclei are arranged in one line and are similar to the nuclei of the neuroblasts from the mantle zone. Sections that were stained by the PAS method for glycogen presentation show a negative reaction. As embryonic development continued, the groups of accessory lobes cells are larger, and small bilateral bulges are visible at the 9th day (Fig. 2). Bulges grow progressively over the following days. The body form of the accessory lobes cells is not so clear on the hematoxylin and eosin sections, but nuclei of these cells correspond to the nuclei of the neuroblasts. It appears that glioblasts differentiate at the 9th day of spinal cord development in the turkey. Elongated, dark coloured cells migrate from the ventricular zone into the intermedial (mantle) zone (Fig. 3), and at the 11th day of embryonic development they can also be seen in the marginal zone (Fig. 4). Change in the structure of the accessory lobes is obvious at the 15th day of embryonic development. Tissue becomes loose, and the neuroblasts differentiate in multipolar neurons (Fig. 5) which, in comparison with multipolar neurons of the ventral horns of the grey matter, are much smaller (Fig. 6). Circular, small nuclei of neuroglia cells are also visible. At the 16th day of development, the PAS positive granules are rare and appear in the accessory lobes cells that are finally formed. At the 18th day of embryonic development of turkey, the accessory lobes tissue consists of vacuolar, oval, neuroglial cells with a circular nucleus located at the periphery. Between these neuroglial cells there are well developed multipolar neurons. In the sections stained by the PAS method for presentation of glycogen, the clearly positive reaction in neuroglial cells can be seen, and a granular product at the margins of the neurons is visible (Fig. 7). Control of the PAS colouring by diastase method after treatment with enzyme is negative, as was expected. PAS-positive reaction is more intensive at the 19th day of development, and the most intensive reaction is seen at the 25th day of embryonic development (Fig. 8). On the same day, a PAS-positive granular product can be visible in dendrites of the neurons of the accessory lobes. In neighbouring multipolar neurons of the ventral horns of grey matter, there are no signs of glycogen accumulation.
Fig. 1. Nuclei of accessory lobe cells (arrow) of turkey embryo arranged at the edge of the marginal zone, at the 8th day of incubation. MZ=marginal zone; H/E; 40×5; bar=30 µm
Fig. 2. Group of accessory lobe cells (AL) of turkey embryo form the bulge, which is visible at the 9th day of incubation. H/E; 20×5; bar=50 µm
Fig. 3. Migration of glioblasts from the ventricular to the intermedial zone (arrow) of turkey embryo is visible at the 9th day of incubation. H/E; 40×5; bar=30 µm
Fig. 4. Migration of glioblasts to the marginal zone of turkey embryo is visible at the 11th day of incubation. H/E; 20×5; bar=50 µm
Fig. 5. Accessory lobe of turkey embryo receives its final form with vacuolar glial cells (GC) and multipolar neurons (MN) at the 15th day of incubation. H/E; 40×3.75; bar=50 µm
Fig. 6. Difference in the size of multipolar neurons of the grey matter ventral horn (VH) and accessory lobes (AL) of turkey embryo is visible at the 15th day of incubation. H/E; 20×3.75; bar=50 µm
Fig. 7. After staining by the PAS method, the small thread of the PAS-positive granular product appear at the edges of accessory lobe cells (arrow) of turkey embryo at the 18th day of incubation. 20×5; bar=50 µm
Fig. 8. More intensive PAS-positive reaction in the accessory lobe cells of turkey embryo is visible at the 25th day of incubation. 20×5; bar=50 µm
Discussion
Results of this investigation proved that the accessory lobes are composed of two cell types differentiated in two periods of development. Neuroblasts or primitive neurons are distinguishable at the edge of the marginal zone at the 8th day of embryonic development. It is probable that they originate from cells of the ventral horn. Their differentiation in the multipolar neurons is somewhat slower than differentiation of multipolar neurons in the ventral horn of the grey matter. Completely developed neurons are visible in accessory lobes at the 16th day, and in the ventral horn at the 14th day, of embryonic development. Development of the neuroglial cells is obvious at the 9th day, when glioblasts begin to migrate from the ventricular zone. Glioblasts were seen through the marginal zone at the 11th day, and at the 15th day they occupied the accessory lobes region. Accessory lobes are completely developed at the 16th day of embryonic development in turkey. Embryos of turkeys of that age correspond with stage 39 of chicken embryo development (HAMBURGER and HAMILTON, 1951; VUKOVIC, 1996). UEHARA and UESHIMA (1982), concluded that cells of the glycogen body begin to synthesize and to accumulate glycogen soon after their differentiation from ventricular cells. As the glycogen granules of the accessory lobes cells were noticed at the 16th day, we can conclude that, according to the length of the glioblasts migration from the ventricular zone, this is the time of their differentiation to astrocytes. PAS-positive reaction is also noticed in neurons of the accessory lobes. The reason for glycogen accumulation is still not clear, but it is interesting to note that there was no sign of glycogen appearance in the neighbouring multipolar cells of the grey matter ventral horns in the avian spinal cord. The glycogen body of the bird species investigated so far is developed from the paired primordia, which appear at the dorsal section of the lumbosacral segment of the spinal cord on both sides of the ependimal septum. Paired primordia are gradually connected on the unique wedge-shaped structure, which extends from the dorsal edge of the spinal cord to its central canal. At the same time, small groups of PAS-positive cells appear ventrally from the central canal (known as the ventral glycogen body). Glycogen body development is completed by connecting dorsal and ventral parts in a unique structure, which surrounds the central canal of the spinal cord.
As the glycogen-rich cells are found, except in the glycogen body and accessory lobes, along the whole length of the spinal cord, UEHARA and UESHIMA (1982) development of the other glycogen reach regions were investigated. The authors concluded that such regions develop through the connection of the paired primordia in all portions of the spinal cord. We can conclude that accessory lobe development is specific because there are two main differences according to the other glycogen-rich regions in the avian spinal cord. The first is that the two cell types are developed in the accessory lobes, and both are capable of accumulating glycogen. The second difference is that each of the paired accessory lobes develop apart one from another, have their own embryonic base, do not develop by connecting the paired primordia like other glycogen-rich regions and, finally, are located ventrolaterally at the avian spinal cord.
References
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Received: 17 February 1999
Accepted: 16 June 1999
VUKOVIC, S., H. LUCIC, S. CURKOVIC: Razvoj dodatnih reznjeva kraljeznicne mozdine purana (Meleagris gallopavo). Vet. arhiv 69, 167-177, 1999.
SAZETAK
Dodatni reznjevi su neobicne strukture lumbosakralnog dijela kraljeznicne mozdine u ptica osebujne po sposobnosti nakupljanja glikogena. Gradeni su od neuroglia i zivcanih stanica nalik na multipolarne zivcane stanice ventralnih rogova sive tvari. Razvoj dodatnih reznjeva razlicit je od razvoja ostalih glikogenom bogatih podrucja kraljeznicne mozdine ptica. Osmog dana embrionalnog razvoja purana na ventrolateralnim rubovima kraljeznicne mozdine uocavaju se neuroblasti koji se tijekom daljnjeg razvoja diferenciraju u multipolarne zivcane stanice. Petnaestog dana embrionalnog razvoja u dodatnim reznjevima se pojavljuju glioblasti. Konacni oblik dodatni reznjevi dobivaju sesnaestog dana kada se pojavljuju i prva PAS-pozitivna zrnca glikogena. Glikogen se nakuplja u neuroglia stanicama i sto je narocito zanimljivo, manja kolicina pojavljuje se i u multipolarnim zivcanim stanicama.
Kljucne rijeci: puran, Meleagris gallopavo, kraljeznicna mozdina, dodatni reznjevi, multipolarne zivcane stanice, neuroni multipolarni, neuroglia stanice, glikogen, zametak