Arch. Histol. Cytol., Vol. 56, No. 5 (1993) p. 501-504

The Occurrence of Rat Spinal Cord Neurons with Strongly

Negative-Charged Surface Coats

Takuro MURAKAMI, Yutaka TSUBOUCHI, Mani TSUBOUCHI, Aiji OHTSUKA, Takehito TAGUCHI and Akio KIKUTA

Section of Human Morphology, Department of Anatomy, Okayama University School of Medicine, Okayama, Japan

Received September 14, 1993

Summary. Light microscopy of tissue sections stained with cationic iron colloid (pH 1.0-1.5) showed that the adult rat spinal cord contains some neurons which are

provided with strongly negative-charged surface coats. These neurons are distributed preferentially in the poste-rior and intermediomedial columns of the grey matter. The present study thus supplements our previous study of the rat brain (MURAKAMI et al., 1993b), and proves that the neurons with strongly negative-charged sur-face coats occur widely in the central nervous system of the adult rat.

Our previous study showed that the rat brain con-tains a considerable number of neurons which are

provided with strongly negative-charged surface coats (MURAKAMI et al., 1993b). The occurrence of such strongly negative-charged neurons has been confirm-ed even in the human brain (MURAKAMI et al., 1993a). The present paper supplementarily demonstrates in the rat that the spinal cord also contains some neu-rons with strongly negative-charged surface coats.

MATERIALS AND METHODS

Adult Wistar rats weighing 300-400 g were anesthet-ized with ethyl ether, and their ascending aorta was cannulated after ligation of the lower end of the abdominal aorta. The animal were then perfused with Ringer's solution and with 2.5% glutaraldehyde or 4.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Immediately after this perfusion fixation, the spinal cord was isolated, cut into approximately 1.0 mm-thick blocks along the frontal planes, and fixed again in the buffered 2.5% glutaraldehyde or

4.0% paraformaldehyde solution for 12 h or longer. The blocks thus fixed were embedded in paraffin,

cut into sections, incubated in our cationic iron col-loid with pH values of 1.0-1.5 (MURAKAMI et al., 1986), immersed in a mixture of K4Fe(CN)6 and HC1 for Prussian blue reaction, counter-stained with nuclear fast red or carbol-thionin mixture (MURAKAMI et al., 1993b), and observed with a light microscope.

RESULTS

Paraformaldehyde fixation as well as glutaraldehyde fixation allowed favorable staining of tissue anionic sites with our cationic iron colloid (Figs. 1-4). The neurons with strongly negative-charged sur-face coats were recognizable in every segment of the spinal cord. More strictly, some of the large-sized neurons in the posterior horn and intermediomedial region of the grey matter of the cervical, thoracic and lumbar segments showed a strong Prussian blue reaction after treatment with our cationic iron col-loid at pH values of 1.0-1.5 (Figs. 1-4). This intense Prussian blue reaction was always observed as thin coats or fine meshworks closely associated with the external surface of the cell body and on the roots of cell processes (Figs. 2-4). The meshwork structures of the reaction were clearly noted in the tangentially cut cases (Fig. 4). The counter-staining with carbol-thionin or nuclear

fast red showed that the neurons with the strongly negative-charged surface-coats were well reactive to thionin, revealed images of Nissl bodies (Fig. 3 Inset), and contained a well developed nucleus and nucleolus

(Figs. 2-4).

501

502 T.MURAKAMI et al.:

Through the spinal cord, few neurons in the ante-

rior horn and intermediolateral region of the grey matter were reactive to our cationic iron colloid at

pH values of 1.0-1.5. Few neurons in the lateral cer-vical nucleus, substantia gelatinosa or other areas of the white matter or related areas were also reactive

to our colloid with such low pH levels.

DISCUSSION

This paper demonstrates that some of the large-sized

neurons, especially their cell bodies and process roots,

in the posterior and intermediomedial columns of the rat spinal cord are coated with certain membrane-

associated substances which are strongly reactive to our cationic iron colloid at pH values of 1.0-1.5. Similar neurons with the same surface features have been demonstrated in the visual cortex, hippocampal subiculum, pontine nuclei, cerebellar nuclei and cer-tain other nuclei of the rat brain (MURAKAMI et al., 1993b) as well as in the visual cortex of the human brain (MURAKAMI et al., 1993a).

It is believed that only sulfate-groups can ionize at such low pH levels as 1.0-1.5 (SEND et al., 1985; SEND, 1987). It is therefore reasonable that the membrane-associated substances or strongly negative-charged sur-face-coats should be sulfated proteoglycans (MURA-KAMI et al., 1993b). Recent biochemical or electro-

phoretic analyses have confirmed that the rat brain

Fig. 1. A frontal section of the adult rat spinal cord, traversing the upper thoracic segment. This section was fixed with glutaraldehyde, incubated in our cationic iron colloid at a pH value of 1.0, treated with a mixture of K,Fe(CN)6 and HC1 for Prussian blue reaction, and stained with nuclear fast red. The portions indicated by the large and small arrows, are shown in Figures 2 and 3. X 60

Fig. 2. A closer view of the part indicated by the large arrow in Figure 1. Note that two large neurons

(arrowheads) are coated with membranous substances (surface coat) showing a strong Prussian blue reaction after incubation in our cationic iron colloid with a pH value of 1.0. X600

1 2

Strongly Negative-Charged Neurons in Adult Rat Spinal Cord 503

contains some sulfated proteoglycans such as chon-droitin and heparan sulfates (OOHIRA et al., 1986, 1988). More recent immunohistochemical experiments have demonstrated that the external surfaces of the Purkinje and Golgi cells in the rat cerebellum are stained with a monoclonal antibody against a 600- to 1000-kDa chondroitin sulfate proteoglycan (MAEDA et al., 1992). However, our previous study has shown that these surfaces of the Purkinje and Golgi cells are not reactive to our cationic iron colloid with pH values of 1.0-2.0, indicating an advantage with our method for detection of sulfate-groups (MURAKAMI et al., 1993b). As is well known, neurons of the posterior grey matter, especially its lateral area, mainly form the spinocervical pathway, and project to the lateral

cervical nucleus which terminates in the medial lem-niscus, thalamus and bulbar brainstem nuclei (DILLY et al., 1968; WEBSTER, 1977). Neurons of the medial area of the posterior grey matter form, together with those of the intermediomedial grey matter, the anter-olateral pathways which terminate in the thalamus and bulbar brainstem nuclei (GWYN and WALDRON, 1968; TRUEX et al., 1970). Judging from their posi-tions, the spinal neurons observed here with their strongly negative-charged surface coats may be in-cluded among those neurons forming the spinocer-vical or anterolateral pathways.

Acknowledgements. We are grateful to Mr. Hiromichi KUSANO for his kind help in tissue preparation and cationic iron colloid staining.

Fig. 3. A closer view of a neuron from the site indicated by the small arrow in Figure 1. Note that this neuron is provided with a marked surface coat which shows a strong Prussian blue reaction (arrowheads) (see also Figure 2). Inset shows two neurons in the posterior grey matter of the adult rat cervical spinal cord. Specimens were fixed with paraformaldehyde, incubated in our cationic iron colloid with a pH value of 1.5, treated for Prusssian blue reaction and stained with carbol-thionin. Note that the neurons with the strongly negative-charged surface coats (arrowheads) are reactive to thionin. X 1,200, Inset: x 700

Fig. 4. Three neurons with strongly negative-charged surface coats (large arrowheads) as observed in the intermediomedial area of the adult rat lumbar spinal cord. Fixed with paraformaldehyde, incubated in cationic iron colloid with a pH value 1.0, treated for Prussian blue reaction, and stained with nuclear fast red. Note that the tangentially cut surface-coats show fine meshwork structures (small arrowheads). x500

3 4

504 T. MURAKAMI et al.

REFERENCES

DILLY, P. N., P. D. WALL and K. E. WEBSTER: Cells of origin of spinothalamic tract in the cat and rat. Exp.

Neurol. 21: 550-562 (1968). GWYN, D. G. and H. A. WALDRON: A nucleus in the dorsolateral funiculus of the spinal cord of the rat. Brain Res. 10: 342-351 (1968). MAEDA, N., F. MATSUI and A. OOHIRA: A chondroitin

sulfate proteoglycan that is developmentally regulated in the cerebellar mossy fiber system. Devel. Biol. 151: 564-574 (1992).

MURAKAMI, T., T. TAGUCHI, A. OHTSUKA, K. SANO, T. KANESHIGE, R. L. OWEN and A. L. JONES: A modified method of fine-granular cationic iron colloid prepara-

tion: its use in light and electron microscopic detection of anionic sites in the rat kidney glomerulus and certain other tissues. Arch. Histol. Jap. 49: 13-23 (1986).

MURAKAMI, T., T. TAGUCHI and A. OHTSUKA: The occur- rence in the human brain of neurons with strongly negative-charged proteoglycans. Arch. Histol. Cytol. 56: 23-26 (1993a).

MURAKAMI, T., T. TAGUCHI, A. OHTSUKA and A. KIKUTA: Neurons with strongly negative-charged surface-coats in adult rat brain as detected by staining with cationic iron colloid. Arch. Histol. Cytol. 56: 13-21 (1993b).

OOHIRA, A., F. MATSUI, M. MATSUDA and R. SHOJI: Developmental change in the glycosaminoglycan com-

position of the rat brain. J. Neurochem. 47: 588-593 (1986).

OOHIRA, A., F. MATSUI, M. MATSUDA, Y. TAKIDA and Y. KUBOKI: Occurrence of three distinct molecular species of chondroitin sulfate proteoglycan in the developing rat brain. J. Biol. Chem. 263: 10240-10246 (1988).

SEND, S.: Ionized groups on the cell surface: their cyto- chemical detection and related cell function. Int. Rev. Cytol. 100: 203-248 (1987).

SEND, S., M. AKITA, T. ONO and T. TSUJII: Fine-granular cationic iron colloid. Its preparation, physicochemical characteristics and histochemical use for detection of ionized anionic groups. Histochemistry 82: 307-312

(1985). TRUER, R. C., M. J. TAYLOR, M. Q. SMYTHE and P. GILDENBERG: The lateral cervical nucleus of cat, dog and man. J. Comp. Neurol. 139: 93-104 (1970).

WEBSTER, K. E.: Somaesthetic pathways. Brit. Med. Bull. 33: 113-120 (1977).

Prof. Takuro MURAKAMI Section of Human Morphology Department of Anatomy Okayama University School of Medicine 2-5-1 Shikata-cho, Okayama 700 Japan

村 上 宅 郎

700岡 山市 鹿 田町2ー5-1

岡 山大 学 医学部

解剖学 第二 講座