ACTA HISTOCHEM. CYTOCHEM. Vol.16, No.2, 1983 CYTOCHEMICAL LOCALIZATION OF ALKALINE PHOS- PHATASE IN THE SPINAL CORD EPENDYMA OF NEWBORN AND ADULT RATS1 TAKAFUMI YOSHIOKA AND KENICHIROU INOMATA Departmentof Anatomy,Shimane Medical University,Izumo 693 Received for publication June 11, 1982 and in revised form September 7, 1982 Localization of alkaline phosphatase (AlPase) in the spinal cord ependyma of newborn and adult rats was studied using cytochemical techniques. When the reaction products were precipitated on the plasma membrane of the ependymal cells, the most intense AlPase activity was apparent on the membrane of the luminal surface of tissues from the newborn rats and this activity was also detected in the neuropil around the central canal. In the axon terminals, reaction products were precipitated on the plasma membrane of the presynapse. These observations warrant further studies on AlPase activity in the ependyma to determine if this enzyme plays a role in the mem- brane transport of the substances from the cerebrospinal fluid to the extra- cellular spaces of the spinal cord. The ependyma of the central nervous system (CNS) forms a selective barrier between the nervous tissue and the cerebrospinal fluid (CSF), and this mechanism has been termed the CSF-brain barrier (5, 12). Morphologically, the ependyma consists of a single layer of closely packed neuro- epithelial cells (or ependymal cells) (3, 7, 11). A varied number of cilia and a large number of short and slender microvilli project from the free or luminal surface of the cell. The ependymal cells form a junctional arrangement, including zonulae occludens, adhaerens and gap junctions, with adjacent cell membranes at the lateral surface. At the basal surface, a long, slender, and branching process descends into the underlying neuropil. Similar cells exist in the absorptive epithelium such as the small intestine and the kidney. It has been reported that alkaline phosphatase (AlPase) activity in these cells is localized on the plasma membrane of the luminal surface, and the possibility that AlPase activity plays an important role in the transport of the substances across the plasma membrane has been suggested (2, 8, 10, 16). There is apparently no documentation of cytochemical studies on the central canal of the spinal cord. We studied the ultrastructural localization of AlPase activity in the ependymal cell of the central canal in newborn and adult rats at the lumbar level and changes with development were observed. MATERIALS AND METHODS Albino rats at the 5th and 7th postnatal day were used for the ultracytochemical 1. This paper is dedicated to the late Prefessor Kazushige Nakamura, Department of Anatomy, Shimane Medical University. 77 78 YOSHIOKA AND INOMATA demonstration of the AlPase activity during the developmental stage and adult Wistar rats (Japan Clea) were used for studies on adult animals. All the rats were anesthetized with sodium pentobarbital (intraperitoneally) and vascular perfusion through the heart was started with physiological saline solution at room temperature for 1 min, and followed by cold 0.1 M cacodylate-buffered 1% glutaraldehyde and 2% paraformaldehyde mixture, pH 7.3-7.4, 920 mOsm. After perfusion for 15 min, the spinal cord at the lumbar level (L1-2) was removed and immersed in the same fixative for 15 min at 0-4•Ž. The materials were sectioned with a Vibra- tome at approximately 40 ƒÊm in thickness, then rinsed with cold 0.1 M cacodylate buffer, pH 7.3-7.4, containing 3% sucrose, 290 mOsm. These sections were incubated in the medium for AlPase activity (10). The omission of substrate from the complete incubation medium, substrate-free medium, and the addition of 5 mM levamisole to the complete medium were the procedures used for the control experiments. After incubation for 30 min at 37•Ž, the sections were postfixed with the cold 1% osmium tetroxide in Caulfield's buffer for 40 min, dehydrated in a series of the graded ethanol solutions, treated with propylene oxide and embedded in Spurr's medium. Thin sections for electron microscopy were obtained with a Porter-Blum MT-2 ultramicrotome or a Reichert-Jung ultracut OmU4 and stained with uranyl acetate or uranyl acetate and lead nitrate. These thin sections were examined under a HS-9 (Hitachi) electron microscope at 75 KV. RESULTS AlPase activity in the ependyma In the 5th and 7th postnatal day rats, ependymal cells had a long-oval nuclei with an irregular shape and were localized in the basal portion of the cells. Re- action products, lead phosphate, were precipitated over the entire plasma membrane of the luminal, lateral and basal surface (Fig. 1). The intense activity was demon- strated on the microvilli and the cilia at the luminal surface, the zonula adhaerens and the gap junction at the lateral surface (Figs. 1, 2a). In addition, the pinocytotic vesicles with intense activity were mainly localized in the lateral and basal portions of the cells. In organelles such as lysosome etc. AlPase activity was negative. In adult rats, AlPase activity revealed a localization pattern rather similar to that seen in the newborn rats, although the activity was comparatively weaker (Figs. 3, 4a). The nucleus of the ependymal cell was oval-shaped and was localized in the central part of the cell. Reaction products for AlPase activity in the luminal surface were evident on the plasma membrane and the activities had no homogenous pattern. However, the activity of the zonula adhaerens and the gap junction in the lateral surface did have a homogenous pattern. Here the number of the pinocytotic vesicles with AlPase activity were more numerous , as compared to findings in the newborn rats. When sections were incubated in a levamisole-containing medium , the reaction FIG. 1. AlPase activity in the ependyma of the lumbar spinal cord of 7-day-old rat . mv: microvilli. FIGS.2 a, b. The luminal surface of the ependymal cell of 5-day-old rat . (a) The plasma membranes of the microvilli (mv) and the cilia (cil) showed the intense activity of AlPase . (b) Control experiment with the substrate-free incubation medium . Note the lack of reaction product. ALPASE ACTIVITY IN EPENDYMA OF SPINAL CORD 79 80 YOSHIOKA AND INOMATA ALPASE ACTIVITY IN EPENDYMA OF SPINAL CORD 81 FIG. 5. A part of the neuropil around the central canal on the 7th postnatal day. Reaction products are apparent in the plasma membranes of many neuronal elements, including soma, dendrites and axons. of AlPase was inhibited (Fig. 4b) and no reaction occurred in specimens incubated in the substrate-free medium (Fig. 2b). AlPase activity in the neuropil around the ependyma. In the neuropil around the ependyma in both newborn and adult rats, the plasma membrane of the neuronal elements exhibited the AlPase activity (Fig. 5). Reaction products were precipitated on the plasma membrane of the neuronal soma, the dendrite, and the axolemma. The most external lammelae of the myelin in the myelinated fiber, also, showed the AlPase activity. In addition, reaction products were associated with the axon terminal (Figs. 6a, b). AlPase activity was localized on the plasma membrane of the presynapse. No difference was found in the activity of the plasma membrane and that of the membrane thickening, thereby indicating probable active sites of the synaptic zone. There was no evidence of reaction products in the control experiments. DISCUSSION Our findings of localization of intense AlPase activity on the plasma membrane FIG. 3. AlPase activity in the ependyma of adult rat. mv: microvilli FIGS.4a, b. (a) An invaginated portion of the luminal surface of the ependymal cell of an adult rat. (b) Control experiment, using the final concentration of 5 mM levamisole. Note the lack of reaction product in any structure of the luminal surface. 82 YOSHIOKA AND INOMATA FIGS. 6a, b. AlPase activity exclusively restricted to the presynaptic membrane of the axon terminal. (a) 5-day-old rat. (b) adult rat. of the luminal surface in the ependymal cell are consistent with findings in case of the absorptive epithelia, in particular the brush border of the small intestine and the proximal convoluted tubules (2, 3, 10, 16). This polarity may suggest the existence of absorptive ability in the ependymal cell at the central canal. In the ependyma of the third ventricle, the tanycytes, which show intense adenosine triphosphatase activity in the luminal microvilli, receive humoral signals by means of uptake of the substances from the CSF (6). However, little is known of the accurate role of AlPase in the ependyma because this enzyme is non-specific, although AlPase is said to be a marker-enzyme for the plasma membrane. The function of AlPase is presumably related to a selective transport of certain phosphorylated metabolites across the plasma membrane. Sakla (14) demonstrated cell migration from the wall of the central canal and the proliferative activity of the cell in the cervical region in mice for up to 30 days after birth. It is considered that enzymes depend to a considerable extent on the processes determining the normal maturation of the brain (13). Therefore, the possibility that AlPase activity may play an important role on the development of the central canal has to be considered. In addition, the number of pinocytotic vesicles showing AlPase activity in tissues from the adult rats is associated with the CSF-brain mechanism and is the result of transependymal transport. There was a remarkable AlPase activity localized on the plasma membrane of the presynapse in the neuropil around the central canal. The communication between neurons is regulated by neurotransmitters, neuromodulators and neuro- ALPASE ACTIVITY IN EPENDYMA OF SPINAL CORD 83 hormones, at the synapse (4, 16). Biochemical studies on the isolated synaptosomes showed that many neuroregulators were incorporated into the synaptosomes from the medium, and that the process of uptake required the presence of sodium or diva- lent cations such as calcium and magnesium (1, 9, 15, 17).