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Correlated Electrophysiology and Morphology of the Ependyma in Rat Hypothalamus

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Correlated Electrophysiology and Morphology of the Ependyma in Rat Hypothalamus The Journal of Neuroscience, October 1988, 8(10): 3691-3702 Correlated Electrophysiology and Morphology of the Ependyma in Rat Hypothalamus C. R. Jarvisa and Ft. D. Andrew Department of Anatomy, Queen’s University, Kingston, Ontario, Canada K7L 3N6 The ependyma lines the ventricular system of the vertebrate and may therefore have similar functions. In the hypothal- brain and spinal cord. Although its embryology and mor- amus, ependyma probably take up K+ released from adja- phology have been studied extensively, little is known of its cent endocrine neurons and shunt it to the ventricular space. physiological properties, particularly in mammals. Tany- cytes are modified ependymal cells that are found predom- The ventricular system of the brain and spinal cord is lined by inantly lining the floor of the third ventricle, overlying the the ependyma, a singlelayer of squamous,cuboidal, or columnar median eminence. Their processes accompany and enwrap epithelial cells. Some ependymal cells, termed tanycytes, have neuroendocrine axons that course from hypothalamic nuclei long basal processesextending into the underlying neuropil. to terminals in the median eminence, but the significance of Tanycytes form the ependymal lining over the circumventricu- this interaction is not yet understood. Intracellular recording lar organs and are found scattered within the ependymal layer and injection techniques were used to study ependymal cells of the ventricles and spinal canal. Most cells lining the third and tanycytes of the rat in the hypothalamic slice preparation ventricle are cuboidal (common ependyma), although tanycytes after differentiating their respective regions morphologically. are clustered along the lateroventral wall and in the floor over- With extracellular [K+] = 6.24 mM, the mean membrane po- lying the median eminence.Tanycyte processesaccompany and tential (*SD) for common ependyma was -79.9 + 1.40 mV enwrap neuroendocrine axons that course from hypothalamic and for tanycytes, -79.5 f 1.77 mV. Input resistances (/Ii,) nuclei to terminate on the portal vasculature of the median were very low (a 1 MD). Single-cell injection of Lucifer yel- eminence(Kozlowski and Coates,1985). Ultrastructurally, some low revealed dye coupling among 2-70 ependymal cells and neuroendocrine axons appear presynaptic to tanycyte processes 5-46 tanycytes. In both freeze-fractured replicas and thin (Card and Moore, 1985) but the significanceof this interaction sections, large numbers of gap junctions were found be- is not yet understood. tween adjacent ependymal cells and between adjacent tan- Although the morphology of common ependymal cells and ycytes. The observations of numerous gap junctions, exten- tanycytes has been thoroughly examined (reviewed by Koba- sive dye coupling and low input resistance demonstrated yashi et al., 1970; Fleischhauer, 1972; Peters et al., 1976; Bruni that both populations are strongly coupled networks. Per- et al., 1985; Flament-Durand and Brion, 1985), the function of haps for this reason, attempts to uncouple these cells using these cells remains unknown. Common ependyma may play a sodium propionate or CO, were unsuccessful. Electrical role in the absorption/secretion of CSF (review by Bruni et al., stimulation of the arcuate nucleus did not elicit any detect- 1985) and the beating of their apical cilia is thought to produce able synaptic response in impaled tanycytes, so that the movement ofthe CSF (Scott et al., 1974). Although it is unlikely functional significance of synaptoid contacts between neu- that bulk fluid movement results, such beating could stir the roendocrine neurons and the postsynaptic tanycytes is not fluid at the CSF-ependyma interface, hasteningthe exchangeof yet apparent. Ependymal cells and tanycytes demonstrated substances.Several functions have been attributed to tanycytes a near-Nernstian response to changes in extracellular [K+] (review by Peters et al., 1976; Bruni et al., 1985; Flament- between 3 and 20 mM. This finding, as well as their high Durand and Brion, 1985) including phagocytosis(Brawer and negative resting potential, low Ri,, extensive coupling and Walsh, 1982) mechanical support, regulation of extracellular absence of spontaneous electrical excitability demonstrate space constituents, and transport of substancesbetween CSF that ependymal cells possess numerous glial characteristics and the portal vasculature (critical review by Pilgrim, 1978). Thesefunctional hypothesesare basedlargely on morphological observations. Of particular interest is the intimate structural relationship between the ependyma of the third ventricle and Received July 8, 1987; revised Feb. 8, 1988; accepted Feb. 15, 1988. neuroendocrineneurons of the hypothalamus (Card and Moore, We thank Dr. R. R. Shivers, University of Western Ontario, for use of his freeze-fracture facilities. Also thanks to Dr. P. K. Rose for helpful discussion, Dr. 1985; Kozlowski and Coates, 1985). B. R. Ransom for supplying a manuscript in press, Ms. A. Doyle for typing the In contrast to the numerous structural studies, electrophys- manuscript, and Mr. H. Verstappen for photographic work. Supported by oper- iological data are restricted to 2 studies examining the ating and equipment grants from the Canadian MRC, the Botterell Foundation, and the Dean’s MRC Fund. properties of ependyma in the turtle (Chan and Nicholson, 1986; Correspondence should be addressed to Dr. R. David Andrew at the above Connors and Ransom, 1987) and one study examining mam- address. a Present address: Division of Neurology, Montreal General Hospital, 1650 malian ependymal cells in culture (Hild et al., 1965). In the Cedar Avenue, Montreal, Quebec H3G lA4 Canada. present study, we intracellularly recorded and dye-injected 2 Copyright 0 1988 Society for Neuroscience 0270-6474/88/103691-12$02.00/O morphologically distinct populations of ependymal cells (cu- 3692 Jaws and Andrew - Electrophysiology and Morphology of Rat Ependyma . P paraformaldehyde and 0.7% glutaraldchydc in 0.1 M sodium cacodylatc. Washing in 0.1 M sodium cacodylate buffer preceded postfxation in 1% P buffered osmium tetroxide for 1.5 hr. Following rinsing in buffer, the tissue was stained en hlocin 1% uranyl acetate overnight and dehydrated through an ethanol series. Using propylene oxide as a transition solvent, the slices were embedded in 100% Epon in Beam capsules and poly- merized at 16°C for l-2 d. Semithin (0.7 Frn) and ultrathin (70 nm) sections were cut with an ultramicrotome. For light microscopy, scmithin sections were mounted on slides, stained with toluidine blue and photographed with a Zeiss Photomicroscope II. For TEM, silver sections were mounted on copper grids, stained with 3% uranyl acetate followed by 2% lead citrate and examined with a Hitachi 500 TEM at 75 kV. Scanning electron microscopy (SEMI. The hypothalamus was blocked, and the dorsal extent of the third ventricle was cut. The adjacent halves of the hypothalamus were gently spread apart and pinned down in a dissecting tray. This exposed the entire ependymal lining of the third ventricle,the walls ofthe third ventricle remaining attached by the floor. Following fixation as for TEM (above), the tissue was washed in 0.1 M sodium cacodvlate buffer, dehydrated through an ethanol series and transferred to Freon-l 13. Using-a Polaron critical-point dryer, the tissue ‘Ii : was dried with liquid CO2 at 38°C and 1200 psi. The blocks were coated with gold in an argon-pure vacuum atmosphere using a Polaron sputter coater set at 15 mA and 2.3 kV, for 3 min. The specimens were examined with a Hitachi S450 SEM fitted with a Polaroid camera. Freeze fracturing. The hypothalamus was blocked and sliced coronally (1 mm). The tissue was fixed at 4°C for 1 hr in a cacodylate-buffered solution of 1% oaraformaldehvde and 1% alutaraldehvde (uH 7.2-7.4) and then washed in 0.1 M cacddylate buffe;containing 4%‘glucose (wti vol). Blocks cryoprotected in 30% glycerol were placed on gold specimen supports (Balzers) and oriented to fracture in the coronal plane. The specimens were immersed in Freon-22 (maintained near its freezing point by liquid NJ and stored in liquid NZ. Tissue was fractured in a Balzers BAF 301 Freeze-Etching Unit (Shivers and Brightman, 1976) while the stage was maintained at ~ 115°C and the vacuum at 10 h Torr. The specimens were coated with platinum (2300 V and 70 mA at 400 Hz for 6-8 set) followed by carbon (2500 V and 110 mA at 400 Hz for 4-8 set). Tissue adhering to the replicas was removed in filtered 10% household bleach. The replicas were rinsed and collected on copper grids and examined with TEM. Electrophysiology Figure 1. Diagram of the rat hypothalamus viewed from a caudal aspect. Slices of the hypothalamus (350-500 Km) were cut in a coronal Tmue preparation. The hypothalamus was blocked as described above (COR) or sagittal (SAG) plane and ependyma impaled with microelec- and placed ventral side down on a tissue slicer. Beginning at the anterior trodes. E, common ependymal cell; T, tanycyte; LT, lamina terminalis; commissure, coronal slices were cut (Fig. 1) and placed in a recording P, endocrine neurons of preoptic area; A, endocrine neurons of arcuate chamber. Slice dimensions were approximately 34 mm x 5 mm x nucleus. Note the close association (1) between common ependyma and 350-500 pm thick (see Fig. 3A). Alternately, 2 sagittal slices were pre- neuroendocrine axons and (2) between tanycyte processes, neuroen- pared by cutting the roof of the third ventricle so that the opposing docrine axons, and the capillaries of median eminence (ME) into which halves of the hypothalamus could be pulled apart. The ventricular floor neurohormone is secreted. (Adapted from Kozlowski and Coates, 1985.) was then cut to separate the halves. With the ependymal layer exposed, each half of the block was sliced approximately 500 pm parasagittal to the midline (Fig. 1) using the tissue slicer. boidal cells and tanycytes) to better understand their function. Slices were weighted with 0.38-mm-diameter silver wire and super- Some of the results have been presented in preliminary form fused with artificial CSF consisting of (in mM): 124 NaCl, 25 NaHCO,, 5 KCl, 1.3 MgSO,, 1.24 KH,PO,, 2.4 CaCl?, and 1 1 glucose.
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