Continuity between the ventricular and subarachnoid cerebrospinal fluid in an amphibian,Rana pipiens

Summary Continuity between the ventricular and subarachnoid cerebrospinal fluid has been investigated in Rana pipiens. The structure of the posterior tela, a deficient membrane situated at the extreme caudal end of the roof of the fourth ventricle, has been studied using whole membrane mounts and by light microscopy of resin embedded tissue. The ependymal component consists of columnar and rounded cells which form a regular syncytium enclosing round and oval fenestrations. Small fenestrations are covered on the subarachnoid side by elongated pial cells and thus do not give total continuity between the fourth ventricle and the subarachnoid space. Large fenestrations, on the other hand, are accompanied by equivalent pial fenestrations giving direct access between the fluid compartments. Towards the caudal end the fenestrations break up and the numbers of ependymal and pial cells decrease, the caudal end itself being characterised by a small remaining clump of ependyma and pia or of pia alone.Flow through the tela has been studied using fluorescein-labelled dextran placed in the intraventricular space. Infusion into the lateral ventricle and subsequent localisation by fluorescence microscopy shows the marker to be in the fourth ventricle, in the fenestrations of the posterior tela and in the subarachnoid space overlying the tela. Infusion of the marker followed by freezing and examination of the cut heads on a freezing microtome, shows fluorescence throughout the ventricular system, in the subarachnoid space adjacent to the posterior tela and also along the dorsal subarachnoid space of the spinal cord.     

Cytodifferentiation in the Rana pipiens oocyte

Summary In early diplotene frog oocytes incubated to illustrate thiamine pyrophosphatase (TPPase) activity, reaction product is uniformly distributed within the compartments of the endoplasmic reticulum and nuclear envelope as well as within the saccules and small vesicles comprising the dictyosomes. With continued oocyte development the reaction product becomes concentrated in localized regions of the dictyosome saccules. Eventually, the enzyme is no longer apparent within the endoplasmic reticulum, but is concentrated in the cisternae of the inner dictyosome saccules. The variations noted suggest that the enzyme is synthesized early in diplotene by the endoplasmic reticulum and is subsequently transported to the Golgi apparatus where it is consistently observed at later developmental stages. TPPase activity is also present in the Golgi apparatus of follicle and theca cells as well as in ovarian epithelial cells. The enzyme is also detected in micropinocytotic vesicles contained within the cells comprising the follicle envelope and in intercellular spaces of the follicle. Horseradish peroxidase injected into the coelomic cavity is transported via micropinocytotic vesicles into and through the cells comprising the follicle envelope and in intercellular spaces. The exogenous protein is not found even after a prolonged time period in early diplotene oocytes. The protein is, however, present in large spherical and tubular vesicles in the cortex of vitellogenic oocytes approximately 500 microns in diameter. The possible functional role of the enzyme TPPase during oogenesis is discussed.This investigation was supported by a research grant from the National Science Foundation (GB-8736).     

Central projections of the frontal organ of Rana pipiens,as demonstrated by the anterograde transport of horseradish peroxidase

Summary The central projections of the frontal organ of Rana pipiens are more widespread, and more similar to those of the epiphysis, than previously realized. Fibers labeled with horseradish peroxidase were traced to the amygdala, the epiphysis, the pretectal region, and several nuclear areas of the mesencephalic and diencephalic central gray. When peroxidase reaction product was carefully distinguished from neuromelanin by means of polarization microscopy, no unequivocally labeled cell bodies of centrifugal fibers could be found.Supported by NIH grants GM-09181, EY-02083, and by BRSG RR-05357 awarded by the Biomedical Research Grant Program, Division of Research Resources, N.I.H.     

High molecular weight-multicatalytic proteinases in premature and mature oocytes of Rana pipiens

High molecular weight, multicatalytic proteinases (named proteasomes) have been for the first time found, on the basis of different protein patterns, in the cytoplasmic soluble fractions of both non-hormone-treated (premature) and progesterone-treated (mature) oocytes of a frog (Rana pipiens).These enzymes, pooled separately as two fractions sedimenting between around 19S and the bottom (over 27S) on glycerol density gradient centrifugation, were composed of several molecular forms with apparent high molecular weights ranging from over 700 kDa, as judged on Sepharose 6B gel filtration. In addition, both the fractions hydrolyzed distinctly a Tyr-containing substrate in the presence of SDS as an activator, and exhibited higher activities toward Arg-containing substrates in the absence of SDS, and activity toward a Glu-containing substrate in the presence and absence of SDS. Immunological experiments using antibodies against proteasomes purified from ovaries of Xenopus laevis clearly revealed characteristic cross-reactivity with both the fractions found in Rana.These data suggest that these enzymes in the two fractions from the respective oocytes in Rana are very similar or identical to the proteasomes of Xenopus. The enzymes in premature oocytes eluted at 0.15–0.18M NaCl on a DEAE-cellulose column disappeared on treatment with TPCK, a well-known chymotrypsin inhibitor, suggesting that the 0.15–0.18M NaCl-eluate contained chymotrypsin-like proteinases probably latent in ovo. The enzymes in mature oocytes had not similar chromatographical patterns to those in premature oocytes.These results suggest that the enzymes already present in premature oocytes may be involved through conformational alterations as to the protein pattern in oocyte maturation following induction by progesterone.Abbreviations AMC 7-Amino-4-methylcoumarin - Boc t-Butyloxycarbonyl - Cbz Carbobenzoxy - GVBD Germinal Vesicle Breakdown - MCA 4-Methylcoumaryl-7-Amide - MPF Maturation Promoting Factor - NA 2-Naphthylamide - SDS Sodium Dodecyl Sulfate - Suc Succinyl - TPCK N-Tosyl-L-Phenylalanine Chloromethyl Ketone     

Directionality of phase locking in auditory nerve fibers of the leopard frog Rana pipiens pipiens

Summary A dorsal approach to the eighth nerve and free-field stimulation were used to investigate the effect of sound direction and intensity on phase locking in auditory nerve fibers of the leopard frog Rana pipiens pipiens.Tuning curves of 75 auditory neurons were analyzed (Fig. 2). Amphibian papillar neurons, but not basilar papillar neurons, exhibit significant phase locking to short tone bursts at the characteristic frequency (CF), the degree of phase locking (vector strength) decreasing with the neuron's CF (Figs. 3, 4 and 10E). Vector strength increases with sound pressure level to saturate about 20 dB above threshold, while the preferred firing phase is only slightly affected (Figs. 5 and 6).In contrast, sound direction hardly affects vector strength (Figs. 7, 8, 9A and 10A and C), but has a strong influence on the preferred firing phase (Figs. 7, 8, 9B and C, 10B and D): With respect to anterior tone presentation there are phase lags for ipsilateral and phase leads for posterior and contralateral presentation. Phase differences between both ears show a sinusoidal or cardioid/ovoidal directional characteristic; maximum differences are found with antero-lateral tone presentation (Fig. 11). The directionality of phase locking decreases with the neuron's CF (Fig. 10F) and only slightly changes with sound pressure level (Fig. 12). Thus, phase locking of amphibian papilla neurons can potentially provide intensity-independent information for sound localization.Abbreviations SPL sound pressure level - FTC frequency threshold curve - CF characteristic frequency - TF test frequency - VS vector strength - AP amphibian papilla - BP basilar papilla     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.     

Influence of changes in composition of the cerebrospinal fluid on the secretory activity of the subcommissural organ in Rana esculenta

Summary Normally the lymphatic sinuses of the lymph node are loosely packed with lymphocytes and free macrophages as well as with macrophages adhering to the fibrocellular trabeculae. After immunization with SRBC cluster formation occurs in the medullary sinuses of rats between a central macrophage and peripherally located lymphocytes. These rosette-like clusters are nearly identical with the clusters found during primary and secondary immune response against SRBC in vitro and seem to be the in vivo equivalent for the same immune response.     

Inheritance of enzymes and blood proteins in the leopard frog,Rana pipiens: Three linkage groups established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.The project was supported by Grant No. RR-00572 from the Division of Research Resources, National Institutes of Health. This paper is contribution No. C-87 from the Amphibian Facility, George W. Nace, Director.     

Afferent and efferent components of the facial nerve in a frog,Rana pipiens

Summary The seventh cranial nerve in Rana pipiens is a slender nerve with limited peripheral distribution. We investigated the afferent and efferent components of this nerve by labeling its major branch, the hyomandibular, with horseradish peroxidase. The efferent portion of the seventh nerve originates from a small cell group in the upper medulla which contains two subdivisions. Afferent fibers carried in nerve VII travel in the solitary tract and the dorsolateral funiculus. The solitary component consists of a small number of ascending fibers that reach the level of the trigeminal nucleus and a large descending component that terminates slightly caudal to the obex in the commissural nuclei of the solitary complex. Afferent fibers also descend in the dorsolateral funiculus; many of these fibers cross dorsal to the central canal in the lower medulla. Most of the fibers in the dorsolateral funiculus terminate in the ipsilateral and contralateral dorsal horns and in nuclei of the dorsal column. A few ipsilateral fibers reach lower thoracic levels of the spinal cord.     

Ultrastructure and hormonal content of the proximal stump of the transected hypothalamo-hypophysial tract of the frog (Rana pipiens)

Summary Several types of neurosecretory fibers were observed in the normal infundibulum of the frog. After transection of the median eminence, these neurosecretory fibers of the proximal stump reacted asynchronously, but followed approximately the same pattern: a passive accumulation of granules observed early after the transection was followed by an active axonal reaction with the appearance of numerous tubular formations which are thought to be related to the Golgi apparatus. They filled the axon almost completely, and then became dilated and filled with an electron dense material. Subsequently these dilatations pinched off and gave origin to new neurosecretory granules. These locally packed granules plus others which were probably formed in more proximal parts of the axon, and the perikaryon and then transported distally, accumulated in the proximal axonal stumps and started to fill the fibers retrogradely.There was a parallelism between the increase of tubular formations and neurosecretory granules larger then 1,500 Å in diameter, on one side, and the vasopressor activity of the proximal stump, on the other. The latter increased at an approximate rate of 1 mU/stump/day.The regeneration of the fibers of the hypothalamo-median eminence system is suggested by the presence in the proximal stump of fibers filled with granules smaller than 1,000 Å in diameter (normally seen in the median eminence) and the fact that 40% of the vasopressor activity of the extracts was not abolished by the thioglycollate treatment, which could be due to the presence of vasopressor amines other than adrenaline. The appearance towards the end of the observation period of a few nerve endings of several types contacting the perivascular basement membrane of vessels of the proximal stump would indicate that the neural lobe and median eminence functions were being reestablished, at least partially.This investigation was supported by grants 5RO1 NB 06641 NEUA and 5RO1 NB 07492 NEUA from the National Institute of Health and by the Space Sciences Research Center of the University of Missouri. The authors wish to thank Mrs. G. Clark, Mr. G. Ribas and Mr. R. Faup for their valuable technical help.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.     

Scanning and transmission electron microscopy of the subfornical organ of the grass frog (Rana pipiens)

Summary The ventricular surface of the subfornical organ of the frog is made up of ependymal cells with numerous apical microvilli, occasional cytoplasmic protrusions and many vacuoles projecting into the lumen of the third ventricle. Between these cells dendrites of cerebrospinal fluid-contacting neurons reach the ventricle to terminate in bulbous enlargements. In addition, flask-shaped encephalo-chromaffin cells, containing granulated vesicles and aggregates of filaments in their cytoplasm, project into the cerebrospinal fluid. Surrounding the centrally located capillaries are enlarged dendrites and axons of heterogeneous morphology, some of which appear to originate within the subfornical organ, intermingled with dendrites and axons of normal structure. The glial cells in this region, especially the microglial cells, often contain large lipofuscin inclusions, suggestive of degeneration and subsequent phagocytosis of some of the enlarged dendrites and axons. The normally scarce neurosecretory peptidergic axons become more evident and form typical Herring bodies in stalk-transected animals. Neuronal perikarya of varying morphology are predominantly located peripheral to the region of enlarged dendrites and axons. Supraependymal macrophages are particularly numerous on the subfornical organ.Abbreviations used CSF cerebrospinal fluid - SEM scanning electron microscope, scanning electron microscopy - SFO subfornical organ - TEM transmission electron microscope, transmission electron microscopy Supported, in part, by NIH grant NB 07492The skillful technical assistance of J.G. Linner and the secretarial assistance of Ann Gerdom are gratefully acknowledged. The SEM studies were made possible through a grant from the Graduate College of Iowa State University and the use of the SEM facility in the Department of Botany     

Immunocytochemical and ultrastructural study of the frog (Rana pipiens) pars distalis with special reference to folliculo-stellate cell function during in vitro superfusion

Summary The colloidal gold immunocytochemical technique was used to determine the ultrastructural features of the glandular cells in the pituitaries of male frogs, Rana pipiens, both in vivo and after superfusion in vitro. Specific reactions to antisera against bullfrog gonadotropins, human prolactin, and synthetic 1–39 corticotropin allowed identification of the 3 corresponding types of glandular cells. No immunoreaction was obtained with antisera against human or ovine-growth hormone, human -thyrotropin hormone, and bovine S-100 protein. General morphological features of these immunocytochemically identified glandular cells were similar to those of equivalent cells previously described in other amphibian species. Non-glandular folliculo-stellate cells were distinctive. In freshly removed pituitaries, these folliculo-stellate cells contained lysosome-like structures, but did not show phagocytic vacuoles in the cytoplasm; they contained many mitochondria, and the Golgi complex and endoplasmic reticulum were relatively undeveloped. After 4 or 18 h of superfusion, some immunoreactive gonadotropic, prolactin, and corticotropic cells showed degeneration and destruction. In the same gland, folliculo-stellate cells retained a viable appearance, but showed phagocytic vacuoles containing secretory granule-like structures which were immunoreactive to gonadotropic, prolactin, and corticotropic antibodies. Some folliculo-stellate cells showed phagocytic vacuoles containing complete glandular cells. These results suggest that superfusion causes a destruction of some of the glandular cells, and that folliculo-stellate cells act as phagocytes when cellular debris or moribund cells are present in the intercellular space in the pituitary parenchyma.Supported by grant DCB 8710462 from the National Science Foundation, grant 2148-83 from the CAICYT (Spain) and the Junta de Andalucia (Spain)     

Esterases in the mosquito Culex pipiens pipiens L.: Formal genetics and polymorphism of adult esterases

The genetics of two esterase loci active in autogenous adults of the mosquito Culex pipiens pipiens L. has been studied by means of starch gel electrophoresis. Three alleles at the Est-1 locus and eight at the Est-2 locus are described. Both loci have a null allele. Active alleles are codominant and there is no hybrid enzyme in heterozygotes. The Est-1 locus codes esterases preferentially hydrolyzing -naphthylacetate and the Est-2 locus esterases preferentially hydrolyzing -naphthylacetate. Strains homozygous for both loci were selected. Linkage studies of the two loci have shown that they are not sex linked but are linked to each other, the crossover frequency being 8.6%. The polymorphism of two laboratory and two natural populations is described for both loci. Phenotypic distributions are in good agreement with Hardy-Weinberg expectations.This work was conducted at the Université des Sciences et Techniques du Languedoc (Laboratoire de Génétique Expérimentale des Populations), Montpellier, France, in partial fulfillment of the requirements for the degree of Docteur de spécialité.     

Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria,Rana esculenta)

Summary The pattern of the vascular supply to the choroid of the frog eye was studied in toto with the use of the injection-replication-SEM technique. The choroid of anuran amphibians is composed mainly of the choriocapillaris. In both species studied (Rana temporaria, Rana esculenta), an independent arterial supply to the choriocapillaris supplemented that from the ciliary arteries. This additional vascular route arises from the optic artery, a separate branch of the arteria infundibularis superficialis. The optic artery, accompanied by its vein within the vascular sheath of the optic nerve, joins the rich arterial capillary network of the choriocapillaris and supplies the posterior pole of the ocular bulb. The superficial capillary network displays a dense collar around the entrance of the optic nerve into the eye and is composed of a circular meshwork of small capillaries, several layers deep. More peripherally, however, it becomes single layered. This capillary network, as a whole, establishes numerous connections with the adjacent choriocapillaris at the posterior pole of the ocular bulb. In anuran amphibians the complex arrangement of both arterial systems supporting the choriocapillaris may be regarded as a more complete equivalent of the short posterior ciliary arteries of mammals.     

Peptidergic regulation of secretory activity in amphibian olfactory mucosa: Immunohistochemistry,neural stimulation,and pharmacology

Summary The role of substance P in the regulation of secretion from sustentacular cells, Bowman's glands and deep glands in the amphibian olfactory mucosa was investigated using immunohistochemical, electrophysiological, and pharmacological methods. Substance P-like immunoreactive varicose fibers extended through the olfactory epithelium, terminating at or near the surface. In addition, immunoreactive varicose fibers innervated Bowman's glands, deep glands, and blood vessels in the lamina propria. Innervation of Bowman's gland was sparse, with fibers terminating on basal acinar cell membranes; deep gland innervation was abundant, with fibers often extending between acinar cells almost to the lumen. Stimulation of the ophthalmic branch of the trigeminal nerve resulted in slow potentials recorded at the surface of the olfactory epithelium. When the olfactory mucosae from trigeminal-stimulated animals were examined histologically, morphological signs of secretory activity were observed, suggesting that substance P was released from the trigeminal nerve terminals. Topical application of 10^-5 to 10^-3 mol substance P resulted in morphological signs of secretion that were very similar to those seen as a result of trigeminal stimulation. Thus, substance P released from trigeminal fibers may modulate secretory activity within the olfactory mucosa.     

Vasoactive intestinal peptide in cerebrospinal fluid

Immunoreactive vasoactive intestinal peptide (VIP) was measured in lumbar and ventricular cerebrospinal fluid (CSF) from patients with various neurological disorders and in 2 hour aliquots of cisternal fluid removed continuously from rhesus monkeys. Although most of the VIP in concentrated pools of human ventricular fluid and of monkey cisternal fluid co-eluted with synthetic porcine VIP28 on a column of Sephadex G-25 superfine, there was evidence that smaller immunoreactive fragments were also present. A circadian pattern of CSF VIP concentration was observed in 2 of the 3 monkeys studied, with highest levels occurring at night and lowest during the day. Ventricular fluid VIP levels were highest in hydrocephalic children and lowest in patients with multiple sclerosis or epilepsy, while VIP was not detectable in ventricular fluid from patients in coma following a severe head injury. There were no significant differences in VIP concentrations in CSF from patients with dystonia. Parkinson's disease, or Alzheimer's disease, suggesting that VIP containing neurons are not affected in these disorders. Lumbar fluid VIP levels were low in patients undergoing aneurysm surgery. Since VIP is a potent vasodilator, these findings may have important implications in relation to the development of vasospasm following subarachnoid hemorrhage.     

Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog,Rana catesbeiana

Summary The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).     

The superficial vascular hyaloid system in the eye of the frogs,Rana temporaria and Rana esculenta

Summary The angioarchitecture of the superficial vascular hyaloid system (membrana vasculosa retinae) of the frog eye was studied by means of scanning electron microscopy of vascular corrosion casts. The terminal vessels form a single-layered sheath intimately adjacent to the vitreal side of the avascular retina. The hyaloid system is subdivided by the ventral venous trunk into three central areas: the dorsal, the temporo-ventral, and the naso-ventral area. Toward the ora serrata, the hyaloid system is bordered by an arterial ring, and by nasal and temporal venous branches forming more or less complete hemicircles. A vascular zone composed of several tongue-like sectors establishes an inter-connection between the peripheral vascular rings and the central areas of the fundus. The arterial blood is supplied from the arterial ring. The drainage of the hyaloid system is provided via two routes: (1) the Y-shaped ventral trunk collects blood from the central areas, (2) the two peripheral venous branches drain the tongue-like sectors. The vessels within the dorsal area follow preferentially a dorso-ventral meridional direction. This densely capillarized territory corresponds in localization to the area centralis retinae. The ultrastructure of microvessels of the hyaloid system is characterized by features typical for capillaries of the central nervous system.     

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

Summary The circulation of the cerebrospinal fluid along the central canal and its access to the parenchyma of the spinal cord of the rat have been analyzed by injection of horseradish peroxidase (HRP) into the lateral ventricle. Peroxidase was found throughout the central canal 13 min after injection, suggesting a rapid circulation of cerebrospinal fluid along the central canal of the rat spinal cord. It was cleared from the central canal within 2 h, in contrast with the situation in the brain tissue, where it remained in the periventricular areas for 4 h. In the central canal, HRP bound to Reissner's fiber and the luminal surface of the ependymal cells; it penetrated through the intercellular space of the ependymal lining, reached the subependymal neuropil, the basement membrane of local capillaries, and appeared in the lumen of endothelial pinocytotic vesicles. Furthermore, it accumulated in the labyrinths of the basement membrane contacting the basolateral aspect of the ependymal cells. In ependymocytes, HRP was found in single pinocytotic vesicles. The blood vessels supplying the spinal cord were classified into two types. Type-A vessels penetrated the spinal cord laterally and dorsally and displayed the tracer along their external wall as far as the gray matter. Type-B vessels intruded into the spinal cord from the medial ventral sulcus and occupied the anterior commissure of the gray matter, approaching the central canal. They represented the only vessels marked by HRP along their course through the gray matter. HRP spread from the wall of type-B vessels, labeling the labyrinths, the intercellular space of the ependymal lining, and the lumen of the central canal. This suggests a communication between the central canal and the outer cerebrospinal fluid space, at the level of the medial ventral sulcus, via the intercellular spaces, the perivascular basement membrane and its labyrinthine extensions.