Ventricles The lateral ventricles are crescentic and a little wider apart ventrally than dorsally. Towards their caudal end they communicate through the interventricular foramina with the narrow vertically-orientated third ventricle. The third ventricle has an infundibular recess which extends caudally to the level of the midbrain and a dorsal recess which contains the paraphysis and choroid plexus. At the caudal end of the third ventricle is the pineal, and at the rostral end of the midbrain ventricle is the sub-commissural organ. Caudal to this the ventricle extends laterally into the optic lobes. The fourth ventricle has a rostral dorsal extension into the cerebellum, and is generally transversely orientated with a shallow lateral wall and an everted rhombic lip.
Hindbrain roof At the rostral end the roof extends dorsally where it forms the choroid plexus, and caudal to this consists of a flat attenuated layer from 620um to 830um in length. Examined in detail, as exemplified by a large number of adult frogs (Rana temporaria) including a series of ten specially sectioned through the hindbrain to study the posterior tela, it appears by light microscopy to be an intact layer just caudal to the fourth ventricle choroid plexus, and beyond this has the appearances of fenestrations i.e. the roof of the fourth ventricle in the frog has a deficiency, the metapore, and there is apparently communication between the ventricular system and an external compartment of the cerebrospinal fluid system.
Meninges The vertebral canal and cranial vault are lined by a layer of fibrous tissue which stains green with Masson's trichrome and contains melanin cells; this is the dura mater. Deep to this there is a network of sinusoids present throughout the spinal canal and cranial vault, up to the level of the midbrain, and most of these are identifiable by their cuboidal epithelial lining as a complicated endolymphatic system with extensive endolymphatic sacs; in fresh specimens these are filled with soft white material, and they lie in the subdural space. Within this is a thick well-defined continuous membrane, disrupted only occasionally in the preparations, which encircles the neuraxis and accompanies nerve roots for a short distance, but not through intervertebral or cranial foramina. This membrane stains light pink and green with Masson's trichrome, but does not stain bright pink with Van Gieson's (i.e. is not collagen) and under greater magnification is seen as a dense laminated structure with regularly interspersed nuclei, and no apparent cells boundaries. This is the arachnoid mater. The arachnoid is separated from the spinal cord and brain, but follows their contours, and at some points, such as the rhombic lip, it is loosely attached to the neuraxis. Around the roof of the hindbrain the arachnoid forms a complete layer over the posterior tela, but is attached to the ependyma which forms the wall of the recess as it extends dorsally from the rhombic lip to the choroid plexus. This attachment of the arachnoid to the ependyma and pia throughout the length of the choroid plexus of the fourth ventricle, forms what can be termed the arachno-choroid junction, by virtue of which the choroid plexus of the fourth ventricle lies dorsal to the arachnoid, and is in the subdural space. There is a similar attachment of the arachnoid along the recess of the third ventricle, leaving both the choroid plexus and paraphysis in an extra-arachnoid position. Ventrally there is a well-defined subarachnoid space around the hindbrain and midbrain, but at the level of the third ventricle the infundibular recess passes through the arachnoid to lie in an extra-arachnoid position over the adenohypophysis.
The neuraxis is closely invested with a thin layer of pia mater which stains green with Masson's trichrome, red with Van Gieson's stain, and follows the blood vessels as a delicate sheath into the neuraxis. Nuclei of this layer are densely staining, there are pigment cells present, and over the posterior tela of the fourth ventricle the pia is fine and partly deficient. At the rhombic lip the pia is prominent, and contains a longitudinally running blood vessel. In the paramedian plane over the roof of the third ventricle the pia contains many blood vessels, and similarly around the infundibulum. The pia is a very distinct fibrous layer around the optic chiasm and extends along the optic nerves.
Ependyma The ependyma of the lateral ventricles is columnar, ciliated, and has basally-placed nuclei. The ventral and lateral ependyma of the third ventricle are similar. In the roof of the third ventricle the ependyma is thin and attenuated caudal to the choroid plexus fold, but caudal to this it forms a cluster of tall columnar cells with investing fibrous tissue which is the pineal gland. Ventrally the ependyma of the infundibular recess is attenuated in the region of the saccus vasculosus, but more distally it extends to the neurohypophysis as an irregular low cuboidal layer. The ependyma of the midbrain ventricle is columnar in the dorsal midline, and at the rostral end forms a distinct collection of dark-staining cells which are ciliated, and this is the sub-commissural organ.
In the fourth ventricle the ependyma of the floor and lateral walls is columnar and ciliated, and at the rhombic lip is somewhat flattened with few cilia, and then dorsal to the attachment of the rhombic lip is a single-celled membrane where it forms the lateral wall of the recess between the rhombic lip and choroid plexus; in this region it is reinforced on the outside at the arachno-choroid junction. Caudal to this the ependyma of the hindbrain roof is deficient at the metapore. The ependyma lining the central canal is high columnar ciliated with regular basally-placed nuclei.
Choroid plexuses There are no choroid plexuses in the lateral ventricles of the frog, and the third ventricle choroid plexus is a densely folded structure consisting of villi of cuboidal epithelium around a core of loose connective tissue and blood vessels, and cytoplasmic vacuoles are visible at higher magnification. The choroid plexus epithelium is continuous in the dorsal and rostral direction with a more crowded columnar epithelium arranged in acini with collagen-lined sinusoids between the epithelial layers and with densely staining nuclei, and no cilia; this is the paraphysis. The choroid plexus of the fourth ventricle is a greatly folded epithelium which protrudes ventrally into the slight recess and the ventricle. It has cores of connective tissue and blood vessels between the epithelial folds. The epithelium is cuboidal with basally-placed nuclei, large cytoplasmic vacuoles and cilia. The connective tissue is continuous with the tela choroidea and adjacent to the subdural blood vessels, sinusoids, and endolymphatic sacs.
CSF-vascular relationships The frog has an internal CSF system, which is continuous through the roof of the fourth ventricle with an external CSF system limited by the well-defined arachnoid. The internal CSF is separated from the blood vessels of the choroid plexus by the choroid plexus epithelium, and from the meningeal blood vessels around the roof and floor of the third ventricle by a thin layer of flattened ependyma only. Within the lateral ventricles the CSF is separated from prominent sub-ependymal blood vessels by ependyma. Within the subarachnoid space the CSF is separated from subdural vessels by the arachnoid itself, and there are no regions of juxtaposition between the arachnoid and thin-walled vessels to form arachnoid villi or granulations. Blood vessels within the arachnoid are separated from the csf by their own covering of fibrous tissue.
Figure 1 TS Rostral forebrain - Masson's trichrome The lateral ventricles are continuous with the third ventricle through the interventricular foramen, and the roof of the third venricle at this level is a thick simple ependymal layer. There is a further extension of the third ventricle ventrally. There are no choroid plexuses in the lateral ventricles. the pia cannot be seen but the arachnoid is well-defined ans the subarachnoid space contains a few blood vessels close to the nerve roots. Ventrally the arachnoid is adherent to the endosteal dura, but elsewhere is separated from it, particularly dorsally, where there are subdural blood vessels.
Figure 2 TS Caudal forebrain - Masson's trichrome The narrow third ventricle is surmounted by the paraphysis at this level, and the blood vessels of the pia can just be seen. The subarachnoid space is clear, the arachnoid well-defined, and outside the arachnoid in the dorsi-lateral position are the forward extensions of the endo-lymphatic sacs. The dura stains green.
Figure 3 TS Midbrain - Masson's trichrome The midbrain ventricle extends laterally into the optic lobes to form the optic ventricles. The surface pia with its blood vessels is just visible. The arachnoid is very well defined and the subarachnoid space with its contained blood vessels is clearly visible. Ventrally the adenohypophysis, surmounted by the caudal extension of the infundibular recess from the thjird ventricle is clearly outside the arachnoid. Similarly, in the dorsi-lateral position the endolymhatic sac is extra-arachnoid.
Figure 4 TS Rostral hindbrain - Masson's trichrome The wide open fourth ventricle is roofed by the convoluted choroid plexus folds which are attached to the rhombic lip by a short thin ependymal layer. The arachnoid, which is well defined, joins this layer at the side forming the arachno-choroid junction and places the choroid plexus and the endolymphatic system on its dorsal aspect outside the arachnoid. It seems likely that, before fixation, these structures were closely adjacent to the vault of the skull. Ventro-laterally the subarachnoid space can be traced into the peri-lymph of the inner ear, and most ventrally the distinction between arachnoid and the endosteal dura can be seen.
Figure 5 TS Caudal hindbrain - Van Gieson's The caudal end of the fourth ventricle is open to the subarachnoid space at this level - the posterior tela is deficient, and a metapore is present. Dorsal to the hindbrain is the arachnoid and on the dorsal aspect of this the endolymphatic sac. Ventrally, in the subarachnoid space outside the pia, are some small blood vessels.
Figure 6 TS Cervical spinal cord - Martius Scarlet Blue the cervical spinal cord has a minute central canal, a layer of pia closely investing it, and, ventrally, it is attached to the pia and surrounding arachnoid, with some median blood vessels in between. Dorsally and laterally the subarachnoid space is quite extensive, the arachnoid is clearly defined and is separate from the endosteum. In the lateral angles of the spinal cord are nerve roots in their exit foramina.
Development of the CSF system in the frog From the series of tadpoles studied the lateral, third and fourth ventricles and central canal are evident at 4 days (7mm) and the optic ventricles appear at 5 days (9mm). The 6 day (11mm) tadpole has a cerebellar ventricle within the cerebellar anlage and the elongated roof of the fourth ventricle has a thin but intact ependyma. In the 7 day tadpole is a well developed third ventricle, choroid plexus and paraphysis, and a primitive choroid plexus fold in the otherwise intact roof of the fourth ventricle. The meninx is a single layer. The 8 day (15 mm) tadpole has well developed choroid plexuses, and an inner meningeal layer forming pia around the brain, and a single external meningeal layer with the pineal gland lying outside this. The 11 day old (18mm) tadpole shows an adult system apart from the roof of the fourth ventricle being intact, and there is no separate defined arachnoid layer. The juvenile frog shows a definite arachnoid membrane. The CSF system of the frog (Rana temporaria) is similar to that of the following amphibians of the order Anura which were studied: toad (Bufo bufo), another frog (Rana pipiens), tree frog (Hyla arborea), bull frog (Rana catesbiana), and the marsh frog (ana ridibunda). The Xenopus toad has a similar system with no choroid plexuses in the lateral ventricles, and optic lobe ventricles in the midbrain, but the posterior tela appears to be a delicate intact single-celled layer of ependyma, surmounted by a layer of pia mater.
Previous literature Little attention has been given to the significent developments in the CSF system found in the amhibians(1) although the development of a defined arachnoid layer, with a fluid-filled subarachnoid space, and the change from a fat or fluid-filled subdural or extradural space occurring in amhibians and reptiles has been noted(2).
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