Chorioid Plexus Research Articles

Histological aspects in the designing of alzheimer's type dementia of vascular origin and the manifestation of the brain tissue cellular regeneration in rats

Background. The brain structure in patients with Alzheimer's disease, caused initially by vascular damage, is poorly known regarding morphological aspects. Objective. To study the histological features of the brain tissue in the designing of Alzheimer's type dementia of vascular origin and the manifestation of cellular regeneration in rats. Methods. The experiment was performed on 32 male WAG rats weighing 180-250 g. Rats from experimental group (group E) were injected with aqueous solution of sodium nitrite at a dosage of 50 mg/kg of body mass intraperitoneally during 14 days. Control animals were injected with 0.9% sodium chloride. The formation of the Alzheimer's disease model caused by vascular injury has been proven and published. Congo-red and gallocyanine-chromium alum according to Einarson's method stained slides of brain were received. The expression of the Ki-67 antigen was determined immunohistochemically (Thermo Fischer Scientific). Results. In gr. E the subendothelial deposition of congophilic masses and endothelium damage were observed in the brain arteries. Small intracerebral vessels had less amyloid changes. The perivascular edema, parietal thrombosis, and an increase in the number of pericytes over endothelial cells by 1.5-2 times and the Ki-67 labeled capillary endothelial cells in all parts of the brain were recorded. Also, it was found the dystrophic processes in neurons, proliferation of glial cells, ependymocytes, epithelial cells of chorioid plexus, the necrosis foci formation in the cortex, subcortical substance, hippocampus, which was accompanied by the inflammatory reactions. Conclusion. The blood vessels and neurons injury in various parts of the brain in rats with model of Alzheimer's type dementia of vascular origin triggered the regeneration at the cellular level which represented the death of these cells.

Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho

Background/Aims: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required for inhibition of 1,25(OH)₂D₃ formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. At least in part by exerting β-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2), Na⁺ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability. Methods: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM)-induced inward current (I_Glu) taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Results: I_Glu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes I_Glu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers. Conclusion: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation.

Upregulation of the Creatine Transporter Slc6A8 by Klotho

Background/Aims: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)₂D₃ formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. β-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. Methods: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. Results: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by β-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. Conclusion: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring β-glucuronidase activity of Klotho protein.

A clinicopathological analysis of papillary endolymphatic sac tumor in inner ear

Background Endolymphatic sac tumor (ELST) is a rare tumor originating from endolymphatic epithelium of inner ear. This tumor exhibits low-grade malignancy with benign histopathological appearance and clinically destructive behavior which occurs in the skull base and frequently invades the posterior petrous bone, the mastoid, semicircular canal, cerebellopontine angle structures and cranial nerve. The presence of intracranial ELST always makes the diagnosis challenge for clinicians and pathologists. Herein we describe a case of ELST in skull base. The clinicopathology of this tumor and its differential diagnosis are discussed. Methods The clinical manifestation of a patient with primary ELST occurring in right cerebellopontine angle was presented retrospectively. Resected mass was routinely paraffin-embedded and stained with hematoxylin and eosin. Dako EnVision immunohistochemical staining system was used to detect the tumor antigen expressions, including cytokeratin (CK), vimentin (Vim), epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), synaptophysin (Syn), chromogranin A (CgA), S-100 protein (S-100), glial fibrillary acidic protein (GFAP), thyroglobulin (TG), thyroid transcription factor-1 (TTF-1) and Ki-67. Results A 32-year-old male patient presented with 20-year history of progressive hearing loss. MRI scan revealed an expansile lytic lesion of the mastoid process of the right petrous bone, measuring 4.20 cm × 3.30 cm × 2.00 cm, occupied the right cerebellopontine angle with infiltration of surrounding dura mater. But the lesion did not break the dura mater and invade the brain parenchyma. Craniotomy was performed and the tumor was removed totally. Histological examination revealed a papillary, cystic or glandular architecture in mass. The papillary and glandular structures were lined by a single layer of flattened cuboidal-to-columnar cells. The stroma of the papillary fronds was richly vascularized and chronically inflamed. There were minimal cellular pleomorphism and rare mitotic activity and necrosis was not observed. There were cystic glandular spaces filled with colloid-like material which was remarkably similar to thyroid tissue. Immunohistochemical staining showed that the tumor cells were diffusely positive for CK (AE1/AE3), Vim, EMA and focally positive for CEA, but negative for Syn, CgA, S-100, GFAP, TG and TTF-1. The Ki-67 index was less than 1%. The colloid-like material showed positive reactivity with periodic acid⁃Schiff (PAS). Based on clinical presentation and histological findings, a final histological diagnosis of primary papillary ELST in cerebellopontine angle was made according to the criteria of WHO classification. The patient had not received radiotherapy or chemotherapy and attended follow-up for 3 months, without any neurological deficit or signs of recurrence. Conclusion ELST is rare skull base tumor originating from endolymphatic epithelium within the vestibular aqueduct, characterized clinically by slow growth with local invasion and bone destruction. Due to the rarity of this tumor, it can easily be confused with other tumors of skull base with papillary architecture such as chorioid plexus papilloma, papillary ependymoma, papillary meningioma, middle ear adenoma, and metastatic papillary carcinoma of the thyroid. Complete excision is the best guarantee against tumor recurrence, while recurrence may occur due to subtotal resection.

Choroid plexus tumors

Introduction. In the article the data of literature are summarized and age-related features of choroid plexus tumors clinical flow, diagnostics, localization and outcomes in children and adults are considered.Materials and methods. The research is based on retrospective analysis of case reports of patients been treated in Institute for period of 30 years.Results. Choroid plexus tumors — are rare primary neoplasms from neuroepithelial tissue. They make from 0.4 to 1% in structure of all intracranial tumors. Most often they are found in infants.Conclusions. Treatment of these patients is complicated by chorioid plexus tumors’ high vascularity, accompanying hydrocephalus and ineffective adjuvant therapy of choroid plexus carcinomas.

Proteomics analysis of prefractionated human lumbar cerebrospinal fluid

Cerebrospinal fluid (CSF) is produced by the chorioid plexus in the ventricles. It surrounds the brain and bone marrow, and reflects several different disorders of the central nervous system (CNS). Proteomics has been used to analyze CSF in order to discover disease-associated proteins and to elucidate the basic molecular mechanisms that either cause, or result from, CNS disorders. However, some disease-associated proteins are of low-abundance and are difficult to detect. A low total-protein concentration, a high amount of albumin and immunoglobins, and a wide dynamic range (several orders of magnitude) of protein concentration cause several difficulties in the identification of low-abundance CSF proteins. In this study, advantage was taken of the range of different hydrophobic properties of CSF proteins, and a reversed-phase solid-phase extraction (SPE) cartridge was used to prefractionate human lumbar CSF proteins into three separate fractions prior to two-dimensional gel electrophoresis resolution of the proteome. A portion of the high-abundance CSF proteins were removed from two (eluted with 35% and 50% acetonitrile) of the three fractions. Some trace CSF proteins were preferentially enriched in the two fractions, and many proteins were detected in the two-dimensional (2-D) gels of the two fractions. Among the novel proteins identified, sixty-two protein spots that represent forty-two proteins were characterized. Most of the proteins have not been annotated in any previous 2-D map of human CSF, and several have been implicated in CNS diseases. The prefractionation of CSF proteins with SPE, followed by proteomics analysis, provides a new method to explore low-abundance, disease-specific CSF proteins.

Intracranial ossifications and microangiopathy at 8 Tesla MRI

Clinical evaluation and MR imaging of microangiopathy associated with hypertension is limited. We describe a case that illustrates sensitivity of MRI at 8 Tesla for imaging of microvasculature, iron, calcium deposits and silent white matter lesions (WML). A 60-year-old black hypertensive woman was evaluated for numbness in the face and extremities. MRI at 1.5 Tesla was unrevealing. MRI at 8 Tesla: Axial and sagittal Gradient Echo images were obtained with an 8T/80 cm human scanner and showed: 1) Large areas of signal voids due to ossifications and fat deposits within the falx. 2) Obstructed small vessels in the periventricular regions and distended cortical veins. 3) Numerous small WML, suggestive of mini-infarcts (<1 cm) and microhemorrhages. 4) Intracranial calcifications in the falx, tentorium, basal ganglia and chorioid plexus that were confirmed by CT scan. Atherosclerotic plaque in right carotid artery and reduced vasomotor reserve in middle cerebral arteries, documented by ultrasound, indicated large and small vessel disease. Conclusions: MRI at 8 Tesla improves visualization of microangiopathy, ossifications and iron deposits due to enhanced magnetic susceptibility at ultra high magnetic field.

Nitric oxide is protective in listeric meningoencephalitis of rats.

The bacterium Listeria monocytogenes causes meningoencephalitis in humans. In rodents, listeriosis is associated with granulomatous lesions in the liver and the spleen, but not with meningoencephalitis. Here, infant rats were infected intracisternally to generate experimental listeric meningoencephalitis. Dose-dependent effects of intracisternal inoculation with L. monocytogenes on survival and activity were noted; 10(4) L. monocytogenes organisms induced a self-limiting brain infection. Bacteria invaded the basal meninges, chorioid plexus and ependyme, spread to subependymal tissue and hippocampus, and disappeared by day 7. This was paralleled by recruitment and subsequent disappearance of macrophages expressing inducible nitric oxide synthase (iNOS) and nitrotyrosine accumulation, an indication of nitric oxide (NO.) production. Treatment with the spin-trapping agent alpha-phenyl-tert-butyl nitrone (PBN) dramatically increased mortality and led to bacterial numbers in the brain 2 orders of magnitude higher than in control animals. Treatment with the selective iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine (L-NIL) increased mortality to a similar extent and led to 1 order of magnitude higher bacterial counts in the brain, compared with controls. The numbers of bacteria that spread to the spleen and liver did not significantly differ among L-NIL-treated, PBN-treated, and control animals. Thus, the infant rat brain is able to mobilize powerful antilisterial mechanisms, and both reactive oxygen and NO. contribute to Listeria growth control.

Human Kallikrein 6 as a Biomarker of Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is a major cause of dementia in the elderly. It is generally difficult to diagnose accurately early AD. A few biomarkers, including τ protein and amyloid β-42, are now used as aids for diagnosis and monitoring of AD. Our aim was to examine the possible use of cerebrospinal fluid, blood and tissue, and human kallikrein 6 (hK6) concentration as a marker of AD. Methods: We have used a highly sensitive and specific immunofluorometric procedure for measuring hK6. We measured hK6 in tissue extracts from AD brain or normal individuals, in cerebrospinal fluids of AD patients or normals and in whole blood of AD patients and normals and compared the findings. We have used ten pairs of AD/normal controls in all cases. Results: We found that hK6 concentration is tissue extracts from AD brain were approximately twofold lower than extracts from normal controls. Further, we found that cerebrospinal fluid hK6 concentration is approximately a threefold increase, in comparison to cerebrospinal fluid controls ( p = 0.001). We have also found that the whole blood hK6 concentration in AD patients is about ten times higher than hK6 concentration in normal controls ( p = 0.002). We have immunohistochemically localized the expression of hK6 in epithelial cells of the chorioid plexus. Conclusions: This is the first report describing significant elevations of cerebrospinal fluid and plasma and whole blood hK6 concentration in AD patients, in comparison to controls. These data suggest that hK6 may constitute a new biomarker for diagnosis and monitoring of AD.

Tissue Distribution and Retinoid-Mediated Downregulation of an FGF-Binding Protein FGF-BP) in the Rat

We showed previously that a secreted fibroblast growth factor-binding protein (FGF-BP) can mobilize and bioactivate locally-stored FGFs from the extracellular matrix. This FGF-BP is upregulated in various cancers and plays a rate limiting role as an angiogenic switch molecule during tumor growth. In this paper, we describe the cloning and sequence analysis of the rat homologue of FGF-BP and show its expression pattern and retinoid-mediated downregulation in normal adult rat tissues. The rat FGF-BP amino acid sequence is 91% and 70% homologous to mouse and human, respectively, and contains 10 cysteine residues whose position is conserved across species. In Northern blots, FGF-BP mRNA was detected in the gut, eye, thymus, skin, lung and tongue. Immunohistochemistry confirmed this tissue distribution with cerebellar Purkinje cells, the cerebral chorioid plexus and the eye showing the most distinctive staining patterns. Oral treatment of animals with all-trans-retinoic acid for one and two days induced a significant decrease of FGF-BP protein in tissues from stomach, eye and lung suggesting that regulation of FGF-BP can be one effector mechanism through which retinoids affect normal and pathological processes.

Expression and functional interaction of hepatocyte growth factor-scatter factor and its receptor c-met in mammalian brain.

Hepatocyte growth factor-scatter factor (HGF-SF) is a pleiotropic cytokine with mito-, morpho-, and motogenic effects on a variety of epithelial and endothelial cells. HGF-SF activity is mediated by the c-met protooncogene, a membrane-bound tyrosine kinase. Here, we demonstrate that both genes are expressed in developing and adult mammalian brains. HGF-SF mRNA is localized in neurons, primarily in the hippocampus, the cortex, and the granule cell layer of the cerebellum, and it is also present at high levels in ependymal cells, the chorioid plexus, and the pineal body. c-met is expressed in neurons, preferentially in the CA-1 area of the hippocampus, the cortex, and the septum, as well as in the pons. In the embryonic mouse, brain HGF-SF and c-met are expressed as early as days 12 and 13, respectively. Neuronal expression of HGF-SF is evolutionary highly conserved and detectable beyond the mammalian class. Incubation of septal neurons in culture with HGF-SF leads to a rapid increase of c-fos mRNA levels. The results demonstrate the presence of a novel growth factor-tyrosine kinase signaling system in the brain, and they suggest that HGF-SF induces a functional response in a neuronal subpopulation of developing and adult CNS.

Immunohistochemistry with keratin monoclonal antibodies in canine tissues: urogenital tract, respiratory tract, (neuro-)endocrine tissues, choroid plexus and spinal cord.

Twelve oligo- or monospecific monoclonal antibodies (MoAbs) directed against human keratin types were used in an immunohistochemical study of the canine male and female urogenital tract, the respiratory tract, the adrenal gland, the (para-)thyroid gland, the choroid plexus and the spinal cord. The keratin MoAbs showed differences in staining patterns in the various epithelial tissues and the diverse epithelial cells. The kidney was characterized by a complex keratin staining pattern and the canine urothelium showed regional differences in keratin staining. Also in the female genital tract different keratin staining patterns were observed. Testicular and adrenal gland cells did not react with any of the keratin MoAbs. The keratin staining patterns in the various canine tissues showed, in addition to similarities, also distinct differences when compared to the staining patterns in corresponding tissues of other species, e.g. of man. These staining dissimilarities indicate that the reactivity patterns of the keratin MoAbs with restricted keratin immunoreactivity can not be always extrapolated from one species to another. Nevertheless, MoAbs directed against human keratin proteins can apparently be used to differentiate between various types of canine epithelia or epithelial compartments.

A technique for removal of an adherent ventricular catheter.

A method of freeing the attachment of the intraventricular catheter to the ventricular ependyma or chorioid plexus is described. A metal ventricular cannula is inserted into the lumen of the ventricular catheter and a cutting current from a unipolar diathermy is applied to the distal end. The cutting effect at the interventricular end of the metal cannula effectively lyses the adhesions and permits safe removal of the ventricular catheter without subsequent bleeding.

Light microscopic autoradiographic localization of [ 3H]oxytocin binding sites in the rat brain, pituitary and mammary gland

An autoradiographical oxytocin (OXT) labeling procedure using frozen, unfixed tissue sections resulted in very dense labeling of the mammary gland. Binding sites for OXT were also found in various forebrain areas, including the hippocampus, especially the ventral subiculum and taenia tecta, central amygdala, posterior part of the anterior olfactory nucleus, claustrum, nucleus accumbens, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, and the posterior pituitary. The ependyma of the lateral ventricle and/or the chorioid plexus near the lateral septum was labeled as well. These data support the hypothesis that OXT plays a role in a number of centrally regulated processes.

Studies on poxvirus infections in irradiated animals

If rabbits were given total body irradiation and infected wih vaccinia virus (strain Elstree) a severe disease developed with a viraemia lasting up to 12 days. The clearance of the virus from the peripheral blood was severely impaired by x-ray doses above 800 R. The attenuated vaccinia virus strain MV did not turn virulent, if it was injected to irradiated rabbits. With caution it can be assumed that live vaccines, containing attenuated viruses, may be given to immunosuppressed persons. Rats are not susceptible to ectromelia-virus (mouse-poxvirus); overt clinical sympatoms, however, with a mortality of 30 per cent developed in irradiated rats. This proofs that specific poxviruses can be transferred to another species. As the experimental conditions are unnatural, this may occur only rarely in immunosuppressed persons. After intracerebral infection of Balb-C-mice with low doses of vaccinia virus two types of infection were seen: 1. a severe cytocidal infection of leptomeninges, chorioid plexus and vessels; 2. a noncytocidal, latent infection of glial cells and neurons. Several animals developed a picture resembling experimental allergic encephalomyelitis. It seems that irradiation altered the antigenic conditions of the cytoplasmic membranes in non-cytocidally infected cells. The model might explain some processes in the pathogenesis of demylinating diseases.

Development of the cerebrospinal fluid pathway in the normal and abnormal human embryos.

The subarachnoid space, the chorioid plexus and the arachnoid villi are microscopically studied in 60 normal human embryos and in 3 abnormal human embryos with rhombencephaloschisis and cervical myeloschisis. The subarachnoid space has been generally considered to be developed by outflow of cerebrospinal fluid (CSF) of the choroid-plexus origin from the IVth ventricle. This generally accepted concept does not meet with our findings: (1) cavity formation in the meninx primitiva is seen before appearance of the choroid plexus; (2) the primitive subarachnoid space is developed earlier in the prepontine region than in the area dorsal to the rhombic roof, and (3) the primitive subarachnoid space is formed in the embryos with dysraphism where the perineural subarachnoid space is separated from the ventricles. Apparently the embryonic pattern of CSF circulation should be much different from the generally believed pattern of adult, since the arachnoid villi are absent in the embryos and the ability of production of CSF in the embryonic choroid plexus is questionable. It is suggested that such embryonic pattern of CSF production and absorption may partly persist in adult human being.

On the histochemistry of N-acetyl-β-D-glucosaminidase in rat central nervous system

The optimal conditions for histochemical demonstration of NAG activity in the cerebrum, diencephalon, midbrain, cerebellum, medulla oblangata, and spinal cord were studied in a series of 37 Wistar rats of either sex. The following more important results were obtained: Each CNS zone required a definite methodlogical approach. Optimal fixation for most structures was achieved after 2 h treatment with formol-calcium and subsequent immersion of tissue blocks in formol-calcium 0,88 M saccharose. By this fixation technique it was possible to preserve high enzyme activity and good tissue structure. Only for the large pyramidal cells of the cerebral cortex the method of Holt provided optimal fixation. Formol-calcium-saccharose mixture and pure 0,88 M saccharose produced the opposide osmotic effect on nervous tissue previously fixed with formol-calcium: the former induced tissue shrinkage, the latter edema. The use of hexazonium p-rosaniline coupler prompted preliminary alcohol treatment of sections and introduction of 0.1 M acetate buffer in the incubation solution. Acetate buffer concentrations lower than 0.2 M diminished the diffuse cytoplasmic coloration and permitted a clear-cut demonstration of the lysosomal reaction. Ample information on the distribution of NAG activity in the CNS was obtained by using fast garnet GBC coupler and 0.1 M citrate buffer. Manganese chloride in a 0.2 mM concentration activates the reaction. The distribution of NAG reaction product in the cells of the different sections of the CNS was studied. The distribution of NAG reaction product in the cells of the different sections of the CNS was studied. The neurons, glial cells, and blood vessels showed positive reaction. Strongest activity was reported for the neurons of the supraoptic and paraventricular necleus, the epithelial cells of the chorioid plexus, nucleus ruber of the mesencephalon, and the vascular wall pericytes.

The origin of (Kolmer's) epiplexus cells. A combined histomorphological and histochemical study.

The morphology and the histochemically proofed enzyme pattern of the (Kolmer's) epiplexus cells found at the telencephalic chorioid plexus of humans indicate that these cells are of monocytogenetic origin, that they are monocytes and monocytogenetic macrophages respectively.

Experience with a shunt system using the third ventricle. Technical note.

The authors present a technique involving placement of a catheter in the third ventricle in shunt systems for hydrocephalus, in order to avoid obstruction of the proximal catheter by particles of chorioid plexus. They emphasize the simplicity of the technique, lack of morbidity, and the good results they have obtained in 30 cases with a follow-up from six to 40 months.

The cellular distribution of aldolase isozymes in rat kidney and brain determined in tissue sections by the immuno-histochemical method.

The use of the immuno-histochemical method permits the localization of aldolase isozymes in tissue sections. Upon incubating a section with a monomer-specific antiserum, isozymes containing that monomer remain in the section, whereas other cytoplasmic enzymes diffuse out of the section. If soluble antigen is added subsequently, it is bound by the tissue-bound antibody. These antibody fixed aldolases can then be stained by the use of a tetrazolium test linked to substrate hydrolysis. In this way it was demonstrated that isozymes of aldolase containing mostly the A monomer are predominantly localized in the distal tubules, the collecting tubules, the vessels and capillaries of the kidney, the ganglia, the Purkinje cells, the neurons, the white matter and the chorioid plexus of the brain. Aldolase containing mostly B-monomers were found in the proximal tubules. Aldolase isozymes particularly rich in C-monomers were seen in the nervus opticus, the pia mater, the vessels of cerebrum and the molecular layer of the cortex cerebelli.