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The meninges of various mammals were prepared for examination with the electronmicroscope by thin sectioning or freeze-fracturing. Particular attention was given to the distribution of tight junctions in order to determine the basis for the meningeal barrier between the blood circulating in dural vessels and the cerebrospinal fluid in the subarachnoid space. While some dural blood vessels are fenestrated, those in the subarachnoid space are not and their component endothelial cells are joined by an extensive system of tight junctions. An extensive and continuous system of tight junctions was also found in a layer of specialized cells at the border of the arachnoid with the dura. This arachnoid barrier layer is apparently the only basis of the meningeal barrier because often cellular layers in the dura and arachnoid lack tight junctions although they are linked by gap junctions and desmosomes. In particular, tight junctions are lacking at the border of the "subdural space" which is actually a fascial plane within the dura. Tight junctions are also lacking between astrocytes at the surface of the brain but these cells are linked by gap junctions and a new type of intercellular junction. The distribution of these junctions, as well as assemblies of intramembranous particles at the astrocytic border, raises the question whether this layer might have a role in the exchange of certain substances between the brain and cerebrospinal fluid.

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Purpose This report describes the NIH Undiagnosed Diseases Program (UDP), details the Program's application of genomic technology to establish diagnoses, and details the Program's success rate over its first two years. Methods Each accepted study participant was extensively phenotyped. A subset of participants and selected family members (29 patients and 78 unaffected family members) was subjected to an integrated set of genomic analyses including high-density SNP arrays and whole exome or genome analysis. Results Of 1191 medical records reviewed, 326 patients were accepted and 160 were admitted directly to the NIH Clinical Center on the UDP service. Of those, 47% were children, 55% were females, and 53% had neurological disorders. Diagnoses were reached on 39 participants (24%) on clinical, biochemical, pathological, or molecular grounds; 21 diagnoses involved rare or ultra-rare diseases. Three disorders were diagnosed based upon SNP array analysis and three others using WES and filtering of variants. Two new disorders were discovered. Analysis of the SNP-array study cohort revealed that large stretches of homozygosity were more common in affected participants relative to controls. Conclusions The NIH UDP addresses an unmet need, i.e., the diagnosis of patients with complex, multisystem disorders. It may serve as a model for the clinical application of emerging genomic technologies, and is providing insights into the characteristics of diseases that remain undiagnosed after extensive clinical workup.

Postnatal development of the cerebellar cortex has been compared in staggerer mutant and unaffected littermate mice. From postnatal day 3 to about day 21 the external granular layer in staggerer mice is decreased in thickness and area, and the number of postmitotic granule cell neurons is reduced. Those granule cells that are generated seem to differentiate normally, with the remarkable exception that they form only primitive junctions with Purkinje cell dendritic shafts. These specialized junctions are not superseded by the normal parallel fiber:Purkinje spine synapses and disappear by the third week. Purkinje cell somata and dendrites are smaller than normal at all stages examined. The dendrites are not confined to the sagittal plane as in the normal and, unique among mutant or other animals described to date, they exhibit virtually no branchlet spines. All other cortical synaptic relations of granule and Purkinje cells, including climbing fiber:Purkinje spine synapses, appear qualitatively normal. However, by 28 days virtually all staggerer granule cells have degenerated. While the primary genetic defect remains unknown, we postulate that the morphological abnormalities may be attributable to a block in the normal developmental relationship between granule cells and Purkinje cells. The small cell size and failure to form branchlet spines suggest that the Purkinje cell abnormality may be closer to the primary effect of the mutant gene than the more flagrant hypoplasia and degeneration of granule cell neurons.

Three sets of filamentous structures were found to be associated with synaptic junctions in slices of cerebellar tissue prepared by rapid-freezing and freeze-etch techniques. The electron-dense fuzz subjacent to postsynaptic membranes corresponds to a web of 4-6-nm-diam filaments that were clearly visualized in rapid-frozen, freeze-etched preparations. Purkinje cell dendritic spines are filled with a meshwork of 5-7-nm filaments that were found to contact the spine membrane everywhere except at the synaptic junction, and extend through the neck of the spine into the parent dendrite. In addition, 8-10-nm microfilaments, possibly actin, were seen to be associated with the postsynaptic web and to extend into the body and neck of the spine. The arrangements and attachments of the filamentous elements in the Purkinje cell dendritic spine may account for its shape.

The structure of membranes at junctions between the plasma membrane and underlying cisterns of endoplasmic reticulum in amphioxus muscle and mouse cerebellar neurons was studied using the freeze-fracture technique. In amphioxus muscle, subsurface cisterns of sarcoplasmic reticulum form junctions with the surface membrane at the level of the sarcomere I bands. On the protoplasmic leaflet of the sarcolemma overlying these junctions were aggregates of large particles. On the protoplasmic leaflet of the membranes of cerebellar basket, stellate, and Purkinje cells there were similar aggregates of large particles. In both tissues, the corresponding external membrane halves had arrays of pits apparently complementary to the aggregates of large particles. Cross fractures through junctions showed that the particle aggregates in neuronal and muscle membranes were consistently located over intracellular cisterns closely applied to the plasma membrane. Thus, a similar plasma membrane specialization is found at subsurface cisterns in mammalian neurons and amphioxus muscle. This similarity supports the hypothesis that subsurface cisterns in neurons, like those in muscle, couple some intracellular activity to the electrical activity of the plasma membrane.Junctions between the surface or transverse tubule membrane and the endoplasmic, or sarcoplasmic, reticulum of muscle are thought to mediate excitation contraction coupling (12,34,39). Junctions between the surface membrane and regions of endoplasmic reticulum (ER), known as subsurface cisterns, are also present in neurons (38). Their similarity to the junctions in muscle has been noted (27, 38), but the functions of subsurface cisterns in neurons are unknown. We have used the freeze-fracture technique to compare the structure of the membranes participating in these junctions in striated muscle from the chordate, amphioxus, to their structure in neurons of the mammalian cerebellar cortex. The morphologic similarities apparent in thin sections are paralleled by similarities in the arrays of membrane particles revealed by freeze-fracturing the membranes at these junctions. These further similarities lend support to the suggestion (19,20,38) that subsurface cistern junctions may function in neurons, as in muscle, to couple excitatory events at the surface membrane with intracellular activities. A preliminary report of our results has been presented (25). MATERIALS AND METHODSAmphioxus Branchiostoma californiensis were obtained from Pacific Bio-marine Supply Co., Venice, Calif., and maintained in cold natural seawater for short periods

Echo-planar diffusion- and perfusion-weighted MR imaging for acute stroke is feasible and applicable before therapy decisions. Ultrafast MR imaging permitted immediate triage of 35 patients with symptoms of hyperacute stroke and thus helped avoid the risks from angiography and thrombolytic agents in some or spurred the judicious use of more aggressive intervention in others.