Disruption of neuronal migration in the neocortex of the dreher mutant mouse.

To analyze developmental abnormalities related to neuronal migration in the dreher mutant mouse, the neocortical cytoarchitecture of dreher and control mice were examined in Nissl-stained serial sections by light microscopy. In general, the dreher neocortex has six layers which are similar in size and thickness to those observed in normal mouse neocortex. However, in dreher neocortex, three types of abnormalities were found: (1) an increase in the number of diffusely distributed neurons in layer I, (2) small, ectopic collections of neurons in layer I, and (3) isolated disturbances of local cytoarchitecture characterized by neuron-free space distributed in areas between layer II to IV. The occurrence of small, punctate deficits in the dreher neocortex may be secondary to disruptions of the radial glial fiber system and neuronal migration. The fact that cytoarchitectonic abnormalities of several types were found in the dreher neocortex may be useful in analyzing the relationship between radial glial fibers and migrating young neurons, the synaptic connections which are formed by ectopically situated neurons, and the mechanism of formation of sporadically distributed neocortical abnormalities.

Abnormal distribution of acetylcholinesterase activity in the hippocampal formation of the dreher mutant mouse.

The distribution of acetylcholinesterase(AChE) in the hippocampal formation of the dreher mutant mouse was studied by comparing homozygous mutant (drsst-J/drsst-J) with littermate control (+/? or +/+). In the control mice, AChE activity was most intense in the inner one-third of the stratum oriens and lacnosum of the hippocampus, and in the inner one-fifth of the molecular layer of the dentate gyrus. In contrast, in homozygous dreher mice, AChE activity in area CA3c of the hippocampus was not restricted to the stratum oriens, and extended upward into the infrapyramidal and suprapyramidal mossy fiber layers, the lower part of the stratum radiatum, the pyramidal cell layer, and downward toward the alveus. In addition, the distribution of AChE activity was modified by accompanying with ectopic pyramidal cells or with disruption of the pyramidal cell layer. AChE activity in the dentate gyrus of the dreher mouse was not confined to the inner one-fifth of the molecular layer. These findings indicated that the cholinergic input to the hippocampal formation is not normal in the dreher mutant mouse. Since the areas of AChE activity correspond to the presence of ectopic pyramidal cells in the dreher mouse, incoming cholinergic fibers may form synapses with these ectopic cells and with the dendrites of normal pyramidal cells that extend into the expanded area of AChE activity.

Prominent expression of type 1 (gamma) protein kinase C in Purkinje cells located in the "central mass" of Reeler mutant mouse cerebellum as revealed by a computer image analysis technique.

In Reeler mutant mice, cerebellar Purkinje cells exhibit abnormal synaptogenesis. In this study, Purkinje cells in the "central mass" with abnormal afferent connections were compared with those located in their normal position in the cortex in terms of immunoreactivity for type 1 (gamma) protein kinase C. A "computer image analysis technique" was developed for the purpose of this quantitative study, and it revealed that the immunoreactivity in the central mass was significantly higher than that in the cortex. The results suggest that type 1 (gamma) protein kinase C may be related to the abnormal synaptic formation of the Purkinje cells.

Cytoarchitectonic abnormalities in cerebellum and hippocampal formation of beige mutant mouse.

Abnormalities in the cerebellum and hippocampal formation of the beige mouse were revealed by histological and immunohistochemical examination. In the cerebellum, Purkinje cells and clusters of granule cells, plus an occasional Bergmann glia cell, were located ectopically in the molecular layer. In the hippocampus, ectopically situated pyramidal cell were found in the stratum oriens of area CA3. The infrapyramidal mossy fiber layer (IPMFL) was formed by fiber bundles emerging from two districtly separated areas of the dentate gyrus. The IMPFL was not compact, but appeared as clumps of scattered fiber bundles. The neuronal heterotopias and cytoarchitectonic disorganization observed in the beige mutant mouse cerebellum and hippocampus may prove to be very useful parameters in analyzing the relationship between the immune system and the development of the central nervous system.

Cytoarchitectonic abnormalities in hippocampal formation and cerebellum of dreher mutant mouse.

The laminated structures in the hippocampal formation and cerebellum of homozygous dreher mice were compared to their littermates and to C57BL/6J mice in Nissl- and myelin-stained preparations. In the dreher dentate gyrus, ectopic granule cells were situated in the molecular layer, and frequently there was either partial or complete absence of the infrapyramidal limb of the granule cell layer. In the dreher hippocampus, the cells of the pyramidal cell layer in area CA3 formed widely dispersed arrangements, and there were ectopically situated pyramidal cells in the stratum radiatum and stratum oriens. In the dreher cerebellum, 3 abnormal patterns were observed: (1) disruptions of foliation with normal cytoarchitectonic structure, (2) foliation with a mixture of normal laminated structure and abnormal laminated structure, and (3) almost complete absence of the cerebellum. In abnormal folia exhibiting the second or third pattern, islands consisting of agglomerations of both granule cells and Purkinje cells or just granule cells were observed. The neuronal heterotopias and cytoarchitectonic disorganization observed in the present study are apparently secondary to disruption of cell proliferation and neuronal migration produced directly or indirectly by the dreher mutation. In addition, the fact that the phenotypic abnormalities in homozygous dreher mice produces different abnormal morphologies in different specimens may be useful for analyzing the development of the hippocampal formation and cerebellum.

Histochemical demonstration of heavy metals in the hippocampal formation embedded in Quetol 523M.

To facilitate improvement of investigations on the distribution of mossy fibers in the hippocampal formation, a method is described using Timm's stained preparations after methacrylate embedding with the hydrophilic resin, Quetol 523M. Fixation with a mixture of formaldehyde and glutaraldehyde yielded satisfactory staining results and good structural preservation. During the course of histochemical experiments employing Timm's staining, examinations revealed that sulfide silver reaction products were consistently present in both the mossy fibers themselves and their terminals associated with the dendrites of pyramidal cells in tissue sections of 1-2 microns in thickness. The results obtained also revealed that variations of the mossy fiber system occurred in the neurological mutant mouse dreher (dr). The bundles of mossy fibers forming the intrapyramidal synaptic field may be considered to reflect genotype-dependent differences in the mutation. The present method is adequate for allowing the histochemical demonstration of mossy fibers and their giant boutons by light microscopy.

Abnormalities of foliation and neuronal position in the cerebellum of NZB/BINJ mouse.

To analyze developmental abnormalities related to neural migration in the NZB/BINJ mouse, the pattern of cerebellar foliation and neural position were compared with that of a normal mouse (C57BL/6J). Three abnormalities of cerebellar foliation--(1) lobe isolated from other cerebellar lobes, (2) lobes imbalanced in relative amounts or ratio of granular cell layer and molecular layer, (3) lobes in which some Purkinje cells and the molecular layer was embedded in the granular cell layer--were observed in NZB/BINJ mice. These morphological abnormalities were not limited to a specific lobe. On the other hand, abnormalities of neural position were observed in both granule and Purkinje cells. The pattern of ectopically-situated granule cells, in general, could be divided into 3 types: (1) large cell clusters extending from granular cell layer to the pia mater or middle part of the molecular layer, (2) clusters of various sizes scattered within the white matter and (3) clusters formed by combination of granule cells extending from two opposed granular cell layers to the molecular layer. The pattern of ectopically-situated Purkinje cells could be divided into 4 types: (1) ectopia of a group of cells from one part of the Purkinje cell layer, (2) ectopia of a single Purkinje cell observed in the molecular layer, (3) single Purkinje cell scattered within the white matter accompanied by clusters of ectopic granule cells and (4) ectopic Purkinje cells embedded in the granular cell layer. The abnormalities in position of both granule cells and Purkinje cells was not limited to a particular cerebellar lobe.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlative light and electron microscopic observations on ectopic neurons in the cerebellum of dreher mutant mouse.

The structure of ectopic neurons in the cerebellum of dreher mutant mouse was investigated by correlative light and electron microscopic observations. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of 2-hydroxypropyl methacrylate, Quetol 523, and methyl methacrylate. Sections at 0.4-0.5 microns in thickness were examined by electron microscopy after observation under a light microscope. By comparing the electron images with those of light microscopy in the same sites, the structures of ectopic cells were confirmed. Ectopic Purkinje cells were arranged with cell bodies that contained an oval, spherical or wrinkled nucleus without deep invagination and the thin layers of endoplasmic reticulum at the perinuclear regions. Granule cells were ectopically matured in the external granular layer and within the cluster at the cortical region. This method provides a useful procedure for understanding structures of the cerebellar neurons of the mutant.

Staining of intestinal goblet cells with ruthenium red in semithin sections.

For a correlative light and electron microscopy of intestinal goblet cells, postembedding staining with ruthenium red (RR) was performed in epoxy-embedded sections. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of Quetol 651, nonenyl succinic anhydride (NSA), methyl nadic anhydride (MNA), and DMP-30. Sections at 0.4-0.5 micron in thickness were mounted on grids and were treated with an aqueous solution of RR followed by osmium tetroxide, uranyl acetate and lead citrate. Postembedding staining of epoxy sections revealed the interaction between RR and anionic groups by both light and electron microscopy. Light and electron microscopic observation of identical sites in semithin sections was successful for the correlations of colored reaction with electron density.

Use of semithin sections embedded in a water-miscible methacrylate for light microscopy of central nerve tissues.

Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogenous block from which it was easy to make sections 1.5 microns thick. Staining could be localized far more precisely in these sections than in paraffin sections owing to the thickness of the semithin sections and to the excellent structural preservation of cellular components.

Quantitative analysis of synaptic boutons on motoneurons of rhesus monkey spinal cord after chronic deafferentation due to cerebral lesions.

The motoneurons in the lateral nucleus of the anterior horn of the rhesus monkey cervical enlargement were reconstructed by light microscopy of semi-thin sections of 2 micro in thickness which were cut serially, alternately with ultra-thin sections for electron microscopy. Four single neurons were reconstructed with a computer graphic system, and simultaneously the synaptic boutons on the soma and each dendrite of single reconstructed neurons were analyzed quantitatively by electron microscopy. The synaptic boutons were classified into 4 types on the basis of the shape of the synaptic vesicles. The F-type boutons with flattened vesicles, S-type with spherical vesicles, G-type with cored vesicles and large L-type boutons with smaller and polymorphic vesicles, were almost identical to the boutons categorized by Bodian (1966b). The F-type boutons represented the major type in terms of the covering ratio and synaptic population. The S-type boutons tended to occur more frequently on dendrites. The motoneurons of the same region were also investigated in a similar manner after chronic deafferentation by the ablation of the upper extremity area of the left cerebral cortex. F-type of boutons were significantly increased, especially on the dendrites, compared with the synaptic distribution of the normal motoneurons.

Postnatal developments of AChE activity in the hippocampus of the reeler mutant mouse.

The acetylcholinesterase (AChE) activity of the hippocampus and the dentate gyrus in the reeler mutant mouse was studied histochemically by the method of Karnovsky and Roots. Developmental studies were done on days 0,3,6,9,12,15,18 and 21 postnatally, and in adults. The adult reeler mouse differs from the normal mouse in that there is no accumulation of the activity at the junction between the stratum lacunosum-moleculare and the stratum radiatum, weaker and divided activity in CA1, and the translocation of the activity to the granule cell zone of the dentate gyrus. The results obtained are considered to be due to the cells' ectopia. But also, other factors such as genesis of the cells in regard to constructing the cytoarchitecture, may influence them. According to the postnatal observations, the developmental pattern of AChE activity in the reeler mouse is about the same as in the normal mouse. AChE-rich cells in the hippocampus showed maximum activity and number from days 12 to 15. The neuropile reactions increased after day 9 in both kinds of mice. The relationship between the movement of the AChE positive neuropiles and cells and their functions are also discussed.

Faulty development of cortical neurons in the Snell dwarf cerebrum.

In the Snell dwarf motor cortex, area 6 of Caviness, the cell number, the stratification of neurons and the portion of layer-widths were absolutely identical to those of the controls. By means of the Golgi-Cox method modified by Ramon-Moliner, however, the pyramidal neuron was found to have small perikarya, short primary dendrites with sparse branchings, and a low spine density on the dendrites. The corpus callosum of the dwarf contained a reduced number of fibers compared to that of the controls, and the staining for myelin basic protein revealed a considerable reduction of positive-fibers of radiation in this area. The content of Thy-1 antigen in the cerebrum, cerebellum and brainstem, was significantly lower than that of controls, but the monoamine content was normal in the cerebrum and brainstem. From these results, it appears that the Snell dwarf cerebrum shows retarded neuronal growth; a reduction in size of neurons, an underdevelopment of axons and dendrites, and a retarded maturation of spine, in addition to arrested glial proliferation. At present, it is unclear which hormone deficient in these mutants, growth hormone or thyroxine, is the essential potentiator for neuronal growth.

Immunohistochemical localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin basic protein in the chick retina.

Antisera against bovine 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and against chick myelin basic protein (MBP) were raised in New Zealand white rabbits. The specificity of CNPase antiserum was examined by Ouchterlony double-immunodiffusion test and immunoadsorption assay. With use of the specific antiserum, immunohistochemical localizations of CNPase and MBP were investigated in the chick retina. Light microscopic immunohistochemical studies have shown that MBP is localized in the optic nerve fiber layer and that CNPase is localized in the optic nerve fiber and photoreceptor layers. Electron microscopic immunohistochemical examinations demonstrated that the myelin-like neural sheaths in the optic nerve fiber layer were clearly stained by both antisera, whereas the membranes of the Müller cell were not stained. In the photoreceptor layer, membranes of the inner and outer segments of rod and cone photoreceptor cells were intensely stained by CNPase antiserum. However, these portions were not stained by MBP antiserum. Membranes of bipolar cells, amacrine cells, horizontal cells, and ganglion cells were not stained by either antiserum.

Histochemical and immunohistochemical studies of the cerebellum from the reeler mutant mouse.

The organization of the fiber connections and architecture of the cerebellum of reeler mutant mice was analyzed by an immunohistochemical and histochemical procedure. By immunohistochemical staining of the myelinated fiber arborization with antiserum against myelin basic protein, it was found in the reeler cerebellum that the fibers ran in all directions throughout the white matter. Some of the fibers surrounded Purkinje cells. The distribution of AChE-positive fibers was abnormal and some Purkinje cells surrounded by AChE-positive fibers were present. Molecular layers and glomeruli showed strong succinic dehydrogenase (SDH) activity. The white matter of the reeler cerebellum showed a mosaic of SDH-positive and -negative sites. The results indicate that changes in the distribution of myelinated fiber arborization and the change in the distribution of SDH and AChE activity are different in each part of the cerebellum as a result of the disorganization of the architecture of the reeler mutation.