Immunocytochemical evaluation of blood-brain barrier to endogenous albumin in scrapie-infected mice

A quantitative immunocytochemical procedure was used for evaluation of the blood-brain barrier (BBB) to endogenous albumin in plaque-forming (PF) and non-plaque-forming (NPF) groups of scrapie-infected mice at the clinical stage of disease. Ultrathin sections of brain samples (cerebral cortex, hippocampus and cerebellum) embedded in resin (Lowicryl K4M) were exposed to anti-mouse albumin antiserum followed by protein A-gold. Using morphometry, the density of immunosignals (gold particles per μm²) was recorded over four compartments: vascular lumen, endothelium, subendothelial space, and brain parenchyma (neuropil). Morphometric and statistical analyses did not reveal significant differences in the barrier function of the microvasculature of the cerebral cortex and hippocampus in either group of mice, although a slight increase in the number of leaking vessels in the PF group was noted. In contrast, in the cerebellum, the permeability of the microvessels to albumin was significantly higher in the PF than in the NPF mouse group, and this was paralleled by the infiltration of the walls of numerous vascular profiles with amyloid deposits (amyloid angiopathy). These data also indicate the existence of distinct regional differences in BBB function in the brain of scrapie-infected mice. The vascular amyloid deposits and the amyloid plaques present in the cerebral cortex of PF mice were labeled with numerous immunosignals suggesting the affinity of extravasated albumin to these deposits. In conclusion, no convincing evidence was obtained indicating that impairment of the BBB, manifested by increased permeability of vascular segments, is directly related to the deposition of amyloid in the vascular wall and in plaques. Segmental impairment of the barrier function seems to be rather the result of disturbed structural integrity of the components of the vascular wall. Received: 8 May 1996 / Revised: 26 August 1996, 13 October 1996 / Accepted: 15 October 1996     

Ultrastructural studies of glycoconjugates in brain micro-blood vessels and amyloid plaques of scrapie-infected mice

Summary Lectin or glycoprotein-gold complexes and samples of scrapie-infected mouse brain embedded in Lowicryl K4M were used for ultrastructural localization of glycoconjugates. The lectins tested recognize the following residues: -D-galactosyl [RCA,Ricinus communis agglutinin (aggl.) 120], N-acetyl and N-glycolyl neuraminic acid (LFA,Limax flavus aggl.), N-acetyl-D-glucosaminyl and sialyl (WGA, Wheat germ aggl.), N-acetyl-D-galactosaminyl (HPA,Helix pomatia aggl., and DBA,Dolichosbiflorus aggl.), -D-mannosyl/-D-glucosyl (Con A, Concanavalin A), -D-galactosyl and -D-galactopyranoside (BSA,Bandeirea simplicifolia aggl., izolectin B₄). Labeling of the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs, however, located inside the plaque area and surrounded directly by amyloid fibers showed attenuation of the endothelium, the surface of which was scarcely and irregularly decorated with RCA, LFA, WGA and Con A. These abnormalities in the composition of glycoconjugates can be associated with previously noted increased permeability of some MBVs in the brains of scrapie-infected mice. Some vessels in the plaque area were encapsulated by perivascular deposits of homogenous or flocculogranular material containing several glycoconjugates. A very intimate structural relation between reactive (microglial-like) cells and amyloid fibers suggests the participation of these cells in elaboration of plaque material. Labeling of the cell surface and adjacent amyloid fibers with the same lectins (RCA, WGA, DBA, Con A) suggests the possibility that the glycosylation of these fibers occurs extracellularly. Only WGA and DBA were occasionally labeling some Golgi elements of the reactive cells.Supported in part by a grant from NINCDS No. 17271-06     

Topographic distribution of scrapie amyloid-immunoreactive plaques in chronic wasting disease in captive mule deer (Odocoileus hemionus hemionus)

Summary Chronic wasting disease (CWD), a progressive neurological disorder of captive mule deer, blacktailed deer, hybrids of mule deer and white-tailed deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation in neuronal perikarya and astrocytic hypertrophy and hyperplasia. We report the topographic distribution of amyloid plaques reactive to antibodies prepared against scrapie amyloid in CWD-affected captive mule deer (Odocoileus hemionus hemionus). Scrapie amyloid-immunoreactive plaques were found in the cerebral gray and white matter, in deep subcortical nuclei, in isolation or in clusters in areas of vacuolation, and perivascularly, in subpial and subependymal regions. In the cerebellum, immunoreactive amyloid plaques were observed in the molecular, pyramidal and granular layers. Scrapie amyloid-immunoreactive deposits were also seen in neuronal perikarya. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Our data corroborate that CWD in captive mule deer belongs to the subacute virus spongiform encephalopathies.     

Spontaneous phagocytosis of C-type synaptic terminals by spinal alpha-motoneurons in newborn kittens. An electron microscopic study.

The cell bodies and proximal dendrites of spinal alpha-motoneurons were studied electron microscopically with the aid of serial sections during the first postnatal week in the cat. The observations suggested that some of the synaptic terminals of the so-called C-type on the cell bodies and dendrites are phagocytosed by the motoneurons during the first few days after birth. This finding is discussed in relation to the earlier demonstrated postnatal loss of synaptic terminals on the motoneurons after birth and differences demonstrated between different functional types of spinal motoneurons with respect to the number and distribution of C-type terminals in the adult cat.     

Evaluation of neurodegeneration in scrapie-infected animals by selective methods that detect cellular degeneration

Scrapie is a fatal neurodegenerative disease of sheep and goats. The precise details of neuronal and neurite degeneration in scrapie-infected animals remain unknown. Using specific silver staining methods, we compared the neurodegeneration caused by treatment of rats with kainic acid (KA) or ibogaine (IBO) to the neuropathology observed in mice infected with the C602 strain of scrapie. As reported previously, KA resulted in extensive silver labeling of neurons, especially in the cortex, putamen and hippocampus. IBO silver labeling was observed only in small clusters of Purkinje neurons in the paravermal region of the cerebellum. However, in scrapie-infected mice, a few silver stained neurons (differing from the dark degenerating neurons observed following neurotoxic exposure) were found in layer II of cortex, cingulate cortex, zona incerta, thalamus and hypothalamus. Some silver grains were observed in glial-like cells, especially those in the paraventricular region. Degenerating axons were positive for silver staining and were found in the cortex, cingulate cortex, corpus callosum, habenulae, septum, fornix, thalamus, caudate putamen and a few in fasciculus retroflexus and substantia nigra. Our results suggest that the limbic system is one of the important loci for the neurodegenerative effect of at least some scrapie strains.     

An electron microscopic study of natural scrapie sheep brain: Further observations on virus-like particles and paramyxovirus-like tubules

Summary Detailed electron microscopic study of cerebellum and medulla of sheepaffected with natural scrapie is reported.Two types of virus-like particles, non-membrane bound and membrane bound occur in neurons, pre-synaptic terminals and axis cylinders. The latter type were seen being apparently formed at the plasma membranes. Their size corresponds with that attributed to the scrapie agent. Three different types of tubules are described which filled a few axis cylinders and pre-synaptic terminals, their structure is compared with tubules found in other conditions.     

Glutamate-positive neurons and terminals in the cat periaqueductal gray matter (PAG): a light and electron microscopic immunocytochemical study

The morphology, distribution, proportion, size, and synaptic organization of periaqueductal gray matter neurons labeled with immunocytochemical techniques by an anti-glutamate (Glu) polyclonal serum were investigated in six adult cats (PAG-GLU 1-6). At the light microscopic level, numerous Glu-positive neurons were found throughout each subdivision of the periaqueductal gray matter. Their proportion and size, calculated in semi-thin sections (1-μm-thick), varied slightly among the subdivisions of the periaqueductal gray matter. The morphology of Glu-positive neurons was similar to that of the multipolar, triangular, and fusiform cells described in previous Golgi studies. Numerous puncta, interpreted as dendrites, axons, and axon terminals were also present in all subdivisions without preferential distribution. At the electron microscopic level, all synaptic contacts made by Glu-positive axon terminals were of the asymmetric type, but not all presynaptic elements making asymmetric synapses were labeled. The vast majority of postsynaptic elements contacted by Glu-positive axon terminals were labeled and unlabeled dendrites. The present results describe for the first time the presence of both Glu-positive neurons and terminals in the feline periaqueductal gray matter and provide further evidence that Glu is the probable neurotransmitter of numerous excitatory neurons of this structure. J. Comp. Neurol. 383:381-396, 1997. © 1997 Wiley-Liss, Inc.     

An electron and light microscopic study of the numbers of dystrophic neurites and vacuoles in the hippocampus of mice infected intracerebrally with scrapie

Summary Numbers of dystrophic neurites, seen with the electron microscope, in CA1 of the hippocampus of either C3H mice infected with 22C or 79A strains of scrapie, or LM mice infected with strain ME7 were greater than in age-matched control mice. Vacuolation, seen by light microscopy in CA1 of the hippocampus of mice infected with either 22C or 79A, preceded the increase in dystrophic neurites by up to about 20 days. In mice infected with ME7, however, the vacuolation followed the increase in dystrophic neurites by some 20 to 40 days. In view of the differences in the times at which dystrophic neurites and vacuolation were seen, no causative relationship between the two lesions appeared to exist.     

Dopaminergic terminals form synaptic contacts with enkephalinergic striatal neurons in pigeons: an electron microscopic study

Medium spiny projection neurons of the striatum consist of two major neuropeptide-specific types, one type containing substance P and another type containing enkephalin. Both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites. However, whether each of these types receives direct dopaminergic input onto distal dendritic shafts and onto dendritic spines has not been explored in depth. In the present study, we used electron microscopic immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto enkephalin-positive (ENK +) striatal neurons in pigeons, in whom ENK + striatal perikarya, dendritic shafts and spines can be readily labeled. Antibodies against tyrosine hydroxylase were used to label dopaminergic terminals using a silver-intensified immunogold method. ENK + neurons were labeled using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts, and dendritic spine necks of ENK + striatal neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto ENK + striatal neurons in a manner similar to that described previously by us for substance P-positive striatal medium spiny neurons.     

Changes in the distribution of anionic sites in brain micro-blood vessels with and without amyloid deposits in scrapie-infected mice

Summary Cationic colloidal gold (CCG) and scrapie-infected mouse brain samples embedded in Lowicryl K4M were used for ultrastructural localization of negatively charged microdomains (anionic sites) in the cerebral microvasculature. The distribution of anionic sites on both fronts (luminal and abluminal) of endothelial cells and in the basement membrane (BM) in the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs (especially capillaries), however, located inside the plaque areas and surrounded directly by amyloid fibers contained attenuated endothelium, the luminal surface of which showed a segmental lack or diminution of anionic sites. In these vessels the BM was frequently infiltrated and replaced by the amyloid fibers. In some vessels located mainly in the areas of the neuropil vacuolization deposits of homogenous material causing the thickening of the BM were noted. These changes were accompanied by irregular labeling of the BM with gold particles. At the sites of bifurcation of some MBVs, predominantly in the area of the venular estuary at the mouth of capillary (at capillary-venular connections), a discontinuity in the distribution of anionic sites was noted. The observed disturbances in the distribution of anionic sites can be associated with a previously noted increased permeability of some MBVs in the brains of scrapie-infected mice.Supported in part by a grant from NINCDS No. 17271-08     

Spontaneous phagocytosis of c-type synaptic terminals by spinal α-motoneurons in newborn kittens. An electron microscopic study

The cell bodies and proximal dendrites of spinal α-motoneurons were studied electron microscopically with the aid of serial sections during the first postnatal week in the cat. The observations suggested that some of the synaptic terminals of the so-called C-type on the cell bodies and dendrites are phagocytosed by the motoneurons during the first few days after birth. This finding is discussed in relation to the earlier demonstrated postnatal loss of synaptic terminals on the motoneurons after birth and differences demonstrated between different functional types of spinal motoneurons with respect to the number and distribution of C-type terminals in the adult cat.     

Rapid alteration of synaptic number and postsynaptic thickening length by NMDA: an electron microscopic study in the occipital cortex of postnatal rats

The N-methyl-D-aspartate (NMDA) receptor has been widely implicated in numerous activity-dependent models of neural plasticity, learning, and memory. The formation of new synapses is a major assumption of the neural basis of learning. The current research was conducted to determine whether NMDA receptor activation could induce synaptic formation and, if so, whether this ability would mirror developmental changes in NMDA receptors. Rats at various developmental ages were given a single intraperitoneal injection of NMDA and sacrificed at various brief postinjection intervals (0.5-2 hr). The rats showed an age-dependent decline in the behavioral response to NMDA, as evidenced by reduced seizure activity and duration. Quantitative electron microscopic observations on the molecular layer of the occipital cortex, an area rich in NMDA receptors, revealed a transient increase in the length of postsynaptic thickenings in 17- and 35-day-old animals, appearing within 0.5 hr of injection. At 1 and 2 hr postinjection, an increase in synaptic density (number of synapses) was observed in 8-day-old animals. These results provide evidence that NMDA administration alone is capable of rapidly inducing alterations in synaptic structure and the formation of new synapses, underscoring the importance of the NMDA receptor in synaptogenesis and synaptic structural plasticity.     

Postnatal developmental changes of tryptophan hydroxylase activity in serotonergic cell bodies and terminals of rat brain

Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis, so its development is very important to the functional maturation of the serotonergic neurons. In the present study, we examined changes of TPH activity in serotonergic cell bodies and terminals of rat brain during postnatal development. TPH activity reached its peak in the cell body regions at 24 days after birth, while the enzyme activity in a terminal region rose to its plateau at day 30. TPH activity in adult rat is the highest in nucleus raphe dorsalis, then nucleus raphe centralis and hypothalamus in a decreasing order.     

Intracytoplasmic inclusion bodies of the thalamus and the substantia nigra,and Marinesco bodies in myotonic dystrophy: a quantitative morphological study

Summary Intracytoplasmic inclusion bodies of the thalamus and the substantia nigra, and Marinesco bodies have been studied in four patients with myotonic dystrophy (MyD), eight patients with other neurological diseases (control A), and eight patients without neurological diseases (control B). The percentages of the affected cells were calculated by dividing the number of neurons including intracytoplasmic inclusion bodies of the thalamus and the substantia nigra, and Marinesco bodies, by the total cell count in these respective regions. Statistical analyses were performed with regard to the frequency of these bodies by using Student'st test. There was a significantly higher incidence of intracytoplasmic inclusion bodies of the thalamus (13.2% versus 0.7%,P<0.001) and the substantia nigra (20.4% versus 2.7%,P<0.001), and Marinesco bodies (37.4% versus 4.1%,P<0.001) in patients with MyD than in controls A and B. From our observations, it is suggested that the presence with a high frequency, in combination, of these bodies is not an incidental finding but may have an intimate and important relationship with the pathogenesis of MyD, and may be a conspicuous and diagnostically important feature of MyD.Supported by Grant-in-Aid for Scientific Research from The Ministry of Education, Science and Culture     

Caudal neurosecretory system synaptic morphology following deafferentation: An electron microscopic degeneration study

The caudal neurosecretory system of Poecilia sphenops (molly) is an isolated population of neurosecretory cells located in the caudal most aspect of the teleost spinal cord. The structure of this neuroendocrine system is favorable for studies on the synaptic control of neurosecretory mechanisms. Little is known about the detailed synaptology of the system. Morphological and electrophysiological reports have shown that the caudal neurosecretory system is linked to higher brain centers by descending spinal projections. To examine the synaptology of the descending synaptic input, surgical deafferentation was performed by microsuction removal of a segment of spinal cord rostral to the caudal system. The degeneration of axon terminals was studied at various times following deafferentation and compared to control synaptology. Based on vesicle content and morphology, three axon terminal types were found in the caudal neurosecretory system. These terminals formed axosomatic, axodendritic, and axoaxonic synaptic contacts. Following deafferentation, axon terminals with dense-cored vesicles and boutons with round clear vesicles degenerated as evidenced by the electron dense dark reaction and the electron lucent reaction respectively. This suggested that at least two different types of axon terminals arise from the descending projection to the caudal neurosecretory system and that two different neurotransmitters may be influencing the neurosecretory activity of these cells.