Distributions of amyloid plaques in four regions of the brains of mice infected with scrapie by intracerebral and intraperitoneal routes of injection

Summary VM mice were inoculated by intracerebral and intraperitoneal routes with brain homogenates containing the 87V strain of scrapie. The distribution and numbers of plaques were found for the parietal cortex, cingulate cortex, corpus callosum and hippocampus/dentate in coronal sections cut at the level of the thalamus and stained with Masson's trichrome. For the intracerebrally injected animals, the greatest numbers were seen on the side of the brain that had been injected. In the four regions, they were most numerous in the corpus callosum and least numerous in the parietal cotex and hippocampus/dentate. Following an intraperitoneal injection, plaques were absent from the corpus callosum and, in the remaining three regions, they were less numerous than in intracerebrally injected animals, although their relative numbers were similar. The distribution of the plaques was suggestive of an inital passive spread of inoculum following intracerebral injection and a tendency for plaques to be associated with myelinated axon tracts.     

Dendritic and synaptic alterations of hippocampal pyramidal neurones in scrapie-infected mice

Neurone damage and eventual loss may underlie the clinical signs of disease in the transmissible spongiform encephalopathies (TSEs). Although neurone death appears to be through apoptosis, the trigger for this form of cell death in the TSEs is not known. Using two different murine scrapie models, hippocampal pyramidal cells were studied through microinjection of fluorescent dye, and synaptic integrity, using p38-immunoreactivity (p38-IR), both visualized using confocal laser scanning microscopy. Intradendritic distensions and dendritic spine loss were found to co-localize to areas of vacuolar and prion protein pathology in the hippocampus of mice infected with ME7 or 87 V scrapie. A significant reduction in p38-IR was found concomitantly in the hippocampus in ME7 scrapie mice. These results indicate that both pre- and post-synaptic sites are altered by scrapie infection; this would disrupt neuronal circuitry and may initiate apoptotic cell death, giving rise to the neurological disturbances manifested in clinical TSE cases.     

On the apparent nonadhesive nature of axospinous dendritic synapses

Synaptosome preparations are rich in synaptic sacs with adherent fragments of postsynaptic membrane and it has been hypothesized that pre- and postsynaptic membranes are strongly adherent across the synaptic cleft. Scanning electron microscopy of tissue specimens subject to preliminary tearing reveals synaptic terminals on neuronal cell bodies and dendrites as well as pits in the plasma membrane from which other terminals have been avulsed. However, neither synaptic terminals nor membrane pits were seen on dendrite spines in our material. These observations support the notion of adhesion between pre- and postsynaptic membranes but suggest an absence of such adhesiveness between the elements of the axospinous synapse, fitting well with recent conceptions about spine lability and the rapid making and breaking of axospinous synapses.     

Spongiform encephalopathy in mice inoculated with scrapie material of sheep origin

The authors report spongy degeneration in experimental scrapie (second passage) in mice. The scrapie agent was originally isolated from Suffolk sheep imported from Canada and diagnosed histopathologically to be infected with scrapie by intracerebral inoculation into JCL/ICR mice. Ten female SIc/ICR mice, 4 weeks of age, were injected intracerebrally in the right frontal lobus with 20 microliter of 10(-1) or 10(-4) dilution of JCR/ICR mice brain homogenate involving scrapie agent. All animals showed signs of the advanced stages of the disease, clinically manifested by lassitude, arched backs, lethargy and paresis of hind quarters. They were sacrificed five to six months post inoculation, and sections of the brain and spinal cord were examined by light and electron microscopy. Focal symmetrical spongiform lesions were seen light microscopically in the cerebral mantle, thalamus, hypothalamus, midbrain, medulla oblongata, cerebellum and cervical mark. There was evidence that these lesions tended to be more intense in the mice inoculated a higher concentration of scrapie agent. Astrocytic proliferation was present in the deep layer of cerebral gray matter, white matter, corpus callosum, dorsal part of hippocampus and thalamus. No leukocytic infiltration was observed. Electron microscopically, the spongiform lesions were shown to be caused by vacuolation or swelling within the neuropil, and vacuolation and focal swelling in the neuronal perikaryon. The changes in the neuronal perikaryon were caused by enlargement of endoplasmic reticulum and cisterns of the Golgi apparatus, accompanied by spherical swelling of a part of the cytoplasm. The vacuolation near or within the neuron produced deformation of the cell contours and displacement of the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus

Numbers of neurones, synapses and axon terminals were quantified in a murine scrapie model with severe hippocampal pyramidal cell loss, in which definite clinical scrapie is evident from 226 days post-infection (dpi) and death occurs around 250 dpi. Disease-specific PrP accumulations were first seen at 70 dpi (28% of the incubation period (IP)) in thalamus and as sparse foci within the stratum pyramidale of CA1. By 98 dpi (39% IP), PrP was seen in the stratum radiatum and was found at later stages throughout all levels of the hippocampus. At the ultrastructural level in the stratum radiatum of CA1, a decrease in the numbers of simple synapses from 84 dpi (34% IP) and in perforated synapses from 98 dpi (42% IP) was found using an unbiased stereological method, the disector analysis. Degeneration of axon terminals was found from 98 dpi (39% IP) onwards. Neuronal loss was detected in CA1 from 180 dpi (72% IP). The results suggest that the fundamental lesion in the hippocampus of ME7-infected mice is associated with PrP release from CA1 pyramidal neurones, which perturbs synaptic function and leads to degeneration of preterminal axons, and that subsequent pathological changes including neurone loss are sequelae to this initial insult.     

A quantitative ultrastructural analysis of the synaptic population in the sacral visceral gray

The synaptic distribution in the sacral visceral gray of the cat was examined. Synaptic complexes exhibiting a distinct membrane specialization were identified and classified according to vesicle type and number. Boutons containing clear spherical vesicles (S type) account for more than 63% of the synaptic contacts on both cell bodies and dendrites. Terminals containing three or more granular vesicles (GS type) comprise between 23 and 31% of the synapses in the nucleus. F boutons are less frequently seen (3–6%) as are T type boutons (1–2%). Presynaptic terminals containing a predominance of dense core vesicles (GG type) constitute less than 1% of the total synapses in this neuropil and are found only on dendrites. Nerve cell bodies receive approximately five synaptic contacts per 100 μm of postsynaptic membrane. Large and medium size dendrites are contacted by an average of seven boutons per 100 μm of postsynaptic membrane and small dendrites have the highest distribution (approximately 12 per 100 μm). Comparison of the percentage distribution of various types of synapses in this nucleus reveals little variation between cell bodies and dendrites. However, expression of the synaptic population as boutons per 100 μm of available postsynaptic membrane clearly illustrates intranuclear differences in synaptic distribution not seen in percentage type comparisons.     

Tubulovesicular particles occur early in the incubation period of murine scrapie

Tubulovesicular bodies are structures, apparently specific to the transmissible spongiform encephalopathies, which are of unknown composition and significance. Prion protein (PrP) is absent from tubulovesicular bodies when tissues are examined by immunogold electron microscopy. In the F1 cross of C57 and VM mice (CVF1) infected with ME7 scrapie there is a marked degeneration of hippocampal CA1 neurons. In this model the earliest changes seen, at about 100 days post inoculation (dpi) are a degeneration of axon terminals and synaptic loss. Terminal disease is around 250 dpi. In blind coded trials we counted the number of tubulovesicular particles and estimated their density in 56–76 electron micrographs taken from the stratum radiatum of each of one or two CVF1 ME7-infected mice at 84, 100, 126, 154 and 181 dpi and from four normal brain inoculated control mice. Tubulovesicular particles were present from 98 dpi and the density of particles increased with increasing incubation period. The very early occurrence of tubulovesicular particles, before the presence of significant pathology, argues that tubulovesicular particles are a part of the primary disease and are not epiphenomena. Received: 28 June 1999 / Revised: 30 August 1999 / Accepted: 6 September 1999     

Immunoelectron microscopic study of synaptic pathology in Alzheimer's disease

Summary Alzheimer's disease (AD) is characterized by an extensive loss of neurons and synapses in the neocortex which correlates strongly with psychometric tests of dementia. To characterize the ultrastructural changes in presynaptic terminals in AD, we studied biopsy material from the frontal cortex. We also examined, at the ultrastructural level, abnormal neurites scattered in the AD neuropil and in the plaque region using sections from autopsy material immunolabeled with anti-synaptophysin. We found that, regardless of amyloid deposits, some presynaptic terminals were distended and contained swollen vesicles and dense bodies. These altered synaptic organelles were similar to those found in dystrophic neurites. The latter structures displayed synaptophysin immunoreactivity, mostly localized to outer membranes of synaptic vesicles and dense bodies. The present study supports the hypothesis of progressive synaptic pathology in AD neocortex and favors the notion that the dystrophic process originates from presynaptic terminals.Supported by National Institute of Health grants AGO8201 and AGO5131, the PEW Charitable Trust and the Alzheimer Disease and Related Disorders Association     

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5–10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.     

Photoreceptor degeneration during infection with various strains of the scrapie agent in hamsters

Hamsters were inoculated intracerebrally with the 22C, 79A, and ME7 strains of the scrapie agent to compare the effects on the retina with those caused by strain 263K. The animals developed clinical signs of encephalopathy. Photoreceptor degeneration occurred in all experimental animals. The changes were similar to those seen in animals infected with the 263K strain of scrapie although somewhat more variable and less extensive.     

Cellular localization of serotonin(2A) (5HT(2A)) receptors in the rat brain

The serotonin(2A) (5HT(2A)) receptors have been shown to play an important role in several psychiatric disorders, including depression, schizophrenia, and alcoholism. This immunohistochemical study examined the cellular localization of 5HT(2A) receptors in various rat brain structures (olfactory, striatum, cortex, hippocampus, and amygdala). The colocalization of 5HT(2A) receptors in astrocytes was performed by double-immunofluorescence staining of 5HT(2A) receptors and of glial fibrillary acidic protein (GFAP) using confocal laser microscopy. 5HT(2A) receptor immunolabeling was observed in olfactory bulbs, neostriatum, hippocampus, amygdala, and neocortex. Somata and dendrites of pyramidal cells in the frontal cortex (layer V) were densely labeled with 5HT(2A) receptors. In several other brain structures (hippocampus, amygdala, striatum, olfactory structures), 5HT(2A) receptor immunolabeling was found in cell bodies and processes of neurons. 5HT(2A) receptor immunolabeling was also observed in GFAP-positive cells of the various brain structures we investigated (layers I/VI of the neocortex, corpus callosum, hippocampal fissure and hilus, and amygdala). These results indicate that 5HT(2A) receptors are expressed in neurons and astrocytes and suggest the possibility that not only neuronal but also glial 5HT(2A) receptors have functional implications in psychiatric disorders.     

Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influence dendritic spine morphology and long-term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4-labeled dendritic spines and axon terminals corresponded to 9-19% and 25-40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre- or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP.     

Astrocytosis and amyloid deposition in scrapie-infected hamsters

In scrapie infection, prion protein (PrP^Sc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrP^Sc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrP^Sc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrP^Sc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrP^Sc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrP^Sc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrP^Sc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrP^Sc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrP^Sc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrP^Sc during some scrapie strain infections. The findings suggest a `snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.     

BDNF enhancement of postsynaptic NMDA receptors is blocked by ethanol

The neurotrophin brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission. Physiological and biochemical evidence implicates the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present studies, murine brain slices containing hippocampus and neocortex were used to study the effects of BDNF on excitatory neurotransmission. Acute exposure to BDNF rapidly and reversibly enhanced the magnitude of NMDA-mediated, but not AMPA receptor-mediated, synaptic currents, specifically enhancing the activity of NMDA receptors containing the NR2B subunit. This effect of BDNF was dependent on activation of trkB neurotrophin receptors because similar effects were not seen with the related neurotrophins NT-3 or NGF. Furthermore, activation of trkB receptors in the postsynaptic neuron was required, as BDNF-induced potentiation was blocked by postsynaptic injection of a trk tyrosine kinase inhibitor. Interestingly, the effect of BDNF was also completely blocked by pretreatment with ethanol, even at concentrations of ethanol that had minimal direct effects on NMDA-mediated responses. These results provide a potential mechanism for the proposed role for BDNF in activity-dependent synaptic plasticity and, potentially, learning and memory processes.     

EFFECT OF ROUTE OF INFECTION ON THE FREQUENCY AND DISTRIBUTION OF CEREBRAL AMYLOID PLAQUES IN SCRAPIE MICE

Cerebral amyloid plaques are a conspicuous pathological feature in mice infected with certain strains of scrapie. The origin of the amyloid protein in these plaques, whether it is locally or systemically synthesized, and the mechanisms leading to its deposition are not known. The frequency of plaques and their distribution in the brain are greatly influenced by the route of injection of the scrapie inoculum. Intracerebral injection consistently results in greater numbers of plaques than are obtained if the same inoculum is introduced peripherally. Following intracerebral injection, plaques are commonly seen in areas close to the lateral ventricles, for example the corpus callosum and hippocampus, whereas they are absent from these areas with peripheral routes. Furthermore, when left- and right- sided intracerebral injections are compared, plaques are more frequent on the side of injection. These results suggest that the distribution of amyloid plaques is influenced either by the localization of some component of the inoculum or by traumatic damage at injection. The most plausible explanation is that amyloid deposition is associated with local concentrations of scrapie infectivity and that the amyloid protein originates in the brain.     

Morphology of layer III pyramidal neurons is altered following induction of LTP in sensorimotor cortex of the freely moving rat

The organization of specific cortical connections can be altered by sensory and motor experience. These changes are believed to result from activity-dependent changes in synaptic connectivity, similar to those induced in the hippocampus by high-frequency stimulation in long-term potentiation (LTP) experiments. If similar mechanisms are involved, then neocortical LTP induction may induce some of the same morphological changes that are seen following learning. We induced LTP in the contralateral sensorimotor cortex by repeated, daily tetanization of the corpus callosum in chronically implanted, freely moving rats. Anatomical results showed that the LTP induction was associated with alterations in dendrite morphology and increased spine density. These changes are qualitatively and quantitatively similar to those commonly observed in studies in which rats are housed in complex environments. The similarity of results following exposure to complex environments and after LTP induction in the neocortex may indicate a reliance on the same cellular mechanisms in both situations.     

Synaptic patterns in the visual cortex of turtle: An electron microscopic study

The part of turtle general cortex that receives afferent fibers from the dorsal lateral geniculate nucleus and that shows evoked potentials to light stimuli has been studied with the electron microscope. This cortex consists of an outer molecular layer, a perikaryal layer, and a subcellular layer lying on a row of ependymal cell bodies. Neurons in the perikaryal lamina are characterized by long spine-bearing apical dendrites ascending through the outer molecular layer and short finer basal dendrites in the subcellular zone. Scattered neurons without apical dendrites occur in both the molecular and subcellular zones. Two types of dendritic spines can be distinguished. Some are large, have a complex irregular shape, contain a variety of membranous sacs and mitochondria, and occasionally, a single bundle of microtubules embedded in an electron-dense background opacity. These large spines are the most common postsynaptic element in the outer third of the molecular layer, where they are located on the distal tips of the apical dendrites. Other spines are small, with a simple spherical distal enlargement that contains only electron-dense fuzz. They are the most common post-synaptic element in the lower two-thirds of the molecular layer where they arise from the proximal portion of apical dendrites. Most synaptic contacts are found on the dendritic spines and are of the “round-asymmetrical” type. Not infrequently “flat-symmetrical” synapses are seen coupled to “round-asymmetrical” contacts on individual large spines. The few contacts present on spine-bearing dendritic shafts are of both types. Axo-somatic contacts are mainly of the “flat-symmetrical” variety. Thus the synaptic patterns on the principal cells of turtle visual cortex are remarkably similar to those found on pyramidal cells of mammalian neocortex. In addition, however, axon terminals, dendrites and glial (ependymal) processes were often seen to give rise to membranous pouches containing large vacuoles and invaginating into dendritic shafts or spines. Rarely, axon terminals were seen to form contacts, identical in appearance to synaptic contacts, on cell bodies in the ependymal lining. More frequently, unusual types of membrane differentiations were present at the site of apposition of the membranes of axon terminals and ependymal processes. They are interpreted as functional neuroependymal contacts.     

Immunocytochemical localization of nicotinic acetylcholine receptor in rat hypothalamus

Immunocytochemical localization of neuronal nicotinic acetylcholine receptor (nAChR) was examined in rat hypothalamus. Monoclonal antibody against α4 ACh-binding subunits of nAChR was used in the avidin-biotin-peroxidase complex (ABC) immunocytochemical method at both the light and electron microscopic levels. By light microscopy nAChR-like immunoreactivity was found in many neuronal cell bodies and their fibers in the paraventricular nucleus (PVN) and in many axons and axon terminals in the median eminence (ME). The immunoreactivity of nAChR was the most intense in the ME. By electron microscopy immunoreaction products occurred on the rough endoplasmic reticulum, nuclear envelope, cytoplasmic matrices and postsynaptic densities of synaptic junctions in some neurons in the parvocellular part of the PVN. In the external layer of the ME, nAChR-like immunoreactivity was found over the entire plasma membranes of many axon terminals. Involvement of nAChRs in the release of neurotransmitters and neuropeptides both in the PVN and the ME is discussed.     

Doublecortin-expressing cells in the ischemic penumbra of a small-vessel stroke

A cortical lesion was induced by disrupting the medium-size pial vessels, which led to a cone-shaped cortical lesion and turned into a fluid-filled cavity surrounded by a glial acidic fibrillary protein-positive (GFAP(+)) glia limitans 21 days after injury. Therefore, it mimics conditions of lacunar infarctions, one of the most frequent human stroke pathologies. Doublecortin (DCX)-positive cells were present in the neocortex and corpus callosum at the base of the lesion. The number of DCX-positive cells in the corpus callosum was significantly increased from day 5 to day 14 compared with the control group. In contrast, there were no DCX-positive cells in neocortex of control animals; the DCX-positive cells appeared in the neocortex after lesioning and were maintained until 14 days postlesioning. Some of the DCX-positive cells were also immunoreactive for beta III-tubulin, another marker of immature neurons. They did not stain positively for markers of glia cells. The presence of these DCX-positive cells near the lesion might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with a lipophilic molecule, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE-containing cells were found in the cortex beneath the lesion. These results do not support the idea that the DCX-positive cells were originating from neural precursors of the SVZ, but they might be generated from local progenitor cells.     

Defined astrocytic expression of human amyloid precursor protein in Tg2576 mouse brain

Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron-specific. Using a novel hAPP-specific antibody in combination with cell type-specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that—in addition to neurons throughout the brain—astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology.