Molecular Cell Layers Research Articles

The level and distribution of the GABA BR2 receptor subunit in the rat's central auditory system

The GABA B receptor is important for the function of auditory neurons. We used Western blotting and immunohistochemical methods to examine the level and localization of GABA BR2, a required subunit of a functional GABA B receptor, in the rat's central auditory system. Results revealed that this subunit was expressed throughout the auditory system with the level being high in the layers I–V of the auditory cortex, medial geniculate nucleus, dorsomedial and lateral parts of the inferior colliculus, and the molecular and fusiform cell layers of the dorsal cochlear nucleus. Labeled cell bodies were found in all the areas showing immunoreactivity. Neuropil labeling was strong in areas with high overall levels of immunoreactivity. Regional distributions of the receptor subunit revealed clear boundaries of some auditory subnuclei including the dorsal and ventral cochlear nuclei and the lateral superior olivary nucleus. Differences in immunoreactivity were found between the central nucleus and the dorsal cortex of the inferior colliculus and between the dorsal and ventral parts of the ventral nucleus of the lateral lemniscus, although no clear boundaries were observed. No differences in immunoreactivity were found between the core and the belt areas of the auditory cortex and among the subdivisions of the medial geniculate nucleus. The regional distribution of the receptor subunit in auditory structures is consistent with inputs to these structures and the cellular localization of the receptor in auditory neurons supports the contribution of the GABA B receptor to synaptic responses in these neurons.

Deposition of Hyperphosphorylated Tau in Cerebellum of PS1 E280A Alzheimer's Disease

Early-onset familial Alzheimer's disease (AD) caused by presenilin-1 mutation E280A (PS1-E280A) presents wide clinical and neuropathological variabilities. We characterized clinically and neuropathologically PS1-E280A focusing in cerebellar involvement and compared it with early-onset sporadic Alzheimer's disease (EOSAD). Twelve E280A brains and 12 matched EOSAD brains were analyzed for beta-amyloid and hyperphosphorylated tau (pTau) morphology, beta-amyloid subspecies 1-40, 1-42 levels, pTau levels, and expression of stress kinases in frontal cortex and cerebellum. The data were correlated to clinical and genetic findings. We observed higher beta-amyloid load, beta-amyloid 1-42 and pTau concentrations in frontal cortex of PS1-E280A compared with EOSAD. High beta-amyloid load was found in the cerebellum of PS1-E280A and EOSAD patients. In PS1-E280A, beta-amyloid localized to the molecular and Purkinje cell layers, whereas EOSAD showed them in Purkinje and granular cell layers. Surprisingly, 11 out of 12 PS1-E280A patients showed deposition of pTau in the cerebellum. Also, seven out of 12 PS1-E280A patients presented cerebellar ataxia. We conclude that deposition of beta-amyloid in the cerebellum is prominent in early-onset AD irrespective of genetic or sporadic origin. The presence of pTau in cerebellum in PS1-E280A underscores the relevance of cerebellar involvement in AD and might be correlated to clinical phenotype.

Predominant Involvement of the Cerebellum in Guinea Pigs Infected with Bovine Spongiform Encephalopathy (BSE)

This study reports the experimental transmission of bovine spongiform encephalopathy (BSE) to guinea pigs and describes the cerebellar lesions in these animals. Guinea pigs were inoculated intracerebrally with 10% brain homogenates from BSE-affected cattle. These animals were designated as the first passage. Second and third passages were subsequently performed. All guinea pigs developed infection at each passage. The mean incubation period of the first passage was 370 days post-infection (dpi) and this decreased to 307 dpi and 309 dpi for the second and third passages, respectively. Mild to severe spongiform degeneration and gliosis were observed in the cerebral cortex, thalamus and brainstem. In addition, the affected animals had marked pathological changes in the cerebellum characterized by severe cortical atrophy associated with Bergmann radial gliosis of the molecular layer and reduction in the width of the granular cell layer. Immunohistochemically, intense PrP(Sc) deposition and scattered plaque-like deposits were observed in the molecular and granular cell layers. Cerebellar lesions associated with severe atrophy of the cortex have not been reported in animal prion diseases, including in the experimental transmission of PrP(Sc) to small rodents. These lesions were similar to the lesions of human kuru or the VV2 variant of sporadic Creutzfeldt-Jakob disease, although typical kuru plaques or florid plaques were not observed in the affected animals.

Ubiquitinated, p62 immunopositive cerebellar cortical neuronal inclusions are evident across the spectrum of TDP-43 proteinopathies but are only rarely additionally immunopositive for phosphorylation-dependent TDP-43

Frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), frontotemporal lobar degeneration with motor neuron disease/amyotrophic lateral sclerosis (FTLD-MND/ALS) and MND/ALS are thought to represent a clinicopathological spectrum of TDP-43 proteinopathies. The cerebellum has been little studied in these conditions, probably because of the lack of cerebellar signs in most cases. We examined p62 immunohistochemistry on cerebellar sections from 43 TDP-43 proteinopathies (including cases of FTLD-TDP, FTLD-MND/ALS and MND/ALS) together with 72 cases of other neurodegenerative diseases, seven controls and three other disease conditions. In 11 of the TDP-43 proteinopathies (26%) there were numerous p62-positive cerebellar inclusions, predominantly within the granular layer, but also the molecular and Purkinje cell layer. Furthermore, only one of the remaining 82 cases (a familial tauopathy) showed similar p62 positivity. Immunohistochemistry for ubiquitin was positive in the granular layer inclusions. The immunohistochemistry for phosphorylation-independent TDP-43, hyperphosphorylated tau, α-synuclein, fusion sarcoma protein (FUS), and neurofilament was negative. In only one case (a case of FTLD-TDP) were the inclusions positive for phosphorylation-dependent TDP43 (p-TDP-43). Those TDP-43 proteinopathy cases that showed the cerebellar inclusions also tended to display other common features, such as a notable excess of p62 pathology when compared to TDP-43 pathology, especially within the pyramidal neurones of the hippocampus but also in some cases within the neocortex. The results suggest that p62-positive inclusions within the cerebellum are seen in a proportion of cases across the range of the TDP-43 proteinopathy spectrum and they appear to be relatively specific for this group of diseases. The question as to whether these cerebellar-positive cases represent a distinct subgroup remains to be answered. Furthermore, the relationship of the p62 positivity in the cerebellum to the underlying pathological processes awaits to be established.

Role of mouse cerebellar nicotinic acetylcholine receptor (nAChR) α4β2- and α7 subtypes in the behavioral cross-tolerance between nicotine and ethanol-induced ataxia

We have demonstrated that nicotine attenuated ethanol-induced ataxia via nicotinic-acetylcholine-receptor (nAChR) subtypes α4β2 and α7. In the present study, ethanol (2g/kg; i.p.)-induced ataxia was assessed by Rotorod performance following repeated intracerebellar infusion of α4β2- and α7-selective agonists. Localization of α4β2 and α7 nAChRs was confirmed immunohistochemically. Cerebellar NOx (nitrite+nitrate) was determined flurometrically. Repeated intracerebellar microinfusion of the α4β2-selective agonist, RJR-2403 (for 1, 2, 3, 5 or 7 days) or the α7-selective agonist, PNU-282987 (1, 2, 3 or 5 days), dose-dependently attenuated ethanol-induced ataxia. These results suggest the development of cross-tolerance between ethanol-induced ataxia and α4β2 and α7 nAChR agonists. With RJR-2403, the cross-tolerance was maximal after a 5-day treatment and lasted 48h. Cross-tolerance was maximal after a 1-day treatment with PNU-282987 and lasted 72h. Pretreatment with α4β2- and α7-selective antagonists, dihydro-β-erythroidine and methyllycaconitine, respectively, prevented the development of cross-tolerance confirming α4β2 and α7 involvement. Repeated agonist infusions elevated cerebellar NOx 16h after the last treatment while acute ethanol exposure decreased it. Pretreatment with repeated RJR-2403 or PNU-282987 reversed ethanol-induced decrease in NOx. The NOx data suggests the involvement of the nitric oxide (NO)–cGMP signaling pathway in the cross-tolerance that develops between α4β2- and α7-selective agonists and ethanol ataxia. Both α4β2 and α7 subtypes exhibited high immunoreactivity in Purkinje but sparse expression in molecular and granular cell layers. Our results support a role for α4β2 and α7 nAChR subtypes in the development of cross-tolerance between nicotine and ethanol with the NO signaling pathway as a potential mechanism.

Balloon cells in the dentate gyrus in hippocampal sclerosis associated with non-herpetic acute limbic encephalitis

The presence of balloon cells, a pathognomonic cellular feature of focal cortical dysplasia type IIB, in a background of hippocampal sclerosis is rare. Here we report the surgical pathologic features of the hippocampus resected from a 32-year-old woman with mesial temporal lobe epilepsy and a precipitating history of non-herpetic acute limbic encephalitis. Histologically, the resected specimen showed features of hippocampal sclerosis with granule cell dispersion. Characteristically, many balloon cells, immunoreactive for nestin, vimentin, glial fibrillary acidic protein (GFAP), GFAP-delta and CD34, were observed in the molecular and granule cell layers of the dentate gyrus. In the present case hippocampal sclerosis was an apparently acquired alteration, rather than a result of maldevelopment. The appearance of balloon cells raises questions regarding their origin and morphogenesis.

5-Lipoxygenase in mouse cerebellar Purkinje cells

It has been suggested that the enzymatic pathway of 5-lipoxygenase (5-LOX) influences brain functioning and pathobiology. The mRNAs for both the enzyme 5-LOX and its activating protein FLAP have been found in the cerebellum. In this work, we investigated the cellular expression of 5-LOX in the adult mouse cerebellar cortex. We used the in situ mRNA hybridization assay, immunocytochemistry, laser capture microdissection, and our previously developed method for assaying the DNA methylation status of a putative mouse 5-LOX promoter. Since both 5-LOX mRNA in situ hybridization signal and FLAP immunoreactivity co-localize with calbindin 28 kD immunoreactivity (a Purkinje cell marker) but not with S-100β immunoreactivity (a Bergmann glia marker), the suggestion is that the 5-LOX pathway is expressed in cerebellar Purkinje cells. We found that methylation in the sites targeted by methylation-sensitive restriction endonucleases AciI and HinP1I but not BstUI and HpaII was greater in DNA samples obtained from a high-5-LOX-expressing cerebellar region (Purkinje cells) versus a low-5-LOX-expressing region (the molecular cell layer), suggesting a possible epigenetic contribution to the cell-specific 5-LOX expression in the cerebellum. We propose that Purkinje cell-localized 5-LOX and FLAP expression may be involved in the cerebellar synthesis of leukotrienes and/or could influence the Dicer-mediated microRNA formation and processes of neuroplasticity.

Antineuronal autoantibodies in paraneoplastic cerebellar degeneration associated with adenocarcinoma of the prostate

Paraneoplastic neurological syndromes are unusual in prostatic cancer, and paraneoplastic cerebellar degeneration associated with adenocarcinoma of the prostate is rare. Here we report a 68 year old man who developed progressive ataxia in the setting of stage D2 adenocarcinoma of the prostate and whose MRI showed cerebellar atrophy. The patient's serum produced a previously undescribed pattern of immunoreactivity, binding to nuclei and cytoplasm of Purkinje cells, deep cerebellar neurons, scattered cells in the molecular and granule cell layers, and neuronal populations in thalamus, cerebral cortex, and hippocampus but not with liver or kidney. The patient's IgG also labeled a 65 kDa protein, discrete from Yo antigen, in Western blots of Purkinje cell lysates and did not react with blotted recombinant HuD, Ri, Yo, or amphiphysin proteins. Sera from neurologically normal patients with adenocarcinoma of the prostate did not contain this antibody, and the patient's serum did not react with normal prostate or with prostatic adenocarcinomas from other individuals. Prostatic adenocarcinoma may occasionally be accompanied by development of anticerebellar antibodies. Adenocarcinoma of the prostate should be considered as a possible underlying malignancy in older males with unexplained progressive cerebellar degeneration.

Cerebellar circuit activation through the mossy fiber – parallel fiber pathway using high-resolution VSD imaging in acute cerebellar slices

Event Abstract Back to Event Cerebellar circuit activation through the mossy fiber – parallel fiber pathway using high-resolution VSD imaging in acute cerebellar slices Daniela Gandolfi1, 2*, Jonathan Mapelli1, 2 and Egidio D‘Angelo1, 2 1 University of Pavia and CNISM, Department of Physiology, Italy 2 Istituto Neurologico IRCCS fondazione C. Mondino, Brain Connectivity Center, Italy It is not yet fully clear how mossy fiber inputs activate the granular layer retransmitting signals through the ascending axon and parallel fibers to Purkinje cells. We have characterized the spatio-temporal properties of cerebellar circuit activation in response to mossy fiber stimulation by using Voltage-Sensitive Dye (VSD) imaging in sagittal and coronal rat cerebellar slices (P20-P25). Fluorescent signals generated by Di4-ANNEPS were sampled at 1 KHz with a Micam-Ultima camera (SciMedia). The granular layer was activated in spots of about 30 micrometer diameter showing distinct intensities of response with delays of 2.1±0.15 ms (n=15). Then activation propagated into the molecular and Purkinje cell layers with an additional 3.6±1.1 ms delay (n=10). Simultaneous patch-clamp recordings from granule cells and Purkinje cells showed a direct correlation between intracellular depolarization and VSD signal. In sagittal slices, mossy fiber stimulation activated the overlaying area supporting vertical transmission, while in coronal slices activation also propagated laterally demonstrating spread of excitation along the parallel fibers. Transmission through the granular layer and to the molecular layer was more pronounced using high-frequency bursts rather than single isolated stimuli (+85.5%, n=8, from 0.1 Hz to 500 Hz), was markedly reduced by NMDA receptor blockers (e.g. -17.9%, n=4, at 200 Hz) and was enhanced by GABA-A receptor blockers (e.g. +36.9 %; n=4 at 200 Hz). VSD recordings reveal therefore (1) that the granular layer activates in spots depending on NMDA and GABA-A receptors, (2) that signals are transmitted toward the molecular layer depending on the frequency of the mossy fiber input and (3) that Purkinje cell excitation through the ascending granule cell axon coexist with that due to parallel fiber transmission. These observations provide the basis for a detailed investigation of spatio-temporal signal processing in the cerebellar circuit. Keywords: Cerebellum Conference: The Cerebellum: from neurons to higher control and cognition, Pavia, Italy, 8 Jul - 9 Jul, 2010. Presentation Type: Poster Presentation Topic: The Cerebellum: from neurons to higher control and cognition Citation: Gandolfi D, Mapelli J and D‘Angelo E (2010). Cerebellar circuit activation through the mossy fiber – parallel fiber pathway using high-resolution VSD imaging in acute cerebellar slices. Front. Neurosci. Conference Abstract: The Cerebellum: from neurons to higher control and cognition. doi: 10.3389/conf.fnins.2010.83.00005 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Aug 2010; Published Online: 29 Aug 2010. * Correspondence: Mr. Daniela Gandolfi, University of Pavia and CNISM, Department of Physiology, Pavia, Italy, daniela.gandolfi@unimore.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Daniela Gandolfi Jonathan Mapelli Egidio D‘Angelo Google Daniela Gandolfi Jonathan Mapelli Egidio D‘Angelo Google Scholar Daniela Gandolfi Jonathan Mapelli Egidio D‘Angelo PubMed Daniela Gandolfi Jonathan Mapelli Egidio D‘Angelo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919–922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95–PMCA2 and syntaxin-1A–PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.

Golgi apparatus localization of ZNT7 in the mouse cerebellum.

We have recently reported that four members of the zinc transporter (ZNT) family, ZNT1, ZNT3, ZNT4, and ZNT6, are abundantly expressed in the mouse cerebellum. In the present study, we reported that ZNT7 was present throughout the cerebellar cortex. ZNT7 immunoreactivity was predominately present in the somas and primary dendrites of the Purkinje cells. ZNT7 was also present in the Bergmann glial cell bodies as well as their radial processes, which extended into the molecular cell layer. Confocal immunofluorescence results demonstrated that the expression of ZNT7 overlapped with that of TGN38 in the somas of the Purkinje cells and granule cells. Immuno-electron microscopic study showed that ZNT7 was localized to the membrane of the Golgi apparatus in the somas of the Purkinje cells, Bergmann glial cells, and granule cells. Western blot analysis demonstrated that a considerable amount of ZNT7 was expressed in the cerebellum. These findings suggest a significant role of ZNT7 in zinc homeostasis in the mouse cerebellum.

Increased Reelin Promoter Methylation Is Associated With Granule Cell Dispersion in Human Temporal Lobe Epilepsy

Mesial temporal sclerosis (MTS) is the most common lesion in chronic, intractable temporal lobe epilepsies (TLE) and characterized by segmental neuronal cell loss in major hippocampal segments. Another histopathological hallmark includes granule cell dispersion (GCD), an architectural disturbance of the dentate gyrus encountered in approximately 50% of patients with mesial temporal sclerosis. Reelin, which plays a key role during hippocampal development and maintenance of laminar organization, is synthesized and released by Cajal-Retzius cells of the dentate molecular layer, and previous studies have shown that Reelin transcript levels are downregulated in human temporal lobe epilepsies specimens. To investigate whether epigenetic silencing by Reelin promoter methylation may be an underlying pathogenetic mechanism of GCD, DNA was harvested from 3 microdissected hippocampal subregions (i.e. molecular and granule cell layers of the dentate gyrus and presubiculum) from 8 MTS specimens with GCD, 5 TLE samples without GCD, and 3 autopsy controls. Promoter methylation was analyzed after bisulfite treatment, cloning, and direct sequencing; immunohistochemistry was performed to identify Cajal-Retzius cells. Reelin promoter methylation was found to be greater in TLE specimens than in controls; promoter methylation correlated with GCD among TLE specimens (p < 0.0002). No other clinical or histopathological parameter (i.e. sex, age, seizure duration, medication or extent, of MTS) correlated with promoter methylation. These data support a compromised Reelin-signaling pathway and identify promoter methylation as an epigenetic mechanism in the pathogenesis of TLE.

Lhermitte-Duclos disease with atypical vascularization – case report and review of the literature

A case of Lhermitte-Duclos disease (LDD, dysplastic gangliocytoma) with atypical vascularization is reported. LDD is a rare cerebellar mass lesion which may be associated with Cowden's syndrome and the PTEN germline mutation. A 61-year-old male presented 15 years before with a transient episode of unspecific gait disturbance. Initial magnetic resonance (MR) imaging revealed a right-sided, diffuse, nonenhancing cerebellar mass lesion. No definitive diagnosis was made at that time, and the symptoms resolved spontaneously. 15 years later, the patient presented with acute onset of vomiting associated with headache and ataxic gait. MR imaging showed a progression of the lesion with occlusive hydrocephalus. The lesion depicted a striated pattern characteristic for LDD with T1-hypointense and T2-hyperintense bands, nonenhancing with contrast. After resection of the mass lesion, the cerebellar and hydrocephalic symptoms improved rapidly. The pathological examination confirmed the diagnosis of dysplastic gangliocytoma (WHO Grade I) with enlarged granular and molecular cell layers, reactive gliosis and dysplastic blood vessels. No other clinical features associated with Cowden's syndrome were present. This case illustrates that LDD with atypical vascularization is a slow-growing posterior fossa mass lesion which may remain asymptomatic for many years. Timing of surgical treatment and extent of resection in patients with LDD is controversial. The typical features on standard T1-/T2-weighted MR imaging allow a diagnosis without surgery in most cases. The authors believe that the decision to treat in these cases should be based on clinical deterioration.

On the origin of the MR image phase contrast: An in vivo MR microscopy study of the rat brain at 14.1 T

Recent studies at high magnetic fields using the phase of gradient-echo MR images have shown the ability to unveil cortical substructure in the human brain. To investigate the contrast mechanisms in phase imaging, this study extends, for the first time, phase imaging to the rodent brain. Using a 14.1 T horizontal bore animal MRI scanner for in vivo micro-imaging, images with an in-plane resolution of 33 μm were acquired.Phase images revealed, often more clearly than the corresponding magnitude images, hippocampal fields, cortical layers (e.g. layer 4), cerebellar layers (molecular and granule cell layers) and small white matter structures present in the striatum and septal nucleus. The contrast of the phase images depended in part on the orientation of anatomical structures relative to the magnetic field, consistent with bulk susceptibility variations between tissues. This was found not only for vessels, but also for white matter structures, such as the anterior commissure, and cortical layers in the cerebellum.Such susceptibility changes could result from variable blood volume. However, when the deoxyhemoglobin content was reduced by increasing cerebral blood flow (CBF) with a carbogen breathing challenge, contrast between white and gray matter and cortical layers was not affected, suggesting that tissue cerebral blood volume (and therefore deoxyhemoglobin) is not a major source of the tissue phase contrast.We conclude that phase variations in gradient-echo images are likely due to susceptibility shifts of non-vascular origin.

Neurodegeneration and plastic changes in parahippocampal regions of the rat after kainic acid-induced epilepsy

The subiculum is the main output region of the hippocampus receiving input from the CA1 region and projecting to deep layers of the entorhinal cortex (EC), septum, mamillary nuclei, and the amygdala. It remains largely preserved in temporal lobe epilepsy (TLE) and therefore may be importantly involved in the generation of epileptic activity arising from the hippocampus. We characterized neurons of the subicular-EC complex and its projections in the kainic acid (KA) model for TLE using neuropeptides, calcium binding proteins, GAD-67 and the vesicular GABA and glutamate transporters as markers. We observed severe losses of interneurons and principal neurons in layer III of the EC and in the proximal subiculum accompanied by signs of reactive gliosis. The number of parvalbumin-ir interneurons was reduced in the subicular pyramidal cell layer and the deep layers of the EC. After 28 d the number of calretinin-ir neurons was reduced in EC layers III to VI, as was the diffuse calretinin-ir in the subiculum molecular layer and the EC, suggesting degeneration of respective calretinin containing projections. In all parahippocampal areas, increases in NPY mRNA in pyramidal cells and of NPY-ir axons were seen at the late intervals after KA. In the molecular layer and layer I of the EC, diffuse labeling for SOM-ir overlapped with that of NPY. The number of GAD67-ir interneurons was reduced in the inner molecular layer and pyramidal layer of the subiculum and in the EC at late timepoints, but also diffuse labeling for GAD67 and VGAT was enhanced in the molecular and pyramidal cell layers of the subiculum and in EC layer I. Our data indicate losses in neurons projecting from the EC to the subiculum. Similarly, projections from the subiculum to the deep layers of the EC seem to degenerate. Pyramidal neurons of the subiculum become immunoreactive for several neuropeptides. Inhibitory neurons projecting to distal dendrites of subicular principal cells sprout as judged by increased numbers of GAD-67, VGAT, SOM and NPY-ir fibers.

Productive Simian Virus 40 Infection of Neurons in Immunosuppressed Rhesus Monkeys

There currently is no animal model of JC virus-associated progressive multifocal leukoencephalopathy (PML). Reactivation of simian virus 40 (SV40) in immunosuppressed rhesus monkeys, however, rarely causes a PML-like illness. We sought to isolate a neurotropic clone of SV40 and determine its pathogenic potential in monkeys. The clone SV40CNS1 was amplified by polymerase chain reaction from the brain DNA of a simian/human immunodeficiency virus-infected monkey that had developed PML and meningoencephalitis. Compared with the SV40 prototype 776, SV40CNS1 had a small number of single-amino-acid mutations and caused a productive infection in monkey fibroblasts. It was inoculated into 2 SV40-negative, simian/human immunodeficiency virus-immunosuppressed monkeys. Both animals developed meningoencephalitis with productive SV40 infection of cerebral cortical neurons and glia in the superficial layers of the cortex and at the gray-white junction. Focal SV40-infected cells were also found in the cerebellar molecular and granule cell layers and white matter. Both animals also developed disseminated SV40 infection with nephritis and pneumonitis. Thus, SV40CNS1 is infectious and pathogenic in immunosuppressed monkeys, but it induces encephalitis with fulminant productive infection in cortical neurons and systemic disease, rather than PML. These findings shed new light on SV40 neurotropism and expand the host cell range of this virus.

Non-tau based neuronal degeneration in Alzheimer's disease — an immunocytochemical and quantitative study in the supragranular layers of the middle temporal neocortex

In Alzheimer's disease (AD), cortical neurons develop neurofibrillary tangles (NFTs) consisting of hyperphosphorylated tau. The neurons eventually die. There are some hints that cortical neurons may also degenerate without the development of cytoskeletal changes. We investigated this possibility by comparing changes in APP staining and neuronal size with respect to the presence or absence of hyperphosphorylated tau. Adjacent sections of the medial temporal neocortex (Brodmann's area 22) of 5 male AD patients aged 60–88 years (Braak V–VI) and 5 age-matched male non-demented control subjects were i) stained with a modified Bielschowsky silver method in order to reveal NFTs and ‘ghost’ tangles, ii) single-stained with anti-APP, and iii) double-labeled with anti-APP and AT8. Anti-APP is directed against the β-amyloid precursor protein and stains virtually all perikarya and proximal neurites of the cortical neurons. AT8 stains pre-tangles, NFTs and extracellular ‘ghost’ tangles due to the recognition of hyperphosphorylated tau. The study was focused on the supragranular cortical layers II–III, since these layers can be clearly delineated from the adjacent molecular and granular cell layers. The results showed that i) APP staining intensity in neurons was variable in the AD cortex, being clearly different from the invariably intense neuronal staining in all controls. Reduced cytoplasmic APP staining was observed, particular in small neurons, while lack of anti-APP staining in proximal neurites, too, was associated with AD. In addition, ii) cross-sectional area measurement on anti-APP-stained neurons revealed that in AD, as compared to controls, a clear decrease in the number of mainly large-sized neurons (> 150 µm 2) was accompanied by a significant increase in the percentage of neurons in the smaller size classes, indicating that many large-sized neurons became smaller in AD. iii) Reduced APP staining and decreased neuronal size were not necessarily associated with the presence or absence of hyperphosphorylated tau in these cells. iv) Twenty-six percent of the neurons contained hyperphosphorylated tau, while the level of NFT-related neuronal loss was low in AD. The present study suggests that non-tau based neuronal degeneration is a major phenomenon in the AD neocortex.

Synaptogenesis in the cerebellar cortex: Differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABA(A) receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABA(A) receptors and gephyrin co-aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABA(A) receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABA(A) receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells.

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

Conventional methods of preparing tissue specimens for morphological investigation of the central nervous system suffer from inevitable artifacts caused by anoxia during the processing. In the present study we performed ultrastructural analyses of mouse cerebellar cortex using the in vivo cryotechnique (IVCr), which minimizes ischemic artifacts of target organs through direct cryofixation in vivo. In molecular and Purkinje cell layers of the mouse cerebellum prepared with IVCr, considerably large extracellular spaces (ECS) were detected among cellular profiles and synaptic clefts. The ECS obtained with IVCr without ischemia were larger than those obtained with IVCr after 8-minute ischemia or a conventional quick-freezing method with fresh resected tissues (FQF), but did not decrease with IVCr after 30-second ischemia. By contrast, the parallel fibers observed with IVCr without ischemia were slightly smaller than those after 30-second ischemia, and significantly smaller than those prepared with IVCr after 8-minute ischemia or FQF. ECS were frequently preserved around synaptic clefts, although the rest were totally or partially enclosed with closely apposed glial processes. The estimated sizes of the ECS around synaptic clefts did not differ between the opened and enclosed synapses, suggesting that the opened synapses might be temporarily surrounded by glial sheaths dynamically extending or retracting throughout the perisynaptic ECS. These findings indicate IVCr to be useful for some morphological analyses of ECS in the central nervous system. The appreciable ECS around synapses would allow morphological and functional changes of neuronal and glial cells dynamically involved in synaptic remodeling or signal transduction.