Divergent Molecular Pathways for Toxicity of Selected Mutant C9ORF72-derived Dipeptide Repeats.

Expansion of a hexanucleotide repeat in a noncoding region of the C9ORF72 gene is responsible for a significant fraction of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) cases, but identifying specific toxic gene products and mechanisms has been difficult. Pathogenesis was proposed to involve the production of toxic RNA species and/or accumulation of toxic dipeptide repeats (DPRs), but distinguishing between these mechanisms has been challenging. In this study, we first use complementary model systems for analyzing pathogenesis in adult-onset neurodegenerative diseases to characterize the pathogenicity of DPRs produced by Repeat Associated Non-ATG (RAN) translation of C9ORF72 in specific cellular compartments: isolated axoplasm and giant synapse from the squid. Results showed selective axonal and presynaptic toxicity of GP-DPRs, independent of associated RNA. These effects involved downstream ASK1 signaling pathways that affect fast axonal transport and synaptic function, a pathogenic mechanism shared with other mutant proteins associated with familial ALS, like SOD1 and FUS. These pathways are sufficient to produce the "dying-back" axonopathy seen in ALS. However, other mutant genes (e.g., SOD1) that activate this mechanism rarely produce FTD. When parallel studies in primary motor neurons from rats were conducted, an additional pathogenic mechanism was revealed. The GR- and PR-DPRs, which had no effect on axonal transport or synaptic transmission, were found to disrupt the nuclei of transfected neurons, leading to "dying-forward" neuropathy. All C9-DRP-mediated toxic effects observed here are independent of whether the corresponding mRNAs contained hexanucleotide repeats or alternative codons. These studies establish the divergent toxicity of C9-DPRs that cause neurodegeneration in ALS and FTD, suggesting that these two independent pathogenic mechanisms may contribute to disease heterogeneity and/or synergize on disease progression in C9ORF72 patients with both ALS and FTD symptoms.

Sex Differences and Estrous Cycle Changes in Synaptic Plasticity-related microRNA in the Rat Medial Amygdala.

The posterodorsal medial amygdala (MePD) is a sex steroid-sensitive and sexually dimorphic subcortical area that dynamically modulates social behaviors in rats. As different microRNA (miRNA) can act as post-transcriptional regulators of synaptic processing, we addressed changes that occur in miRNA expression in the MePD of males and females along the estrous cycle. The expression of miR25-3p, miR132-3p, miR138-5p, miR181a-5p, miR195-5p, and miR199a-5p, involved in neuronal cytoskeleton remodeling and synaptic plasticity, were evaluated by RT-qPCR. We found that the expression of miR138-5p was higher in males than in females along the different phases of the estrous cycle. Males also showed higher levels of miR-181a when compared to females in diestrus and estrus. On the other hand, when compared to females in proestrus, males presented lower levels of miR132-3p and miR199a-5p. The expression of miR25-3p was higher in diestrus females than in proestrus females. In addition, diestrus females showed higher values of miR25-3p, miR181a-5p, and miR195-5p when compared to estrus females. These miRNA expression profiles indicate a variable and fine-tuned protein regulation in the adult MePD. It is likely that these miRNA can be involved in structural and functional synaptic features and plasticity characteristic of males and cycling females and for the MePD regulation of mammalian reproduction.

Lipofuscin Granules in the Epileptic Human Temporal Neocortex with Age.

Lipofuscin granules (LGs), the "age pigments", are autofluorescent cell products from lysosomes that diverge in number and size among brain regions. Human temporal cortex from 20- to 55-year-old epileptic subjects were studied with the fat soluble dye Sudan Black, under confocal and electron microscopy. Ultrastructural analysis showed that with age LGs increase in area, but not in number. Proportionally to the LGs area, the electron lucid portion increases and the electron dense reduces over time. The robust increase in lipid components is possibly due to modifications in the neuronal metabolism with age in physiological and pathological conditions.

The human medial amygdala: structure, diversity, and complexity of dendritic spines.

The medial nucleus of the amygdala (Me) is a component of the neural circuit for the interpretation of multimodal sensory stimuli and the elaboration of emotions and social behaviors in primates. We studied the presence, distribution, diverse shape, and connectivity of dendritic spines in the human Me of adult postmortem men. Data were obtained from the five types of multipolar neurons found in the Me using an adapted Golgi method and light microscopy, the carbocyanine DiI fluorescent dye and confocal microscopy, and transmission electron microscopy. Three-dimensional reconstruction of spines showed a continuum of shapes and sizes, with the spines either lying isolated or forming clusters. These dendritic spines were classified as stubby/wide, thin, mushroom-like, ramified or with an atypical morphology including intermediate shapes, double spines, and thorny excrescences. Pleomorphic spines were found from proximal to distal dendritic branches suggesting potential differences for synaptic processing, strength, and plasticity in the Me neurons. Furthermore, the human Me has large and thin spines with a gemmule appearance, spinules, and filopodium. The ultrastructural data showed dendritic spines forming monosynaptic or multisynaptic contacts at the spine head and neck, and with asymmetric or symmetric characteristics. Additional findings included en passant, reciprocal, and serial synapses in the Me. Complex long-necked thin spines were observed in this subcortical area. These new data reveal the diversity of the dendritic spines in the human Me likely involved with the integration and processing of local synaptic inputs and with functional implications in physiological and various neuropathological conditions.

Distribution of glutamate receptors in the posterodorsal medial amygdala of adult male rats.

The rat posterodorsal medial amygdala (MePD) has a remarkable neuronal plasticity and responds to olfactory/pheromonal stimuli to modulate emotional and reproductive behaviors. Glutamate is locally released by incoming sensorial pathways to establish and enforce synaptic inputs. Here, we combined DiI dye and immunolabeling procedure under confocal microscopy to describe the presence and distribution of glutamate receptors on neurons of the MePD of adult male rats. Western blot analysis interrogated binding specificity. Both AMPA (GluA1-4 subunits) and NMDA (GluN1 subunit) receptors were immunolabeled on cell bodies and along proximal and distal dendritic shafts. AMPA receptors were mainly observed on mushroom and stubby/wide spines, whereas NMDA receptors were found on thin spines. Colocalization of AMPA and NMDA receptors occurred in some spines. Filopodium did not show immunolabeled puncta on it. Our results are different from the distribution of glutamate receptors in the amygdaloid lateral nucleus, an upstream area involved with emotional processing, and suggest a region-specific excitatory transmission at proximal and distal dendritic branches. Altogether, these data provide new information for synaptic processing in the MePD likely related to the modulation of social behavior in rats.

Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline.

Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa(++) amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5-10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.

Inhibitory and multisynaptic spines, and hemispherical synaptic specialization in the posterodorsal medial amygdala of male and female rats.

The density of dendritic spines is sexually dimorphic and variable throughout the female estrous cycle in the rat posterodorsal medial amygdala (MePD), a relevant area for the modulation of reproductive behavior in rats. The local synaptic activity differs between hemispheres in prepubertal animals. Here we used serial section transmission electron microscopy to produce 3D reconstructions of dendritic shafts and spines to characterize synaptic contacts on MePD neurons of both hemispheres in adult males and in females along the estrous cycle. Pleomorphic spines and nonsynaptic filopodia occur in the MePD. On average, 8.6% of dendritic spines received inputs from symmetric gamma-aminobutyric acid (GABA)-immunoreactive terminals, whereas 3.6% received two synaptic contacts on the spine head, neck, or base. Presynaptic terminals in female right MePD had a higher density of synaptic vesicles and docked vesicles than the left MePD, suggesting a higher rate of synaptic vesicle release in the right MePD of female rats. In contrast, males did not show laterality in any of those parameters. The proportion of putative inhibitory synapses on dendritic shafts in the right MePD of females in proestrus was higher than in the left MePD, and higher than in the right MePD in males, or in females in diestrus or estrus. This work shows synaptic laterality depending on sex and estrous cycle phase in mature MePD neurons. Most likely, sexual hormone effects are lateralized in this brain region, leading to higher synaptic activity in the right than in the left hemisphere of females, mediating timely neuroendocrine and social/reproductive behavior.

Cellular components of the human medial amygdaloid nucleus.

The medial nucleus (Me) is a superficial component of the amygdaloid complex. Here we assessed the density and morphology of the neurons and glial cells, the glial fibrillary acidic protein (GFAP) immunoreactivity, and the ultrastructure of the synaptic sites in the human Me. The optical fractionator method was applied. The Me presented an estimated mean neuronal density of 1.53 × 105 neurons/mm³ (greater in the left hemisphere), more glia (72% of all cells) than neurons, and a nonneuronal:neuronal ratio of 2.7. Golgi-impregnated neurons had round or ovoid, fusiform, angular, and polygonal cell bodies (10-30 µm in diameter). The length of the dendrites varied, and pleomorphic spines were found in sparsely spiny or densely spiny cells (1.5-5.2 spines/dendritic µm). The axons in the Me neuropil were fine or coarsely beaded, and fibers showed simple or notably complex collateral terminations. The protoplasmic astrocytes were either isolated or formed small clusters and showed GFAP-immunoreactive cell bodies and multiple branches. Furthermore, we identified both asymmetrical (with various small, clear, round, electron-lucent vesicles and, occasionally, large, dense-core vesicles) and symmetrical (with small, flattened vesicles) axodendritic contacts, also including multisynaptic spines. The astrocytes surround and may compose tripartite or tetrapartite synapses, the latter including the extracellular matrix between the pre- and the postsynaptic elements. Interestingly, the terminal axons exhibited a glomerular-like structure with various asymmetrical contacts. These new morphological data on the cellular population and synaptic complexity of the human Me can contribute to our knowledge of its role in health and pathological conditions.

Dendritic spines of the medial amygdala: plasticity, density, shape, and subcellular modulation by sex steroids.

The medial nucleus of the amygdala (MeA) is a complex component of the "extended amygdala" in rats. Its posterodorsal subnucleus (MePD) has a remarkable expression of gonadal hormone receptors, is sexually dimorphic or affected by sex steroids, and modulates various social behaviors. Dendritic spines show remarkable changes relevant for synaptic strength and plasticity. Adult males have more spines than females, the density of dendritic spines changes in the course of hours to a few days and is lower in proestrous and estrous phases of the ovarian cycle, or is affected by both sex steroid withdrawal and hormonal replacement therapy in the MePD. Males also have more thin spines than mushroom-like or stubby/wide ones. The presence of dendritic fillopodia and axonal protrusions in the MePD neuropil of adult animals reinforces the evidence for local plasticity. Estrogen affects synaptic and cellular growth and neuroprotection in the MeA by regulating the activity of the cyclic AMP response element-binding protein (CREB)-related gene products, brain-derived neurotrophic factor (BDNF), the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and the activity-regulated cytoskeleton-related protein (Arc). These effects on signal transduction cascades can also lead to local protein synthesis and/or rearrangement of the cytoskeleton and subsequent numerical/morphological alterations in dendritic spines. Various working hypotheses are raised from these experimental data and reveal the MePD as a relevant region to study the effects of sex steroids in the rat brain.

Blocking Effects of Human Tau on Squid Giant Synapse Transmission and Its Prevention by T-817 MA.

Filamentous tau inclusions are hallmarks of Alzheimer's disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the squid giant synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.

O ciclo da vesícula sináptica, espinhos dendríticos e a transdução de sinal / Synaptic vesicle cycle, dendritic spines and signal transduction

No sistema nervoso, a sinapse é a estrutura que permite a um neurônio passar um sinal elétrico ou químico a outro neurônio ou outra célula (muscular ou glandular). A palavra sinapse vem de "synaptein", palavra que Sir Charles Scott Sherrington e seus colegas acunharam do grego "syn" (junto) e "haptein"(afivelar). As sinapses podem ser separadas entre elétricas e químicas, porém a maior parte da transmissão sináptica é realizada através das sinapses químicas. Apesar das sinapses químicas terem uma resposta mais lenta que as elétricas, elas possuem a vantagem da amplificação do sinal gerada através de uma cascata de segundos mensageiros. As sinapses químicas podem ser excitatórias ou inibitórias e são caracterizadas por um terminal pré-sináptico (onde estão presentes as vesículas que contêm os neurotransmissores) em contato com um terminal pós-sináptico (onde estão presentes os receptores ionotrópicos e metabotrópicos para esses neurotransmissores) separados pela fenda sináptica. As sinapses típicas acontecem sobre axônios (axo-axônicas), sobre dendritos (axo-dendríticas), sobre o soma de outro neurônio (axo-somáticas) e sobre os espinhos dendríticos...

In the nervous system, the synapse is the structure that allows a neuron pass an electrical or chemical signal to another neuron or another cell (muscle or glandular). The word synapse comes from "synaptein" that Sir Charles Scott Sherrington and his colleagues minted from the Greek "syn" (together) and "haptein"(buckling). Most part of the synaptic transmission is performed through chemical synapses. Chemical synapses have a slower response than the electric ones; they have the advantage of amplifying the signal generated through a cascade of second messengers. Chemical synapses can be excitatory or inhibitory and are characterized by a presynaptic terminal (where there are vesicles that contain the neurotransmitters) in contact with a postsynaptic terminal (where there are the ionotropic and metabotropic receptors) separated by the synaptic cleft. Synapses can occur on axons (axo-axonal), on dendrites (axodendritic), on soma (axo-somatic) and on dendritic spines...

Neurotransmissão glutamatérgica e plasticidade sináptica: aspectos moleculares, clínicos e filogenéticos / Glutamatergic neurotransmission and synaptic plasticity: molecular, clinical, and phylogenetic aspects

A comunicação entre neurônios é passível de constantes modificações, até mesmo no encéfalo adulto. Esta capacidade de circuitos neuronais fortalecerem ou enfraquecerem suas interações sinápticas específicas (fenômeno conhecido como plasticidade sináptica) pode ocorrer de acordo com as diferentes demandas ambientais, o que favorece a noção de que alterações dinâmicas na comunicação entre neurônios estão na base da flexibilidade comportamental (i.e., processos de aprendizagem e memória). Nas últimas décadas, o avanço das neurociências tem permitido uma melhor compreensão a respeito da plasticidade sináptica, especialmente a plasticidade de sinapses glutamatérgicas, cujos processos moleculares de modificação sináptica parecem estar entre os mais comuns de todo o sistema desse progresso na ciência básica tem contribuído para uma melhor compreensão acerca dos processos patológicos envolvendo as sinapses glutamatérgicas, como a doença de Alzheimer. Além disso, a crescente compreensão sobre o funcionamento da comunicação glutamatérgica tem ajudado a esclarecer como as sinapses, em geral, teriam se originado e evoluído na escala filogenética do reino animal (Metazoa)...

Communication between neurons is subject to constant changes, even in the adult brain. This ability of neural circuits to strengthen or weaken their specific synaptic interactions (a phenomenon known assynaptic plasticity) may occur according to different environmental demands, which favors the idea that dynamic changes in the communication between neurons underlie behavioral flexibility (i.e., learning and memory processes). In recent decades, advances in neuroscience has allowed a better understanding of synaptic plasticity, specially the plasticity of glutamatergic synapses, whose molecular processes of synaptic change appear to be among the most common throughout the central nervous system.Much of this progress in basic science has contributed to a better understanding of pathological processes involving the glutamatergic synapses, such as Alzheimer's disease. Furthermore, the growing understanding about the physiology of glutamatergic communication has helped explain how synapses, in general, would have originated and evolved in the phylogenetic scale of the Metazoa...

Glomerular nucleus of the weakly electric fish, Gymnotus sp.: cytoarchitecture, histochemistry, and fiber connections--inisights from neuroanatomy to evolution and behavior.

The present study provides a detailed description of morphological and hodological aspects of the glomerular nucleus in the weakly electric fish Gymnotus sp., and explores the evolutionary and functional implications flowing from this analysis. The glomerular nucleus of Gymnotus shows numerous morphological similarities with the glomerular nucleus of percomorph fish, although cytoarchitectonically simpler. In addition, congruence of the histochemical acetylcholinesterase (AChE) distribution with cytoarchitectonic data suggests that the glomerular nucleus, together with the ventromedial cell group of the medial subdivision of the preglomerular complex (PGm-vmc) rostrally, and the subglomerular nucleus (as identified by Maler et al. [1991] J Chem Neuroanat 4:1­38) caudally, may form a distinct longitudinally organized glomerular complex. Our results show that an important source of sensory afferents to the glomerular nucleus originates in the pretectal and electrosensorius nuclei. The glomerular nucleus in turn projects to the hypothalamus (inferior lobe and anterior hypothalamus), to the anterior tuberal nucleus, and to the medial region of the preglomerular nucleus (PGm). These data suggest that visual and electrosensory information reach the glomerular nucleus and are relayed to the hypothalamus and, via PGm, to the pallium. Such connections are similar to those of the glomerular nucleus in percomorphs and the posterior pretectal nucleus in osteoglossomorph, esocids, and salmonids, where they comprise one component of a visual processing pathway. In Gymnotiform fish, however, the pretectal region that projects to the glomerular nucleus is dominated by electrosensory input (visual input is minor), which is consistent with the dominant role of electroreception in these fish.

Descriptive findings on the morphology of dendritic spines in the rat medial amygdala.

The rat posterodorsal medial amygdala (MePD) is a brain area in which gonadal hormones induce notable plastic effects in the density of dendritic spines. Dendritic spines are post-synaptic specializations whose shape and spacing change neuronal excitability. Our aim was to obtain new data on the dendritic spines morphology and density from MePD neurons using the carbocyanine dye DiI under confocal microscopy. In adult male rats, the dendritic spine density of the medial branches of the left MePD (mean+/-SD) was 1.15+/-0.67spines/dendritic microm. From the total sampled, approximately 53% of the spines were classified as thin, 22.5% as "mushroom-like", and 21.5% as stubby/wide. Other spine shapes (3%) included those ramified, with a filopodium-like or a gemule appearance, and others with a protruding spinule. Additional experiment joining DiI and synaptophysin (a pre-synaptic protein) labeling suggested synaptic sites on dendritic shafts and spines. Dendritic spines showed synaptophysin puncta close to their head and neck, although some spines had no evident labeled puncta on them or, conversely, multiple puncta appeared upon one spine. These results advance previous light microscopy results by revealing features and complexities of the dendritic spines at the same time that give new insight on the possible synaptic organization of the adult rat MePD.

The "single-section" Golgi method adapted for formalin-fixed human brain and light microscopy.

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The "single-section" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, "sandwiched" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the "single-section" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results.

Increased sarcolemmal permeability as an early event in experimental septic cardiomyopathy: a potential role for oxidative damage to lipids and proteins.

This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.

Decreased number of caveolae in endothelial cells impairs the relaxation induced by acetylcholine in hypertensive rat aortas.

The present study was designed to investigate the contribution of endothelial cell caveolae to vascular relaxation in aortas from a normotensive (2K) and renal hypertensive (2K-1C) rat. For that purpose, concentration-effect curves to acetylcholine were constructed in 2K and 2K-1C intact endothelium aortic rings, in the absence or in the presence of the caveolae disassembler methyl-beta-ciclodextrin. The potency (pD(2)) and the maximum relaxant effect to acetylcholine were greater in 2K than in 2K-1C aortas. Methyl-beta-ciclodextrin reduced the pD(2) in 2K and the maximum relaxant effect in both 2K and 2K-1C. The quantification of the caveolae number by electronic microscopy has shown a larger number of caveolae in 2K than in 2K-1C endothelial cells, which was reduced by methyl-beta-ciclodextrin in both 2K and 2K-1C. The production of NO stimulated with acetylcholine was greater in 2K than in 2K-1C endothelial cells, and this effect was impaired by methyl-beta-ciclodextrin in both 2K and 2K-1C. The cytosolic Ca(2+) concentration ([Ca(2+)]c) was simultaneously measured in endothelial and smooth muscle cells stimulated with acetylcholine by confocal image of aortic slices. Acetylcholine produced a greater [Ca(2+)]c increase in 2K than in 2K-1C endothelial cells, which response was inhibited by methyl-beta-ciclodextrin only in 2K cells. In smooth muscle cells the reduction of [Ca(2+)]c was higher in 2K than in 2K-1C. This effect was inhibited by methyl-beta-ciclodextrin only in 2K cells. Taken together, our results suggest that the decreased number of caveolae in the endothelial cells from 2K-1C rat aortas is involved in the impaired effect of acetylcholine on [Ca(2+)]c and NO.

Synaptic transmission block by presynaptic injection of oligomeric amyloid beta.

Early Alzheimer's disease (AD) pathophysiology is characterized by synaptic changes induced by degradation products of amyloid precursor protein (APP). The exact mechanisms of such modulation are unknown. Here, we report that nanomolar concentrations of intraaxonal oligomeric (o)Abeta42, but not oAbeta40 or extracellular oAbeta42, acutely inhibited synaptic transmission at the squid giant synapse. Further characterization of this phenotype demonstrated that presynaptic calcium currents were unaffected. However, electron microscopy experiments revealed diminished docked synaptic vesicles in oAbeta42-microinjected terminals, without affecting clathrin-coated vesicles. The molecular events of this modulation involved casein kinase 2 and the synaptic vesicle rapid endocytosis pathway. These findings open the possibility of a new therapeutic target aimed at ameliorating synaptic dysfunction in AD.

Role of Rab27 in synaptic transmission at the squid giant synapse.

Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

Hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hantavirus pulmonary syndrome.

Despite clinical evidence of myocardial dysfunction, there is no pathological evidence of myocardial injury in hantavirus pulmonary syndrome (HPS). The dominant opinion is that the primary cardiac lesion is functional rather than structural. The present study describes hantaviral antigen and particles in the cardiac endothelium and interstitial macrophages in association with a typical myocarditis in HPS. Human hearts from 14 individuals who died of HPS were compared with hearts from 14 individuals who died of acute necrotizing pancreatitis associated with acute lung injury and 4 individuals who died accidental deaths without thoracic injury (as controls); all cases were selected from autopsies. Transmural blocks of myocardial tissue were excised from the middle portion of the left-ventricular free wall and fixed in formalin. Small samples of myocardial tissue from 4 HPS cases and 4 non-HPS controls were fixed in glutaraldehyde for electron microscopic study. Histomorphometric, immunohistochemical, and ultrastructural methods were employed to detect the presence of hantavirus in the myocardium and to evaluate interstitial edema and the minor diameter of myocytes, to characterize the immunophenotype, and to estimate the number of inflammatory cells and in situ cytokine-producing cells and the T helper cell subset 1 and 2 immune responses (tumor necrosis factor [TNF]-alpha, interferon-gamma, interleukin [IL]-10, and IL-4). Cardiac remodeling; hantaviral antigen and particles in the endothelium and macrophages; scattered foci of myofiber necrosis; greater interstitial cellular infiltration, mainly composed of macrophages and memory T lymphocytes and a significant number of T helper and B lymphocytes; and TNF-alpha protein expression in macrophage-type cells and cardiomyocytes were observed to a greater extent in HPS myocardium than in normal and acute pancreatitis control myocardium. These findings give support to the opinion that structural changes could be responsible for myocardial depression and shock in HPS, and it should be properly named as "hantavirus cardiopulmonary syndrome" (HCPS).