Mapping the subcellular distribution of α-synuclein in neurons using genetically encoded probes for correlated light and electron microscopy: implications for Parkinson's disease pathogenesis.

Modifications to the gene encoding human α-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of α-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human α-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of α-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of α-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, α-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human α-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that α-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that α-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.

TxBR montage reconstruction for large field electron tomography.

Electron tomography (ET) has been proven an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. Large-field high-resolution views of biological specimens span more than four orders of magnitude in spatial scale, and, as a consequence, are rather difficult to generate directly. Various techniques have been developed towards generating those views, from increasing the sensor array size to implementing serial sectioning and montaging. Datasets and reconstructions obtained by the latter techniques generate multiple three-dimensional (3D) reconstructions, that need to be combined together to provide all the multiscale information. In this work, we show how to implement montages within TxBR, a tomographic reconstruction software package. This work involves some new application of mathematical concepts related to volume preserving transformations and issues of gauge ambiguity, which are essential problems arising from the nature of the observation in an electron microscope. The purpose of TxBR is to handle those issues as generally as possible in order to correct for most distortions in the 3D reconstructions and allow for a seamless recombination of ET montages.

Cocaine- and morphine-induced synaptic plasticity in the nucleus accumbens.

The critical brain areas and molecular mechanisms involved in drug abuse and dependence have been extensively studied. Drug-induced persistent behaviors such as sensitization, tolerance, or relapse, however, far outlast any previously reported mechanisms. A challenge in the field of addiction, therefore, has been to identify drug-induced changes in brain circuitry that may subserve long-lasting changes in behavior. This study examined behavioral changes and electron microscopic evidence of altered synaptic connectivity within the nucleus accumbens (NAc) following repeated administration of cocaine or morphine. The unbiased quantitative stereological physical disector method was used to estimate the number of synapses per neuron. Increases in the synapse-to-neuron ratio were found in the NAc shell of cocaine-treated (49.1%) and morphine-treated (55.1%) rats and in the NAc core of cocaine-treated animals (49.1%). This study provides direct ultrastructural evidence of drug-induced synaptic plasticity and identifies synaptic remodeling as a potential neural substrate underlying drug-induced behavioral sensitization.

Dopamine D5 receptor localization on cholinergic neurons of the rat forebrain and diencephalon: a potential neuroanatomical substrate involved in mediating dopaminergic influences on acetylcholine release.

The study of dopaminergic influences on acetylcholine release is especially useful for the understanding of a wide range of brain functions and neurological disorders, including schizophrenia, Parkinson's disease, Alzheimer's disease, and drug addiction. These disorders are characterized by a neurochemical imbalance of a variety of neurotransmitter systems, including the dopamine and acetylcholine systems. Dopamine modulates acetylcholine levels in the brain by binding to dopamine receptors located directly on cholinergic cells. The dopamine D5 receptor, a D1-class receptor subtype, potentiates acetylcholine release and has been investigated as a possible substrate underlying a variety of brain functions and clinical disorders. This receptor subtype, therefore, may prove to be a putative target for pharmacotherapeutic strategies and cognitive-behavioral treatments aimed at treating a variety of neurological disorders. The present study investigated whether cholinergic cells in the dopamine targeted areas of the cerebral cortex, striatum, basal forebrain, and diencephalon express the dopamine D5 receptor. These receptors were localized on cholinergic neurons with dual labeling immunoperoxidase or immunofluorescence procedures using antibodies directed against choline acetyltransferase (ChAT) and the dopamine D5 receptor. Results from this study support previous findings indicating that striatal cholinergic interneurons express the dopamine D5 receptor. In addition, cholinergic neurons in other critical brain areas also show dopamine D5 receptor expression. Dopamine D5 receptors were localized on the somata, dendrites, and axons of cholinergic cells in each of the brain areas examined. These findings support the functional importance of the dopamine D5 receptor in the modulation of acetylcholine release throughout the brain.

Localization of dopamine D2 receptors on cholinergic interneurons of the dorsal striatum and nucleus accumbens of the rat.

Striatal cholinergic interneurons located in the dorsal striatum and nucleus accumbens are amenable to influences of the dopaminergic mesolimbic pathway, which is a pathway involved in reward and reinforcement and targeted by several drugs of abuse. Dopamine and acetylcholine neurotransmission and their interactions are essential to striatal function, and disruptions to these systems lead to a variety of clinical disorders. Dopamine regulates acetylcholine release through dopamine receptors that are localized directly on striatal cholinergic interneurons. The dopamine D2 receptor, which attenuates acetylcholine release, has been implicated in drug relapse and is targeted by therapeutic drugs that are used to treat a variety of neurological disorders including Tourette Syndrome, Parkinson's disease and schizophrenia. The present study provides the first direct evidence for the localization of dopamine D2 receptors on striatal cholinergic interneurons of the rat brain using dual labeling immunocytochemistry procedures. Using light microscopy, dopamine D2 receptors were localized on the cell somata and dendritic and axonal processes of striatal cholinergic interneurons in the dorsal striatum and nucleus accumbens of the rat brain. These findings provide a foundation for understanding the specific roles that cholinergic neuronal network systems and interacting dopaminergic signaling pathways play in striatal function and in a variety of clinical disorders including drug abuse and addiction.

Three-dimensional reconstruction of serial mouse brain sections: solution for flattening high-resolution large-scale mosaics.

Recent advances in high-throughput technology facilitate massive data collection and sharing, enabling neuroscientists to explore the brain across a large range of spatial scales. One such form of high-throughput data collection is the construction of large-scale mosaic volumes using light microscopy (Chow et al., 2006; Price et al., 2006). With this technology, researchers can collect and analyze high-resolution digitized volumes of whole brain sections down to 0.2 µm. However, until recently, scientists lacked the tools to easily handle these large high-resolution datasets. Furthermore, artifacts resulting from specimen preparation limited volume reconstruction using this technique to only a single tissue section. In this paper, we carefully describe the steps we used to digitally reconstruct a series of consecutive mouse brain sections labeled with three stains, a stain for blood vessels (DiI), a nuclear stain (TO-PRO-3), and a myelin stain (FluoroMyelin). These stains label important neuroanatomical landmarks that are used for stacking the serial sections during reconstruction. In addition, we show that the use of two software applications, ir-Tweak and Mogrifier, in conjunction with a volume flattening procedure enable scientists to adeptly work with digitized volumes despite tears and thickness variations within tissue sections. These applications make processing large-scale brain mosaics more efficient. When used in combination with new database resources, these brain maps should allow researchers to extend the lifetime of their specimens indefinitely by preserving them in digital form, making them available for future analyses as our knowledge in the field of neuroscience continues to expand.

The atypical cadherin flamingo regulates synaptogenesis and helps prevent axonal and synaptic degeneration in Drosophila.

The formation of synaptic connections with target cells and maintenance of axons are highly regulated and crucial for neuronal function. The atypical cadherin and G-protein-coupled receptor Flamingo and its orthologs in amphibians and mammals have been shown to regulate cell polarity, dendritic and axonal growth, and neural tube closure. However, the role of Flamingo in synapse formation and function and in axonal health remains poorly understood. Here we show that fmi mutations cause a significant increase in the number of ectopic synapses on muscles and result in the formation of novel en passant synapses along axons, and unique presynaptic varicosities, including active zones, within axons. The fmi mutations also cause defective synaptic responses in a small subset of muscles, an age-dependent loss of muscle innervation and a drastic degeneration of axons in 3rd instar larvae without an apparent loss of neurons. Neuronal expression of Flamingo rescues all of these synaptic and axonal defects and larval lethality. Based on these observations, we propose that Flamingo is required in neurons for synaptic target selection, synaptogenesis, the survival of axons and synapses, and adult viability. These findings shed new light on a possible role for Flamingo in progressive neurodegenerative diseases.

Comparación de olor, cuerpo, sabor y sensación posterior a la ingesta que producen cinco ampicilinas y dos trimetoprim-sulfametoxazol en presentaciones líquidas con sabor distinto / Comparison of the smell, body, taste and sensibility produced by five oral ampicillins and two trimetoprim-sulfametoxazol liquid formulations with different taste

Una de las características para el cumplimiento adecuado del tratamiento con un antibiótico líquido en la edad pediátrica es que el gusto y la sensación posterior a la ingestión sean acetables al paladar del niño. Se comparan siete suspensiones de antimicrobianos (cinco ampicilinas y dos trimetoprim-sulfametoxazol) de uso común en pediatría en un estudio doble ciego con 50 médicos pediatras voluntarios a determinar diferencias en la aceptabilidad del olor, textura, gusto, sensación posterior a la ingestión y el total de los antibióticos orales. en el recuento total el mejor es Pentrexyl, con diferencias significativas (p< 0.05), seguidos de omnipen, Binotal y pembritin sin diferencias significativas entre ellos, seguido de Lampicin y al final se encuentran Septrin y Bactrim. En el gusto el mejor fue Pentrexyl con diferenias significativa al resto. Muchos clínicos pueden considerar lo resultados del presente estudio, para seleccionar una suspensión antimicrobiana y así obtener un mejor cumplimiento del tratamiento

Cefalotina amikacina vs. cefalotina isepamicina en el tratamiento inicial del paciente neutropénico febril / Cephalotine-amikacin vs. cephalotine isepamicine in the initial treatment of neutropenic patient febrile

Para evaluar la eficacia y seguridad de isepamicina y cefalotina vs. amikacina cefalotina en el tratamiento del paciente neutropénico febril, se realizó un estudio prospectivo longitudinal, aleatorizado, en donde se incluyeron 60 episodios de neutropenia y fiebre, en 58 pacientes menores de 18 años con diagnóstico de base hemato-oncológico, hospitalizados en el Instituto Nacional de Pediatría durante el periodo de abril de 1996 a febrero de 1997. Treinta y seis fueron asignados al grupo 1 (cefalotina + amikacina) y 24 en el grupo 2 (cefalotina isepamicina). No se encontraron diferencias estadísticamente significativas en relación con la edad, género, padecimiento hematooncológico de base y cuenta de neutrófilos absolutos al ingreso. Treinta y nueve pacientes (65 por ciento) tenían diagnóstico de leucemia aguda o linfoma, mientras que 21 niños (35 por ciento) tenían diagnóstico de tumor sólido. Se documentó clínicamente foco infeccioso aparente en 37 episodios (61 por ciento) y hubo documentación bacterilógica sólo en 2/60 (3.3 por ciento), el resto presentó fiebre de origen oscuro. La curación clínica fue similar en ambos grupos: 29/36 pacientes en el grupo 1 (80 por ciento) y 19/24 en el grupo 2 (79 por ciento) (Z=0.1311 y P 0.8966). Se modificó el tratamiento antimicrobiano en 7 pacientes del grupo 1 y cinco del grupo 2 por persistencia de la fiebre; dos pacientes fallecieron, uno en cada grupo, sin evidencia microbiológica de infección. En un solo paciente del grupo 1 se documentó en audiometría hipoacusia leve. Con esto podemos concluir que el tratamiento con isepamicina y cefalotina es una alternativa eficaz y segura en el tratamiento empírico inicial del paciente neutropénico febril

Complicaiones de la varicela en niños inmunocompetentes / Varicella complications in immunocompromised children

El objetivo de este estudio fue conocer la evolución y tipo de complicaciones de la varicela en niños inmunocompetentes del Hospital Infantil Privado. Se revisaron los expedientes de 100 pacientes con diagnóstico de egreso de varicela. Se analizó el tiempo de duración de la fase vesicular, la duración e intensidad de la fiebre, la biometría hemática, la presencia y tipo de complicaciones, así como su evolución. Se realizaron pruebas estadísticas de tendencia central y para la correlación de variables la prueba de chi cuadrada (p < 0.05). Ocho pacientes presentaron más de una complicación, 54 por ciento correspondieron al sexo masculino y 46 por ciento al femenino. La mediana de edad fue 65 meses, (límites 1 a 207 meses). Sólo se reportó una defunción (1 por ciento). Se concluye que la varicela puede condicionar complicaciones graves, independientemente del estado de inmunidad. Las complicaciones supurativas, infecciones de piel y tejidos blandos son más frecuentes que las complicaciones no supurativas. La linfopenia y/o neutropenia no influyen en la presencia de complicaciones ni en la severidad de las mismas en niños inmunocompetentes con varicela