Immunohistochemical distribution of calcium binding proteins in the cochlear nuclear complex of circling mice / Distribución inmunohistoquímica de proteínas de unión a calcio en el complejo nuclear coclear de ratones circulantes

SUMMARY: The term "circling mouse" refers to an animal model of deafness, in which the mouse exhibits circling, head tossing, and hyperactivity, with pathological features including degenerated spiral ganglion cells in the cochlea, and the loss of the organ of Corti. The cochlear nuclear (CN) complex, a part of the auditory brain circuit, is essential to process both ascending and descending auditory information. Considering calcium's (Ca2+) importance in homeostasis of numerous biological processes, hearing loss by cochlear damage, either by ablation or genetic defect, could cause changes in the Ca2+ concentration that might trigger functional and structural alterations in the auditory circuit. However, little is known about the correlation of the central nervous system (CNS) pathology in circling mice, especially of the auditory pathway circuit and Ca2+ changes. This present study investigates the distribution of Ca2+- binding proteins (CaBPs), calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) by using a free floating immunohistochemical method inthe CN of the wild-type mouse (+/+), the heterozygous mouse (+/cir), and the homozygous (cir/cir) mouse. CaBPs are well known to be an important factor that regulates Ca2+ concentrations. Compared with the dorsal and ventral cochlear nuclei of +/+ and +/ cirmice, prominent decreases of CaBPs' immunoreactivity (IR) in cir/cirmice were observed in the somas, as well as in the neuropil. The present study reportson the overall distribution and changes in the immunoreactivity of CaBPs in the CN of cir/cirmice because ofa hearing defect. This data might be helpful to morphologically elucidate CNS disorders and their relation to CaBPs immunoreactivity related to hearing defects.

RESUMEN: El término "ratón circulante" se refiere a un modelo animal con sordera, en el que el ratón exhibe hiperactividad, movimientos circulares y movimientos de la cabeza, con características patológicas que incluyen células ganglionares espirales degeneradas en la cóclea, un canal de Rosenthal vacío y la pérdida del órgano de Corti. El complejo nuclear coclear (CN), una parte del circuito cerebral auditivo, es esencial para procesar la información auditiva tanto ascendente como descendente. Considerando la importancia del calcio (Ca2+) en la homeostasis de numerosos procesos biológicos, la hipoacusia por daño coclear, por ablación o por defecto genético, podría provocar cambios en la concentración de Ca2+que pueden desencadenar alteraciones funcionales y estructurales en el circuitoauditivo. Sin embargo, existe poca información de la correlación de la patología del sistema nervioso central (SNC) en ratones circulantes, especialmente del circuito de la víaauditiva y los cambios de Ca2+. Este estudio nvestiga la distribución de proteínas de unión a Ca2+ (CaBP), calbindina D-28k (CB), parvalbúmina (PV) y calretinina (CR) mediante el uso de un método inmunohistoquímico de flotaciónlibre en el CN del ratón de tiposalvaje (+/+), el ratón heterocigoto (+/cir) y el ratón homocigoto (cir/cir). Se sabe que los CaBP son un factor importante que regula las concentraciones de Ca2+. En comparación con los núcleos cocleares dorsal y ventral de los ratones +/+ y +/ cir, se observaron disminuciones prominentes de la inmunorreactividad (IR) de CaBPs en los ratonescir/cir en los somas, asícomo en el neuropilo. El presente estudio informa sobre la distribución general y los cambios en la inmunorreactividad de CaBP en el CN de ratones cir/cir debido a un defecto auditivo. Estos datos podrían ser útiles para dilucidar morfológicamente los trastornos del SNC y su relación con la inmunorreactividad de CaBP relacionada con los defectosauditivos.

Isolation and culture of porcine primary fetal progenitors and neurons from the developing dorsal telencephalon

The development of long-term surviving fetal cell cultures from primary cell tissue from the developing brain is important for facilitating studies investigating neural development and for modelling neural disorders and brain congenital defects. The field faces current challenges in co-culturing both progenitors and neurons long-term. Here, we culture for the first time, porcine fetal cells from the dorsal telencephalon at embryonic day (E) 50 and E60 in conditions that promoted both the survival of progenitor cells and young neurons. We applied a novel protocol designed to collect, isolate and promote survival of both progenitors and young neurons. Herein, we used a combination of low amount of fetal bovine serum, together with pro-survival factors, including basic fibroblast growth factor and retinoic acid, together with arabinofuranosylcytosine and could maintain progenitors and facilitate in vitro differentiation into calbindin 1+ neurons and reelin+ interneurons for a period of 7 days. Further improvements to the protocol that might extend the survival of the fetal primary neural cells would be beneficial. The development of new porcine fetal culture methods is of value for the field, given the pig's neuroanatomical and developmental similarities to the human brain.

Entry of rabies virus in the olfactory bulb of mice and effect of infection on cell markers of neurons and astrocytes / Ingreso del virus de la rabia en el bulbo olfatorio de ratones y efecto de la infección en marcadores celulares de neuronas y astrocitos

There are few studies of infection by rabies virus in the olfactory bulb (OB). This work was carried out with the purpose of establishing the time required to detect rabies antigens in the OB of mouse, after the intramuscular inoculation of the virus and to evaluate the effect of the infection on the expression of three proteins: calbindin (CB), parvalbumin (PV) and the glial fibrillary acidic protein (GFAP). Mice were inoculated with rabies virus intramuscularly in the hind limbs. Every 8 hours, after 72 hours postinoculation (p.i.), animals were sacrificed by perfusion with paraformaldehyde and coronal sections of OB were obtained for immunohistochemical study. These cuts were used to reveal the entry and spread of viral antigens. Tissue sections obtained in the terminal phase of the disease (144 hours p.i.), and controls of the same age were also processed for immunohistochemistry of CB, PV and GFAP. Rabies virus antigens were initially detected at 80 hours p.i. in a few mitral cells. At 88 hours p.i. the antigens had spread through most of these neurons but until the terminal phase of the disease there was little dispersion of the virus towards other cellular layers of the OB. The CB protein was expressed in cells of the glomerular stratum, the PV in cells of the outer plexiform layer and the GFAP was expressed in all the layers of the OB. Viral infection generated loss of CB expression and increase of PV expression. Immunoreactivity to GFAP was increased in the outer plexiform layer of the OB as a response to infection.

Son escasos los estudios de la infección por virus de la rabia en el bulbo olfatorio (OB). Este trabajo se realizó con el objetivo de establecer el tiempo requerido para detectar antígenos de rabia en el OB del ratón, luego de la inoculación intramuscular del virus y evaluar el efecto de la infección en la expresión de tres proteínas: calbindina (CB), parvoalbúmina (PV) y la proteína ácida fibrilar glial (GFAP). Los ratones fueron inoculados con virus de la rabia por vía intramuscular en las extremidades posteriores. Cada 8 horas, después de 72 horas de inoculación (p.i.), los animales se sacrificaron por perfusión con paraformaldehído y se obtuvieron secciones coronales de OB para el estudio inmunohistoquímico. Estos cortes se usaron para revelar la entrada y propagación de antígenos virales. Las secciones de tejido obtenidas en la fase terminal de la enfermedad (144 horas p.i.), y los controles de la misma edad también se procesaron para inmunohistoquímica de CB, PV y GFAP. Los antígenos del virus de la rabia se detectaron inicialmente a las 80 horas p.i. en unas pocas células mitrales. A las 88 horas p.i. los antígenos se habían diseminado a través de la mayoría de estas neuronas, pero hasta la fase terminal de la enfermedad había poca dispersión del virus hacia otras capas celulares del OB. La proteína CB se expresó en las células del estrato glomerular, la PV en células de la capa plexiforme externa y la GFAP se expresó en todas las capas del OB. La infección viral generó pérdida de expresión de CB y aumento en la expresión de PV. La inmunorreactividad a GFAP aumentó en la capa plexiforme externa del OB como respuesta a la infección.

GFAP-Positive Progenitor Cell Production is Concentrated in Specific Encephalic Regions in Young Adult Mice / 神经科学通报·英文版

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP cells mainly generated NeuN neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP cells at the young adult stage is significantly different from that at the adult stage.

Treadmill Exercise Improves Motor Function by Suppressing Purkinje Cell Loss in Parkinson Disease Rats / 대한배뇨장애요실금학회지

PURPOSE: Rotenone is the most widely used neurotoxin for the making Parkinson disease (PD) animal model. The neurodegenerative disorder PD shows symptoms, such as slowness of movements, tremor at resting, rigidity, disturbance of gait, and instability of posture. We investigated whether treadmill running improves motor ability using rotenone-caused PD rats. The effect of treadmill running on PD was also assessed in relation with apoptosis of cerebellar Purkinje cells. METHODS: Treadmill running was applied to the rats in the exercise groups for 30 minutes once a day for 4 weeks, starting 4 weeks after birth. We used rota-rod test for the determination of motor coordination and balance. In this experiment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, immunohistochemistry for calbindin, glial fibrillary acidic protein (GFAP), Iba-1, and western blot analysis for Bax and Bcl-2 were performed. RESULTS: Treadmill running enhanced motor balance and coordination by preventing the loss of Purkinje cells in the cerebellar vermis. Treadmill running suppressed PD-induced expression of GFAP-positive reactive astrocytes and Iba-1-positive microglia, showing that treadmill running suppressed reactive astrogliosis and microglia activation. Treadmill running suppressed TUNEL-positive cell number and Bax expression and enhanced Bcl-2 expression, demonstrating that treadmill running inhibited the progress of apoptosis in the cerebellum of rotenone-induced PD rats. CONCLUSIONS: Treadmill running improved motor ability of the rotenone-induced PD rats by inhibiting apoptosis in the cerebellum. Apoptosis suppressing effect of treadmill running on rotenone-induced PD was achieved via suppression of reactive astrocyte and inhibition of microglial activation.

Differential Expression of Ca²⁺-buffering Protein Calretinin in Cochlear Afferent Fibers: A Possible Link to Vulnerability to Traumatic Noise

The synaptic contacts of cochlear afferent fibers (CAFs) with inner hair cells (IHCs) are spatially segregated according to their firing properties. CAFs also exhibit spatially segregated vulnerabilities to noise. The CAF fibers contacting the modiolar side of IHCs tend to be more vulnerable. Noise vulnerability is thought to be due to the absence of neuroprotective mechanisms in the modiolar side contacting CAFs. In this study, we investigated whether the expression of neuroprotective Ca²⁺-buffering proteins is spatially segregated in CAFs. The expression patterns of calretinin, parvalbumin, and calbindin were examined in rat CAFs using immunolabeling. Calretinin-rich fibers, which made up ~50% of the neurofilament (NF)-positive fibers, took the pillar side course and contacted all IHC sides. NF-positive and calretinin-poor fibers took the modiolar side pathway and contacted the modiolar side of IHCs. Both fiber categories juxtaposed the C-terminal binding protein 2 (CtBP2) puncta and were contacted by synaptophysin puncta. These results indicated that the calretinin-poor fibers, like the calretinin-rich ones, were afferent fibers and probably formed functional efferent synapses. However, the other Ca²⁺-buffering proteins did not exhibit CAF subgroup specificity. Most CAFs near IHCs were parvalbumin-positive. Only the pillar-side half of parvalbumin-positive fibers coexpressed calretinin. Calbindin was not detected in any nerve fibers near IHCs. Taken together, of the Ca²⁺-buffering proteins examined, only calretinin exhibited spatial segregation at IHC-CAF synapses. The absence of calretinin in modiolar-side CAFs might be related to the noise vulnerability of the fibers.

Astrocyte Specificity and Coverage of hGFAP-CreERT2 Tg(GFAP-Cre/ERT2)13Kdmc Mouse Line in Various Brain Regions

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.

The expression of Calbindin and Parvalbumin in auditory pathway of kit gene mutated C57BL/6J mouse / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the expressions of Calbindin(CB) and Parvalbumin (PV), the two calcium-binding protein, in auditory pathway in mice of wild type C57BL/6J and kit⁺/kitW⁻ ²Bao, a kit gene mutant.</p><p><b>METHODS</b>Six mutated kit gene kit⁺/kitW⁻ ²Bao mice and 6 wild type C57BL/6J (B6) mice were anaesthetized i. p. with chloral hydrate. After the mice were fixed by heart perfusion, the brains were removed and coronal sections were cut with a freezing microtome.</p><p><b>RESULTS</b>We found that wild type mice had significant expressions of PV on ventral cochlear nucleus, anterior part (AVCN), ventral cochlear nucleus, posterior part (PVCN), inferior colliculus (IC) and auditory cortex (AC). CB was expressed in wild type mice on PVCN and nucleus of the trapezoid body (Tz). The mutant of kit gene induced the less expression of PV on PVCN, IC and AC (P < 0.01), but increased the expression of Tz (P < 0.01). CB could not be observed on PVCN in mutant mice, and the expression of AC was increased( P < 0.01).</p><p><b>CONCLUSION</b>CB and PV has differential expression level in auditory pathway. Since mutated kit gene can affect expression of PV on PVCN, IC, Tz and AC, as well as CB on PVCN and AC, it suggests that the mutation of kit gene can affect the advanced function of central nervous system in auditory pathway.</p>

Human prefrontal layer II interneurons in areas 46, 10 and 24 / Interneuronas de la lámina II en las áreas prefrontales 46, 10 y 24 del humano

Background: Prefrontal cortex (PFC) represents the highest level of integration and control of psychic and behavioral states. Several dysfunctions such as autism, hyperactivity disorders, depression, and schizophrenia have been related with alterations in the prefrontal cortex (PFC). Among the cortical layers of the PFC, layer II shows a particular vertical pattern of organization, the highest cell density and the biggest non-pyramidal/pyramidal neuronal ratio. We currently characterized the layer II cytoarchitecture in human areas 10, 24, and 46. Objective: We focused particularly on the inhibitory neurons taking into account that these cells are involved in sustained firing (SF) after stimuli disappearance. Methods: Postmortem samples from five subjects who died by causes different to central nervous system diseases were studied. Immunohistochemistry for the neuronal markers, NeuN, parvalbumin (PV), calbindin (CB), and calretinin (CR) were used. NeuN targeted the total neuronal population while the rest of the markers specifically the interneurons. Results: Cell density and soma size were statically different between areas 10, 46, 24 when using NeuN. Layer II of area 46 showed the highest cell density. Regarding interneurons, PV+-cells of area 46 showed the highest density and size, in accordance to the proposal of a dual origin of the cerebral cortex. Interhemispheric asymmetries were not identified between homologue areas. Conclusion: First, our findings suggest that layer II of area 46 exhibits the most powerful inhibitory system compared to the other prefrontal areas analyzed. This feature is not only characteristic of the PFC but also supports a particular role of layer II of area 46 in SF. Additionally, known functional asymmetries between hemispheres might not be supported by morphological asymmetries.

Antecedentes: La corteza prefrontal (CPF) representa el nivel más alto de integración y control de funciones psíquicas y comportamentales. Varias patologías como autismo, desórdenes de hiperactividad, depresión y esquizofrenia se han relacionado con alteraciones de la CPF. La lámina II de las áreas que constituyen la CPF posee un patrón de organización vertical, una alta densidad celular y la mayor proporción de neuronas no-piramidal/piramidal. Sin embargo, la distribución del componente inhibitorio en estas regiones no se ha descrito. Objetivo: En el presente estudio nos propusimos caracterizar la lámina II de las áreas 10, 24 y 46 del humano, particularmente su componente inhibitorio teniendo en mente su participación en procesos de actividad sostenida relevantes cuando desaparece el estímulo. Métodos: Se utilizaron muestras de cinco sujetos que fallecieron por causas diferentes a enfermedades del sistema nervioso. Se tomaron secciones de las áreas 10, 24 y 46 de Brodmann y se procesaron con los anticuerpos contra NeuN para determinar la población neuronal total y contra Parvalbumina (PV), Calbindina (CB) y Calretinina (CR) para analizar la población de interneuronas. Resultados: Los resultados no mostraron diferencias interhemisféricas entre las áreas. Sin embargo, las tres áreas seleccionadas son significativamente diferentes entre sí en todos los parámetros analizados. El área 46 posee la mayor densidad y tamaño de interneuronas positivas para PV. Conclusiones: La ausencia de asimetrías morfológicas no permite explicar las asimetrías funcionales. La lámina II del área 46 posee el sistema inhibitorio más poderoso. Teniendo en cuenta la arquitectura modular de las capas supragranulares, este sistema inhibitorio subyace a la actividad sostenida, eje fundamental de la memoria operativa.

The expression of α2A-adrenoceptors in the calcium-binding protein immunoreactive interneurons in rat prefrontal cortex / 生理学报

The α2A adrenoceptors (α2A-ARs) are the most common adrenergic receptor subtype found in the prefrontal cortex (PFC). It is generally accepted that stimulation of postsynaptic α2A-ARs on pyramidal neurons are key to PFC functions, such as working memory. However, the expression of α2A-ARs in interneurons is largely unknown. In the present study using double-labeling immunofluorencence technique, we investigated the expression of α2A-ARs in major types of rat PFC interneurons expressing calcium-binding proteins parvalbumin (PV), calretinin (CR), and calbindin (CB). Our data demonstrated that α2A-ARs are highly expressed in calcium-binding protein immunoreactive interneurons of rat PFC, suggesting that stimulation of α2A-ARs may alter neural networks comprising pyramidal neurons and interneurons, thereby exerting a beneficial effect on PFC cognitive functions. The present study provides the morphological basis for a potential mechanism by which stimulation of α2A-ARs induces cognitive improvement.

Distribución de calbindina y parvoalbúmina y efecto del virus de la rabia sobre su expresión en la médula espinal de ratones / Calbindin and parvalbumin distribution in spinal cord of normal and rabies-infected mice

Introducción. Aunque se trata de una enfermedad infecciosa del sistema nervioso, poco se conoce sobre los mecanismos patogénicos de la infección con el virus de la rabia. En particular, son escasos los estudios sobre su histopatología en la médula espinal. Objetivo. Estudiar la distribución de las proteínas calbindina y parvoalbúmina, en la médula espinal de ratones y evaluar el efecto de la infección con el virus de la rabia sobre su expresión. Materiales y métodos. Se inocularon ratones con virus de la rabia, por vía intracerebral o intramuscular, y se extrajo la médula espinal para hacer cortes transversales, los cuales se sometieron a tratamiento inmunohistoquímico con anticuerpos monoclonales para revelar la presencia de las dos proteínas en ratones normales y en animales infectados. Se llevó a cabo el análisis cualitativo y cuantitativo de la inmunorreacción de las dos proteínas. Resultados. Las proteínas calbindina y parvoalbúmina se distribuyeron de manera diferencial en las láminas de Rexed. La infección con el virus de la rabia produjo una disminución en la expresión de calbindina. Por el contrario, la infección provocó un incremento en la expresión de parvoalbúmina. El efecto de la rabia sobre las dos proteínas fue similar al comparar las dos vías de inoculación. Conclusión. El efecto diferencial de la infección con el virus de la rabia sobre calbindina y parvoalbúmina en la médula espinal de ratones, es similar al reportado anteriormente para áreas encefálicas. Esto sugiere uniformidad en su respuesta a la infección en todo el sistema nervioso central y es un aporte importante para el conocimiento de la patogénesis de la rabia.

Introduction: Rabies is a fatal infectious disease of the nervous system; however, the knowledge about the pathogenic neural mechanisms in rabies is scarce. In addition, there are few studies of rabies pathology of the spinal cord. Objective: To study the distribution of calcium binding proteins calbindin and parvalbumin and assessing the effect of rabies virus infection on their expression in the spinal cord of mice. Materiales y methods: Mice were inoculated with rabies virus, by intracerebral or intramuscular route. The spinal cord was extracted to perform some crosscuts which were treated by immunohistochemistry with monoclonal antibodies to reveal the presence of the two proteins in normal and rabies infected mice. We did qualitative and quantitative analyses of the immunoreactivity of the two proteins. Results: Calbindin and parvalbumin showed differential distribution in Rexed laminae. Rabies infection produced a decrease in the expression of calbindin. On the contrary, the infection caused an increased expression of parvalbumin. The effect of rabies infection on the two proteins expression was similar when comparing both routes of inoculation. Conclusion: The differential effect of rabies virus infection on the expression of calbindin and parvalbumin in the spinal cord of mice was similar to that previously reported for brain areas. This result suggests uniformity in the response to rabies infection throughout the central nervous system. This is an important contribution to the understanding of the pathogenesis of rabies.

Survival of calbindin, calretinin and parvalbumin positive neurons in mouse hippocampal CA area at chronic stage of pilocarpine-induced epilepsy / 中南大学学报(医学版)

OBJECTIVE@#To analyze the survival and the changes of proportions of Calbindin, Calretinin and Parvalbumin positive neurons in mouse hippocampal CA area at chronic stage of Pilocarpine-induced epilepsy.@*METHODS@#Calbindin, Calretinin and Parvalbumin immunofluoresence staining were done 2 months after Pilocarpine-induced epilepsy in mice or saline injection.@*RESULTS@#Two months after Pilocarine-induced epilepsy, the number of Calbindin, Calretinin and Parvalbumin positive neurons in the CA area decreased significantly compared with the control (P<0.01), especially the Calbindin positive neurons had a great drop and Pavalbumin positive neurons had a least drop. At the chronic stage of epilepsy, the proportion of Calbindin, Calretinin and Parvalbumin positive neurons in the CA area was changed. The content of Pavalbumin positive neurons increased whereas the content of Calbindin positive neurons decreased significantly compared with the control (P<0.01).@*CONCLUSION@#The changes of proportions of Calbindin, Calretinin and Parvalbumin positive neurons in the CA area of mouse hippocampus may be a factor in the ongoing epileptic activity at chronic stage of Pilocarpine-induced epilepsy.

Immunocytochemical Study of Calbindin D(28k) Positive Cells in the Developing Rat Kidney / 대한해부학회지

Calbindin D(28k),a calcium binding protein,is found in various tissues,including some cells in the distal nephron.It plays an important role in the regulation of calcium reabsorption. We previously reported the expression of calbindin D(28k) in adult rat kidney.However,the exact time of expression during differentiation in the embryonic kidney is not known.During development,intercalated cells are deleted from the medullary collecting duct by two distinct mechanisms.However,the reason for the different modes of cell death is not known.As calbindin is reported to protect cells against apoptosis,we examined the expression of calbindin D(28k) in the developing rat kidney.Kidneys from 16-,17-,18-and 20-day-old fetuses and 1-,3-,5-,7-,14-and 21-day-old pups and adult Sprague awley rats were processed for immunohistochemistry using a monoclonal antibody against calbindin D(28k) .Intercalated cells were identified by immunostaining for H+ -ATPase and by electron microscopy.Calbindin D(28k) immunoreactivity first appeared in subpopulations of cells in the connecting tubule and medullary collecting duct in the 17-day-old fetus.In the connecting tubule,calbindin D(28k) was expressed only in H+ -ATPase negative connecting tubule cells,and there was no labeling of intercalated cells.In the medullary collecting duct,calbindin D(28k) immunostaining was observed in a few cells with apical H+ -ATPase,characteristic of type A intercalated cells.The numbers of calbindin D(28k) -positive type A intercalated cells increased from day 18 of gestation.In contrast,there was little or no calbindin D(28k) immunoreactivity in the type B intercalated cells or principal cells.During the first two weeks after birth,calbindin D(28k) -positive type A intercalated cells were lost from the terminal part of the medullary collecting duct by simple extrusion. After two weeks,calbindin D(28k) immunostaining decreased in the type A intercalated cells throughout the medullary collecting duct.However,the immunoreactivity of calbindin D(28k) in the cortical collecting duct was increased in some of the type A intercalated cells and the adult pattern was observed in 21-day-old pups.Thus,we propose that the different expression of calbindin D(28k) in type A and type B intercalated cells may be responsible-at least partly-for the different modes of cell death demonstrated in these cells during kidney development.

Astragalus membranaceus promote expression of insulin-like growth factor 1 in rat model of olivo-cerebellar degeneration / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of Astragalus membranaceus (AM) on insulin-like growth factor 1 (IGF-1) expression in a rat model of olivo-cerebellar degeneration and assess the neuroprotective actions of AM meanwhile.</p><p><b>METHOD</b>Rats model of olivo-cerebellar degeneration was established by using 3-acetylpyridine. The effect of AM on the expression of Calbindin D-28K in inferior olive (IO) neurons by immunohistochemistry, the serum IGF-1 level by Elisa, the IGF-1 mRNA level in the cerebellum by RT-PCR were detected respectively.</p><p><b>RESULT</b>AM effectively improve the serum IGF-1 level, Cerebellar IGF-1 mRNA level and the survival of the 10 neurons in a rat model of olivo-cerebellar degeneration, even at a lower dose (9 g x kg(-1)), and the effect was in a dose-dependent manner.</p><p><b>CONCLUSION</b>AM could effectively upregulate the IGF-1 expression in the rat model of olivo-cerebellar degeneration, and have neuroprotective effect on IO neurons.</p>

Co-existence of calcium-binding proteins and gamma-aminobutyric acid or glycine in neurons of the rat medullary dorsal horn / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We investigated the co-expression of calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV, a combination of the three is referred to as CaBPs) with gamma-aminobutyric acid (GABA) or glycine in neurons of the rat medullary dorsal horn (MDH).</p><p><b>METHODS</b>Immunofluorescence histochemical double-staining for CaBPs and GABA or glycine was performed on the sections from rat MDH.</p><p><b>RESULTS</b>CB-, CR-, PV-, GABA- and glycine-like immunoreactive (LI) neurons were differentially observed in all layers of the MDH, but particularly in lamina II. Neurons that exhibited immunoreactivity for both CaBPs and GABA or glycine were also observed mainly in lamina II. A few of them were found in laminae I and III. The percentages of neurons which co-expressed CB/GABA or CB/glycine out of the total numbers of CB- and GABA-LI neurons or CB- and glycine-LI neurons were 5.3% and 12.1% or 4.1% and 10.0%, respectively. The ratios of CR/GABA or CR/glycine co-existing neurons out of the total numbers of CR- and GABA-LI neurons or CR- and glycine-LI neurons were 5.8% and 7.6% or 4.4% and 7.1%, respectively. The rates of PV/GABA or PV/glycine co-localized neurons out of the total numbers of PV- and GABA-LI neurons or PV- and glycine-LI neurons were 11.1% and 5.1% or 9.9% and 5.1%, respectively.</p><p><b>CONCLUSION</b>The results indicate that some neurons in the MDH contain both CaBPs and GABA or glycine.</p>

Somatic and visceral nociceptive inputs from the orofacial area and the upper alimentary tract converge onto CB-containing neurons in interstitial nucleus of the spinal trigeminal tract in rats / 生理学报

The interstitial nucleus of the spinal trigeminal tract (INV) contains many calbindin-D28k-containing neurons (CB-neurons) receiving convergence information from the somatic and visceral structures. The purpose of the present study was to confirm whether the primary afferent terminals from the inferior alveolar nerve (IAN) make close contact and synaptic connections with the same CB-neurons receiving visceral nociceptive signals in INV. Biotinylated dextran amine (BDA) and horseradish peroxidase (HRP) tracing combined with CB and Fos proteins immunohistochemistry were used. After injections of BDA and formalin into unilateral IAN and upper alimentary tract, respectively, the transganglionic labeled afferent fibers and terminals from IAN were observed in the ipsilateral INV, especially in its enlarged part. A large number of CB- and Fos-like immunoreactive (LI) neurons were found in bilateral INV. These CB- and Fos-LI neurons mostly overlapped with BDA-labeled terminals in the enlarged part of INV. About one half of the CB-LI neurons were double labeled with Fos-LI nuclei (74/153). The terminals from IAN were to made close contacts with many CB/Fos-double labeled or CB-single labeled neurons. After injection of HRP into IAN, HRP-labeled fibers and terminals in INV were similar to that labeled with BDA. Under the electron microscope, a large number of CB-LI dendrites and a few soma in the enlarged part of INV were found to form asymmetrical axo-dendritic and axo-somal synapses with the HRP-labeled axon terminals. These results indicate that the orofacial somatic inputs from IAN and the visceral nociceptive inputs from the upper alimentary tract converge onto the same CB-containing neurons in INV. These CB-containing neurons in INV probably play an important role in information integration as well as visceral and cardiovascular activity.

Expression of an Anion Exchanger Pendrin in Human Kidney / 대한해부학회지

It has been reported that new apical anion exchanger perndrin, encoded by the pendred syndrome (PDS/pds, Slc26A4) gene, was expressed in the AE1-negative intercalated cells of rat and mouse kidneys. The purpose of this study was performed that expression of pendrin in the subtypes of intercalated cells in human kidney. The normal human renal tissues obtained from nephrotomized kidneys for renal cell carcinoma were fixed in periodate-lysine-paraformalde-hyde, and processed for immunohistochemistry. Subtypes of intercalated cells were identified by using antibodies for H(+)-ATPase and AE1, and connecting tubule cells and principal cells of collecting duct were identified using antibodies for calbindin D28K and AQP2, respectively. In human kidney, pendrin was expressed in the apical domain of AE1-negative intercalated cells including type B cells with diffuse and/or basolateal H(+)-ATPase, non A-non B (non -A/B) type intercalated cells with apical H(+)-ATPase and bipolar type of intercalated cells with apical and basolateral H(+)-ATPase. The AQP2-positive principal cells of cortical collecting duct were also had apical pendrin immunoreactivity. However, there was no pendrin immunoreactivity in AE1-positive type A intercalated cells, calbindin D28K-positive connecting tubule cells, and AQP2-positive medullary collecting duct. These results suggest that pendrin is an apical anion exchanger not only in the AE1-negative intercalated cells (type B, non-A/B and bipolar cells) but also in the principal cells of cortical collecting duct, and has an essential role in HCO3-secretion in human kidney.

Morphological Studies on the Calbindin D-28K and Parvalbumin Immunoreactive Neurons in the Medulla Oblongata and Ventral Horn of the Spinal Cord Gray Matter after Spinal Cord Injury in Rats / 체질인류학회지

This study was examined and compared the immunocytochemical distribution of the two calcium-binding proteins calbindin D-28K and parvalbumin immunreactive neurons in the medulla oblongata and spinal cord after transection of spinal cord in rats. In this experiment, calbindin D-28K immnunoreactive neurons were mainly found in many pyramidal cells distributed medulla oblongata and spina1 cord of rats. Parvalbumin immunoreactive cells were demonstrated in all lamina of the gray matter of the spinal cord. These immunoreactive cells had the most high density in the severa1 nuclei of the ventra1 horn of the all segments of the spina1 cord. Calbindin D-28K neuropil labeling was strongly noted in spina1 all segments of the spinal cord. In contrast parva1bumin immunoreactive, little differences were found in distribution, size and morphology of calbindin D-28K cell body or neuropil staining in the spinal cord. The number of parvalbumin immunoreactive cells were more than twice in the medulla oblongata and spinal cord compared to the calbindin D-28K immunoreactive cells. Calbindin D-28K and parvalbumin-immmoreactive somata were round, ova1, spind1e and polygona1 in shape, and the immunoreactive neurons were unipolar, bipolar, multipolar and horizontal in shape. The diameters of the somata of the two immunoreactive neurons were 40 ~50 micrometer, respectively. Also dendrites of two immunoreactive neurons were densely arrayed in network. These results suggest that CB-IR and PV-IR most high density in the of the VII~X layers in the ventra1 horn of the all segments of the spina1 cord.

Roles of Stem Cell Factor and c-kit Receptor in the Development of Cerebellar GABAergic Neurons / 대한해부학회지

Evidence that Stem cell factor (SCF) and c-Kit receptor tyrosine kinase are expressed in the cerebellum during postnatal development, suggests a possible contribution of the SCF/Kit signaling pathway in the cerebellar development. In the present study, we prepared cerebellar cultures from C57Bl/6J mouse at postnatal day 1and 7 to investigate the role of c-kit receptor and SCF in regulation of growth and differentiation in the postnatal cerebellar GABAergic cells. SCF increased the number of survival cerebellar cells and density of glutamic acid decarboxylase 65/67 (GAD65/67) and calbindin D-28K expression in the immunoblot analysis. SCF also improved the neurite extension of the interneuron neuritis and dendritogenesis of Purkinje cells. Treatment with c-Kit antibody accelerated cellular loss in serum-free media and decreased the growth ability and dendritogenesis of Purkinje cells and cerebellar inhibitory interneurons. Our data suggest that SCF and c-kit receptor are required for the normal growth of postnatal cerebellum and a possible involvement of functional regulation through the SCF/c-kit receptor pathways in the postnatal cerebellar development.

Comparative proteomics research of apoptosis initiation induced by homoharringtonine in HL-60 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the related proteins of apoptosis initiation induced by homoharringtonine (HHT) in HL-60 cells.</p><p><b>METHODS</b>After establishment of an apoptosis initiation model induced by HHT in HL-60 cells, proteins of untreated and HHT treated HL-60 cells were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching.</p><p><b>RESULTS</b>Proteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by HHT.</p><p><b>CONCLUSION</b>The present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.</p>