Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.     

Changes in TRPC channel expression during postnatal development of cerebellar neurons

In the brain, classical (canonical) transient receptor potential (TRPC) channels are thought to be involved in different aspects of neuronal development. We investigated the developmental expression profile of TRPC channels in rat cerebellum during the first 6 weeks after birth. TRPC3 expression is significantly up-regulated whereas TRPC4 and TRPC6 expression are significantly down-regulated over this period of time. TRPC3 expression is mainly found on Purkinje cells and their dendrites, suggesting that the increase in TRPC3 expression reflects development of the dendritic tree of Purkinje cells. TRPC4 expression was restricted to granule and their precursor cells. TRPC6 expression is found on Purkinje cell bodies, on mature granule cells in the internal granule cell layer (but not their precursors) and interneurons in the molecular layer. The decrease in TRPC4 expression suggests that it is required for proper granule cell development whereas the decrease in TRPC6 expression is presumably correlated with interneuron development. Moreover, we demonstrate the presence of functional TRPC channels on Purkinje cell dendrites that are activated following stimulation of metabotropic glutamate receptors. Our results reveal cell-specific expression patterns for different TRPC proteins and suggest that developmental changes in TRPC protein expression may be required for proper postnatal cerebellar development.     

Distribution of neuronal nitric oxide synthase-immunoreactive neurons in the cerebral cortex and hippocampus during postnatal development

Although many reports have argued a role for nitric oxide (NO) during postnatal development, there has been no combined demonstration in the cerebral cortex and hippocampus. We have investigated the distribution and morphology of neurons and fibers expressing neuronal NO synthase (nNOS) in the cerebral cortex and hippocampal formation of rats during the postnatal development, and correlated these findings with developmental events taking place in these regions. In the cerebral cortex, the nNOS-immunoreactive cells could be divided into two classes : heavily stained neurons and lightly stained neurons. For the lightly stained nNOS-positive neurons, only the cell bodies were observed, whereas for the heavily stained neurons, the cell bodies and their dendrites were visible. During the postnatal days, heavily stained neurons reached their typical morphology in the second week and appeared in all layers except for layer I. In the hippocampus, there was a transient expression of nNOS in the pyramidal cell layer at P3â€P7, and this expression disappeared during following days. The adult pattern of staining developed gradually during the postnatal period. This study suggested that these alterations might reflect a region-specific role of NO and a potential developmental role in the postnatal cerebral cortex and hippocampus     

Glucose-6-phosphatase activity in mouse small intestine during postnatal development.

The postnatal development of phosphohydrolase activity of glucose-6-phosphatase has been examined in the different parts of the small intestine of the mouse and compared with that of trehalase and glucoamylase. After birth, glucose-6-phosphatase is present in all parts of the small intestine and the activity is already two to three times the adult values. In the course of the first 2 weeks, the activity increases and the different parts of the small intestine show distinct patterns. One day after birth, glucoamylase activity is present in all segments, while trehalase activity is confined to the first intestinal third. During the first 2 weeks, these activities remain rather stable. In the course of the third week, when trehalase and glucoamylase are increasing, glucose-6-phosphatase activity is declining toward adult values.     

p38 MAPK在小鼠睾丸不同发育阶段的表达和定位

为探讨丝裂原活化蛋白激酶p38 MAPK在小鼠睾丸不同发育阶段的表达,应用蛋白质免疫印迹杂交技术和免疫组织化学SABC法检测1至7周龄小鼠睾丸p38 MAPK的表达、定位及发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。免疫印迹杂交发现,p38 MAPK在2～7周龄小鼠睾丸中均有表达。免疫组织化学结果显示,在2周龄小鼠睾丸曲细精管上皮中即可观察到p38 MAPK免疫阳性反应,免疫反应阳性细胞为精原细胞;3、4、5周龄小鼠睾丸仅有个别曲细精管上皮可见p38 MAPK免疫阳性反应;6、7周龄小鼠睾丸中p38 MAPK表达较丰富,免疫反应阳性细胞为精原细胞和初级精母细胞,免疫阳性反应物均主要位于细胞核内。在7周龄小鼠睾丸中还可见到部分间质细胞的细胞质亦呈p38 MAPK阳性。这些结果提示,p38 MAPK可能对生精细胞的增殖分化具有调控作用。     

Expression of rat, renal NHE2 and NHE3 during postnatal development

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.     

Expression of epidermal growth factor in the kidney and submandibular gland during mouse postnatal development

Earlier work has demonstrated that the salivary glands and kidneys are the major sites of epidermal growth factor (EGF) synthesis in adult mice. The precise timing of the onset of endogenous EGF synthesis in these tissues is not yet clear. In the present study we assessed the ontogenesis of EGF expression in the Swiss-Webster mouse. Paraformaldehyde-fixed frozen sections of neonatal kidneys and salivary glands were probed with proEGF cRNA labelled with 35S for in situ hybridization and with rabbit antisera to mouse EGF for immunocytochemistry. Both EGF mRNA and immunoreactivity were first detected in the developing distal nephron between days 3 and 5 postpartum. Juxtamedullary nephrons underlying the superficial nephrogenic zone were the first to express EGF. During the 2nd week after birth, EGF-expressing tubules became more abundant and distributed to medullary as well as cortical regions, corresponding to the thick ascending limb of Henle and distal convoluted tubule. Initial EGF mRNA and immunoreactivity in the submandibular gland were first detected between days 18 and 20 postpartum and increased notably during the following weeks.     

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.     

Structural stabilization of CNS synapses during postnatal development in rat cortex

CNS synapses are produced rapidly upon pre- and post-synaptic recruitment. However, their composition is known to change during development and we reasoned that this may be reflected in the gross biochemical properties of synapses. We found synaptic structure in adult cortical synaptosomes to be resistant to digestion with trypsin in the presence and absence of calcium ions, contrasting with previous observations. We evaluated the divalent cation dependence and trypsin sensitivities of synapses using synaptosomes from different developmental stages. In contrast to adult synapses, at postnatal day (P) 10 EDTA treatment eliminated approximately 60% of the synapses, and trypsin and EDTA, together, eliminated all junctions. Trypsinization in the presence of calcium eliminated approximately 60% of the junctions at P10. By P35, all synapses were calcium independent, whereas full trypsin resistance was not attained until P49. To compare the calcium dependence and trypsin sensitivity of synapses in another region of the adult brain, we examined synapses from adult (P50) hippocampus. Adult hippocampus maintained a population of synapses that resembled that of P35 cortex. Our results show that synapses are modified over a long time period in the developing cortex. We propose a model in which the addition of synergistic calcium-dependent and -independent adhesive systems stabilize synapses.     

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.     

Expression of low-molecular-weight neurofilament (NF-L) mRNA during postnatal development of the mouse brain

A regional Northern blot analysis demonstrated that the highest levels of NF-L mRNA in the adult mouse brain are present in brain stem followed by mid-brain, with lower levels found in neocortex, cerebellum, and hippocampus. The study was extended to the cellular level over the time course of postnatal development using in situ hybridization. This developmental analysis revealed that the expression of NF-L mRNA closely follows the differentiation pattern of many large neurons during postnatal neurogenesis. Neurons which differentiate early such as Purkinje, mitral, pyramidal, and large neurons of brain stem and thalamic nuclei, expressed high levels of NF-L mRNA at postnatal day 1. Early expression of NF-L mRNA may be required for the maintenance of the extensive neurofilament protein networks that are detected within the axons of larger neurons. Smaller neurons which differentiate later, such as dentate gyrus granule cells, small pyramidal and granule cells of the neocortex, and granule cells of the cerebellum, exhibit a delayed expression of NF-L mRNA.To whom to address reprint requests.     

Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development

Spermatogonial stem cells (SSCs) are stem cells of the male germ line. In mice, SSCs are quiescent at birth but actively proliferate during the first postnatal week, while they rarely divide in adult, suggesting an age-dependent difference in SSC characteristics. As an approach to evaluate this possibility, we studied the expression pattern of cell-surface molecules on neonatal, pup, and adult mouse SSCs. Using immunomagnetic cell sorting, testis cells were selected for the expression of alpha(6) integrin, alpha(v) integrin, c-kit receptor tyrosine kinase (Kit), or a binding subunit of glial-cell-line-derived neurotrophic factor (GDNF) receptor, GFRalpha1. Selected cells were assayed for their stem cell activity using spermatogonial transplantation. The results showed that SSCs expressed alpha(6) integrin, but not alpha(v) integrin and Kit, regardless of age. The SSC activity in pup GFRalpha1(+) cells was higher than that in adult and neonatal cells, indicating that the expression pattern of GFRalpha1 varied age-dependently. To evaluate if SSCs show an age-dependent difference in their response to GDNF, we cultured highly enriched pup and adult SSCs with GDNF: we could not observe such an age-dependent difference in vitro. In addition, we failed to immunologically detect the expression of two types of GDNF receptor signaling subunits on SSCs. These results indicate that SSCs may change the expression patterns of cell-surface molecules during postnatal development, and suggest that GDNF receptor molecules may not be abundantly or specifically expressed in the in vivo population of mouse SSCs.     

Ultrastructural localization of intestinal glucose-6-phosphatase activity during the postnatal development of the mouse

Summary The development of the endoplasmic reticulum (ER) and the ultrastructural localization of glucose-6-phosphatase activity have been studied in the proximal jejunum and distal ileum during the postnatal period. One day after birth, the amount and the repartition of ER in the jejunal enterocytes are similar to that observed in postweaning period. In the following days an extensive proliferation of SER is noted in the supranuclear zone of the absorbing cells. From day 7 till postweaning period a gradual decrease of the amount of SER is observed and after weaning, the ultrastructure of the enterocytes is similar to that in the adult mouse enterocytes. At all time, a positive reaction for G-6-Pase activity is observed in the cisternae of the endoplasmic reticulum and in the nuclear envelope. In the distal ileum, the SER is poorly developed one day after birth. During the first two weeks, the ER increases but no extensive proliferation of SER can be noted as in the jejunum. The G-6-Pase activity can be visualized in the rough and smooth endoplasmic reticulum as well as in the nuclear envelope. It appears that the proliferation of SER could be interpreted as the morphologic expression of an increased G-6-Pase activity.This work was supported by research grant from the Medical Research Council of CanadaD. Ménard, Ph. D. is «Chercheur-boursier du Conseil de la recherche en santé du Québec»     

Dynamics of muscle fibre growth during postnatal mouse development

Background  

Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle.     

Ultrastructural localization of intestinal glucose-6-phosphatase activity during the postnatal development of the mouse

The development of the endoplasmic reticulum (ER) and the ultrastructural localization of glucose-6-phosphatase activity have been studied in the proximal jejunum and distal ileum during the postnatal period. One day after birth, the amount and the repartition of ER in the jejunal enterocytes are similar to that observed in postweaning period. In the following days an extensive proliferation of SER is noted in the supranuclear zone of the absorbing cells. From day 7 till postweaning period a gradual decrease of the amount of SER is observed and after weaning, the ultrastructure of the enterocytes is similar to that in the adult mouse enterocytes. At all time, a positive reaction for G-6-Pase activity is observed in the cisternae of the endoplasmic reticulum and in the nuclear envelope. In the distal ileum, the SER is poorly developed one day after birth. During the first two weeks, the ER increases but no extensive proliferation of SER can be noted as in the jejunum. The G-6-Pase activity can be visualized in the rough and smooth endoplasmic reticulum as well as in the nuclear envelope. It appears that the proliferation of SER could be interpreted as the morphologic expression of an increased G-6-Pase activity.     

Expression of DPP6 in Meckel's cartilage and tooth germs during mouse facial development

Dipeptidyl peptidase-like protein 6 (DPP6), a member of the dipeptidyl aminopeptidase family, plays distinct roles in brain development, but its expression in embryonic Meckel's cartilage and tooth germs development is unknown. We analyzed the expression pattern of DPP6 in Meckel's cartilage and tooth germs development using in situ hybridization. DPP6 was detected in different patterns in Meckel's cartilage and tooth germs during mouse facial development from 11.5 to 13.5 days post-coitus (dpc) embryos. The expression pattern of DPP6 suggests that it may be involved in mandible and tooth development.     

Critical role of TRPC6 channels in the development of human renal cell carcinoma

Renal cell carcinoma (RCC) is the most common tumor arising from the cells in the lining of tubules in the kidney. Some members of the Ca²⁺-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of some types of cancer cells. In this study, we investigated the role of TRPC6 in the development of human RCC. RT-PCR and Western blotting were used to investigate TRPC6 expression in 1932 and ACHN cells. Immunohistochemical techniques were applied to study TRPC6 expression in 60 cases of RCC primary tissue samples and 10 cases of corresponding normal renal tissues. To inhibit TRPC6 activity or expression, RNA interference was used. The effects of TRPC6 channels on RCC cell viability and cell cycle progression were investigated by MTT and flow cytometry. TRPC6 was expressed in 1932 and ACHN cells. TRPC6 protein was detected in 73.3 % of RCC samples, and there was a significant difference compared with the normal renal samples (30 %) (p < 0.05). Moreover the level of TRPC6 expression was associated with RCC Fuhrman grade (p < 0.01). Blockade of TRPC6 channels in ACHN cells suppressed basal cell proliferation and partially inhibited HGF-induced cell proliferation. Furthermore, inhibition of TRPC6 channels expression prolonged the transition through G₂/M phase in ACHN cells. In summary, expression of TRPC6 is markedly increased in RCC specimens and associated with RCC histological grade. TRPC6 plays an important role in ACHN cells proliferation.