Primary cultured neuronal networks and type 2 diabetes model mouse fatty liver tissues in aqueous liquid observed by atmospheric SEM (ASEM): Staining preferences of metal solutions.

Neural networking, including axon targeting and synapse formation, is the basis of various brain functions, including memory and learning. Diabetes-mellitus affects peripheral nerves and is known to cause fatty liver disease. Electron microscopy (EM) provides the resolution required to observe changes in fine subcellular structures caused by such physiological and pathological processes, but samples are observed in vacuum. Environmental capsule EM can directly observe cells in a more natural aqueous environment, but the size-limited capsules restrict cell culturability. The recently developed atmospheric scanning electron microscope (ASEM) has an open, 35 mm sample dish, allowing the culture of primary cells, including neurons, on the electron-transparent film window fabricated in its base. The system's inverted scanning electron microscope observes aldehyde-fixed cells or tissues from below through the silicon nitride film; the optical microscope located above allows direct correlation of fluorescence labeling. To observe fixed biological samples, damage due to low dose electron radiation is minimized in three ways. First, knock on damage that pushes out atoms is decreased by the low accelerating voltage of 10-30 kV. Second, increased radical generation due to the decreased acceleration voltage is countered by the addition of a radical scavenger, glucose or ascorbic acid, to the sample solution. Third, the large volume (max. 2 ml) of aqueous buffer surrounding the sample has a high specific heat capacity, minimizing the temperature increase caused by irradiation. Using ASEM, we have developed protocols for heavy metal staining in solution to selectively visualize intracellular structures. Various EM staining methods served as a starting point. Uranyl acetate preferably stains proteins and nucleic acid, and prior tannic acid treatment enhances membranes. Osmium tetroxide is suggested to enhance lipids, especially oil droplets. Imaging primary-culture neurons stained with platinum blue or uranyl acetate revealed growth cones, synapses, and 50-500 nm spines, together with neurite backbones and their associated structures. Correlative microscopy with immuno-fluorescence labeling suggested that these were mainly microtubule associated objects; some showed signs of a fission process and were, thus, possibly mitochondria. Liver tissue excised from the ob/ob type 2 diabetes model mouse, was stained with osmium tetroxide and observed using ASEM. Swollen bright balls occupied a large area of the cytoplasm and could be distinguished from vacuoles, suggesting that they are oil droplets. In some of the images, oil-like droplets were pressing surrounding structures, including sinusoids, significant for blood circulation in the liver. Based on these studies, ASEM combined with metal staining methods promises to allow the study of various mesoscopic-scale phenomena of cells and tissues immersed in natural aqueous environment in the near future. The quick nature of ASEM could facilitate not only the precise imaging for neuroscience but also the diagnosis of fatty liver disease and related diseases.

Roles of the serotonin 5-HT4 receptor in dendrite formation of the rat hippocampal neurons in vitro.

Serotonin (5-HT) is involved in various aspects of hippocampal development, although the specific roles of 5-HT receptors are poorly understood. We investigated the roles of 5-HT receptors in the dendrite formation of hippocampal neurons. We focused on the 5-HT4 receptor, which is coupled with Gs protein, and compared the effects with those of the Gi-coupled 5-HT1A receptor. Neurons from rat hippocampi at embryonic day 18 were dissociated and treated for 4 days with the 5-HT4 receptor agonist BIMU8 or the 5-HT1A receptor agonist 8-OH DPAT. The formation of primary dendrites and dendrite branching were promoted by BIMU8, whereas the dendrite branching was inhibited by 8-OH DPAT. BIMU8-induced promotion of dendrite formation was neutralized by concomitant treatment with the 5-HT4 receptor antagonist, confirming the specific actions of the 5-HT4 receptor. We then examined the signaling mechanisms underlying the actions of the 5-HT4 receptor by using a protein kinase A (PKA) inhibitor. The BIMU8-induced promotion of dendrite formation was reversed partially by the PKA inhibitor, suggesting involvement of PKA signaling downstream of the 5-HT4 receptor. Finally, we examined the contribution of brain-derived neurotrophic factor (BDNF) to the promotion of dendrite formation by BIMU8. Quantitative RT-PCR analysis showed that BIMU8 increased the BDNF mRNA expression and that treatment of cultured neurons with the TrkB antagonist reversed the BIMU8-induced increase in dendrite formation. In summary, the present study suggests a novel role for the 5-HT4 receptor in facilitation of dendrite formation in which intracellular signaling of PKA and the BDNF-TrkB system may be involved.

Effects of prenatal stress and neonatal handling on anxiety, spatial learning and serotonergic system of male offspring mice.

Environmental factors during perinatal period have various effects on behavior. The present study examined the effects of prenatal stress and neonatal handling on anxiety and spatial learning of offspring. Prenatal stress increased anxiety-related behavior of adult offspring, whereas neonatal handling had no effect. In contrast, spatial learning was not affected by prenatal stress, but improved by neonatal handling in both prenatally stressed and non-stressed mice. Next, to elucidate possible brain mechanisms mediating effects of environmental factors on behavior, we focused on serotonin (5-HT) system in the frontal cortex and hippocampus which is involved in anxiety and learning. We examined effects of environmental factors on the mRNA expression of 5-HT1A, 5-HT2A and 5-HT2C receptors in the frontal cortex and hippocampus during postnatal period and adulthood. Both prenatal stress and neonatal handling altered the mRNA expression of 5-HT receptors. These effects were dependent on environmental factors, brain regions and developmental stages. In summary, the present study revealed that prenatal stress and neonatal handling had differential effects on anxiety and spatial learning of offspring, and concomitantly the expression of 5-HT receptors. It was also shown that the effects of prenatal stress on 5-HT system were recovered partially by neonatal handling.

Serotonin 2A receptor regulates microtubule assembly and induces dynamics of dendritic growth cones in rat cortical neurons in vitro.

Serotonin (5-HT) regulates the development of cerebral cortex, but 5-HT receptors mediating the effects are poorly understood. We investigated roles of 5-HT2A receptor in dendritic growth cones using dissociation culture of rat cerebral cortex. Neurons at embryonic day 16 were cultured for 4 days and treated with 5-HT2A/2C receptor agonist (DOI) for 4h. DOI increased the size of growth cone periphery which was actin-rich and microtubule-associated protein 2-negative at the dendritic tip. The length increase of the growth cone periphery may be mediated by 5-HT2A receptor, because the 5-HT2A receptor antagonist reversed the effects of DOI. Moreover, the time-lapse analysis demonstrated the increase of morphological dynamics in dendritic growth cones by DOI. Next, to elucidate the mechanisms underlying the actions of 5-HT2A receptor in dendritic growth cones, we examined the cytoskeletal proteins, tyrosinated α-tubulin (Tyr-T; dynamic tubulin) and acetylated α-tubulin (Ace-T; stable tubulin). DOI increased the fluorescence intensity of Tyr-T, while decreased that of Ace-T in the dendritic growth cone periphery. These effects were reversed by the 5-HT2A receptor antagonist, suggesting that 5-HT2A receptor promotes microtubule dynamics. In summary, it was suggested that 5-HT2A receptor induces morphological changes and dynamics of dendritic growth cones through regulation of microtubule assembly.

Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase-immunoreactive neurons in the sexually dimorphic nucleus in female mice.

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.

Receptor-dependent regulation of dendrite formation of noradrenaline and dopamine in non-GABAergic cerebral cortical neurons.

The present study characterized the receptor-dependent regulation of dendrite formation of noradrenaline (NA) and dopamine (DA) in cultured neurons obtained from embryonic day 16 rat cerebral cortex. Morphological diversity of cortical dendrites was analyzed on various features: dendrite initiation, dendrite outgrowth, and dendrite branching. Using a combination of immunocytochemical markers of dendrites and GABAergic neurons, we focused on the dendrite morphology of non-GABAergic neurons. Our results showed that (1) NA inhibited the dendrite branching, (2) ß adrenergic receptor (ß-AR) agonist inhibited the dendrite initiation, while promoted the dendrite outgrowth, (3) ß1-AR and ß2-AR were present in all the cultured neurons, and both agonists inhibited the dendrite initiation, while only ß1-AR agonist induced the dendrite branching; (4) DA inhibited the dendrite outgrowth, (5) D1 receptor agonist inhibited the dendrite initiation, while promoted the dendrite branching. In conclusion, this study compared the effects of NA, DA and their receptors and showed that NA and DA regulate different features on the dendrite formation of non-GABAergic cortical neurons, depending on the receptors.

Subtype specific roles of serotonin receptors in the spine formation of cortical neurons in vitro.

Dendritic spines are postsynaptic structures which are formed from filopodia. We examined roles of serotonin (5-HT) receptors in the spine formation. Embryonic rat cortical neurons were cultured for 10 or 14 days and treated by 5-HT receptor agonists for 24 h. At 11 days in vitro, 5-HT(1A) agonist increased filopodia density, whereas 5-HT(2A/2C) agonist increased the density of puncta and spines. At 15 days in vitro, 5-HT(1A) agonist decreased the density of puncta and spines, whereas 5-HT(2A/2C) agonist decreased filopodia density with increase of spines. In conclusion, the present study shows 5-HT receptors have subtype-specific effects on the spine formation.

Differential roles of calcitonin family peptides in the dendrite formation and spinogenesis of the cerebral cortex in vitro.

We examined roles of calcitonin family peptides in the initial stages of dendrite formation and the maturation of dendritic spines in the rat cerebral cortex in vitro. Embryonic day 18 cortical neurons were dissociated and cultured for 2-3days in the presence of calcitonin gene-related peptide (CGRP), calcitonin, amylin or adrenomedullin. The treatment of cortical neurons with CGRP promoted the formation of primary dendrites of non-GABAergic neurons. In contrast, the treatment with amylin and adrenomedullin for 3days inhibited the dendritic elongation of non-GABAergic neurons. Calcitonin had no effect on the initial dendrite formation. Next, we examined roles of the peptides in the spine formation. Embryonic day 16 cortical neurons were cultured for 14days and then treated acutely with CGRP, amylin or adrenomedullin for 24h. The density of filopodia, puncta/stubby spines and spines were increased by the CGRP treatment, whereas decreased by amylin. Therefore, CGRP and amylin showed opposite effects on the formation of dendritic filopodia, puncta and spines. Adrenomedullin had no effects on the spine formation. In conclusion, the present study showed that calcitonin family peptides have differential effects both in the dendrite formation during the initial stages and the spine formation of cortical neurons in vitro.

Roles of serotonin 5-HT3 receptor in the formation of dendrites and axons in the rat cerebral cortex: an in vitro study.

The serotonin type 3 (5-HT(3)) receptor is an only ligand-gated ion channel among 14 serotonin receptors. Here, we examined the roles of the 5-HT(3) receptor in the formation of dendrites and axons, using a dissociation culture of embryonic rat cerebral cortex. Cortical neurons at embryonic day 16 were cultured for 4 days in the presence of a selective 5-HT(3) receptor agonist with or without an antagonist. Neurons were then immunostained by antibodies against microtubule-associated protein 2 (MAP2) and glutamic acid decarboxylase (GAD) 65. All cells expressed MAP2, whereas only limited number of cells expressed GAD65. From the immunoreactivity and the cell shape, we tentatively divided neurons into 3 types; GAD-positive multipolar, GAD-positive bipolar/tripolar and GAD-negative neurons. The total length of axons and dendrites, the number of primary dendrites and the dendritic branching of GAD-negative neurons were decreased by the agonist (10 or 100nM), most of which were reversed by the concomitant treatment of the antagonist. In contrast, no or little effect was observed on the formation of dendrites and axons of GAD-positive multipolar neurons, and the neurite formation of GAD-positive bipolar/tripolar neurons. The present study revealed differential roles of the 5-HT(3) receptor in the formation of dendrites and axons of subtypes of cortical neurons.

Neurotensin promotes the dendrite elongation and the dendritic spine maturation of the cerebral cortex in vitro.

We examined roles of neurotensin in the dendrite formation and the maturation of dendritic spines in the rat cerebral cortex. Embryonic day (E) 18 cortical neurons were cultured for 2 or 4 days in the presence of neurotensin. The chronic treatment of cortical neurons with neurotensin for 4 days increased the dendritic length of non-GABAergic neurons. In addition, the acute treatment of cortical neurons for 24h at 3 days in vitro also increased the dendritic length of non-GABAergic neurons similarly but more strongly than the chronic treatment. In contrast, the acute treatment for 4h had no effects on the dendrite formation. Next, we examined the effects of neurotensin on the maturation of dendritic spines. E16 cortical neurons were cultured for 10 or 14 days in a basal medium and then treated with neurotensin for 24h. At 11 days in vitro, neurotensin increased the postsynaptic density (PSD) 95-positive dendritic protrusions (filopodia, puncta and spines) together with the increase of spine density and the decrease of puncta density. At 15 days in vitro, neurotensin decreased the puncta density. In addition, the immunohistochemical localization of neurotensin type 1 and type 3 receptors in cultured neurons suggested the differential contribution of the receptors in these effects. These findings suggest that neurotensin promotes the dendrite outgrowth and the maturation of dendritic spines of cultured cortical neurons, although further studies are needed to conclude that these roles of neurotensin are also the case in vivo.

Expression and characterization of the active molecular forms of choline/ethanolamine kinase-alpha and -beta in mouse tissues, including carbon tetrachloride-induced liver.

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.