A possible new mechanism for the control of miRNA expression in neurons

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration,which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR,we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs “make sense” if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were oncurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression.

Leptin signaling in Kiss1 neurons arises after pubertal development

The adipocyte-derived hormone leptin is required for normal pubertal maturation in mice and humans and, therefore, leptin has been recognized as a crucial metabolic cue linking energy stores and the onset of puberty. Several lines of evidence have suggested that leptin acts via kisspeptin expressing neurons of the arcuate nucleus to exert its effects. Using conditional knockout mice, we have previously demonstrated that deletion of leptin receptors (LepR) from kisspeptin cells cause no puberty or fertility deficits. However, developmental adaptations and system redundancies may have obscured the physiologic relevance of direct leptin signaling in kisspeptin neurons. To overcome these putative effects, we re-expressed endogenous LepR selectively in kisspeptin cells of mice otherwise null for LepR, using the Cre-loxP system. Kiss1-Cre LepR null mice showed no pubertal development and no improvement of the metabolic phenotype, remaining obese, diabetic and infertile. These mice displayed decreased numbers of neurons expressing Kiss1 gene, similar to prepubertal control mice, and an unexpected lack of re-expression of functional LepR. To further assess the temporal coexpression of Kiss1 and Lepr genes, we generated mice with the human renilla green fluorescent protein (hrGFP) driven by Kiss1 regulatory elements and crossed them with mice that express Cre recombinase from the Lepr locus and the R26-tdTomato reporter gene. No coexpression of Kiss1 and LepR was observed in prepubertal mice. Our findings unequivocally demonstrate that kisspeptin neurons are not the direct target of leptin in the onset of puberty. Leptin signaling in kisspeptin neurons arises only after completion of sexual maturation.

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding

P2Y1 receptors expressed by C1 neurons determine peripheral chemoreceptor modulation of breathing, sympathetic activity, and blood pressure

Catecholaminergic C1 cells of the rostral ventrolateral medulla (RVLM) are key determinants of the sympathoexcitatory response to peripheral chemoreceptor activation. Overactivation of this reflex is thought to contribute to increased sympathetic activity and hypertension; however, molecular mechanisms linking peripheral chemoreceptor drive to hypertension remain poorly understood. We have recently determined that activation of P2Y1 receptors in the RVLM mimicked effects of peripheral chemoreceptor activation. Therefore, we hypothesize that P2Y1 receptors regulate peripheral chemoreceptor drive in this region. Here, we determine whether P2Y1 receptors are expressed by C1 neurons in the RVLM and contribute to peripheral chemoreceptor control of breathing, sympathetic activity, and blood pressure. We found that injection of a specific P2Y1 receptor agonist (MRS2365) into the RVLM of anesthetized adult rats increased phrenic nerve activity (≈55%), sympathetic nerve activity (38±6%), and blood pressure (23±1 mm Hg), whereas application of a specific P2Y1 receptor antagonist (MRS2179) decreased peripheral chemoreceptor–mediated activation of phrenic nerve activity, sympathetic nerve activity, and blood pressure. To establish that P2Y1 receptors are expressed by C1 cells, we determine in the brain slice preparation using cell-attached recording techniques that cells responsive to MRS2365 are immunoreactive for tyrosine hydroxylase (a marker of C1 cells), and we determine in vivo that C1-lesioned animals do not respond to RVLM injection of MRS2365. These data identify P2Y1 receptors as key determinants of peripheral chemoreceptor regulation of breathing, sympathetic nerve activity, and blood pressure.

Effects of ischemia and reperfusion on subpopulations of rat enteric neurons expressing the P2X7 receptor

BACKGROUND: Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM: To analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of rat ileum. METHODS: The ileal artery was occluded for 35 min and animals were euthanized 6, 24, and 72 h, and 1 week later. Immunohistochemistry was performed with antibodies against the P2X7 receptor as well as nitric oxide synthase (NOS), calbindin, calretinin, choline acetyltransferase (ChAT), or the pan-neuronal marker anti-HuC/D. RESULTS: Double immunolabeling demonstrated that 100% of NOS-, calbindin-, calretinin-, and ChAT-immunoreactive neurons in all groups expressed the P2X7 receptor. Following I/R, neuronal density decreased by 22.6% in P2X7 receptor-immunoreactive neurons, and decreased by 46.7, 38, 39.8, 21.7, and 20% in NOS-, calbindin-, calretinin-, ChAT-, and HuC/D-immunoreactive neurons, respectively, at 6, 24, and 72 h and 1 week following injury compared to the control and sham groups. We also observed a 14% increase in the neuronal cell body profile area of the NOS-immunoreactive neurons at 6 and 24 h post-I/R and a 14% increase in ChAT-immunoreactive neurons at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% at 6 h post-I/R and increased by 8% at 24 h post-I/R. CONCLUSIONS: This work demonstrates that I/R is associated with a significant loss of different subpopulations of neurons in the myenteric plexus accompanied by morphological changes, all of which may underlie conditions related to intestinal motility disorder

Glutamate-induced potentiation of calcium influx in primary hippocampal culture neurons

ABSTRACT This works aim was to test whether LTP-like features can also be measured in cell culture and by methods that allow to analyse a alrger number of cells. A suitable method for this purpose is calcium imaging. The rationale for this approach lies in the fact that LTP/LTD are dependent on changes in intracellular calcium concentrations. Calcium levels have been measured using the calcium sensitive dye fura-2, whose fluorescence spectrum changes upon formation of the [fura-2-Ca2+] complex. Our LTP-inducing protocol comprised of two glutamate stimuli of identical size and duration (50 mM, 30 s) which were separated by 35 min. We could demonstrate that such a stimulation pattern gives rise to approx. 25% larger calcium influx at the second stimulus. It has been shown than such a stimulation pattern gives rise to an average of 25% augmentation (potentiation) of the second response, with 69% of potentiated cells. This experimental paradigm shows the pharmacological properties of LTP, established by previous electrophysiological studies:- blocking of NMDARs and mGluRs eliminates LTP induction;- blocking of AMPARs and L-type VGCCs does not eliminate LTP induction. Having obtained a system for induction and following of LTP-like changes, a preliminary application example was performed. Its purpose was to investigate possible influence of nicotine and galanthamine on our potentiation effect. Nicotine (100 mM) was shown both to increase and to eliminate glutamate-induced potentiation. Galanthamine coapplication (0.5 mM) with nicotine and glutamate exerted no effect on nicotinic modulation. However, galanthamine coapplied with glutamate alone seems to augment glutamate-induced potentiation. An LTP model system presented here could be additionally refined, by variation of glutamate application times, and testing for dependence on various forms of protein kinases. Galanthamine effect would probably be better addressed by cell-to-cell measurements instead of statistical approach, with subsequent identification of the cell type. Alternatively, combined calcium imaging – electrophysiological experiments could be performed. Spatial and temporal properties of intracellular ion dynamics could be utilised as diagnostic tools of the physiological state of the cells, thereby finding its application in functional proteomics.

Neurons derived from P19 embryonic carcinoma cells as a platform for biosensor applications - optimisation and characterisation

P19 is a mouse-derived embryonal carcinoma cell line capable of differentiation toward ectodermal, mesodermal and endodermal lineages and could thus be differentiated into neurons. Different culture conditions were tested to optimise and increase the efficiency of neuronal differentiation since the population of P19-derived neurons was reported to be heterogeneous with respect to the morphology and neurotransmitters they synthesise. P19-derived neurons were cultured on microelectrode arrays as cell aggregates and as dissociated cells. Improved neuronal maturation was shown by the presence of microtubule associated protein 2, neurofilament and synaptophysin formation when initiation of neuronal differentiation was prolonged. High initial cell density cultures and coating of surfaces with polyethylenimine-laminin further improved neuronal maturation of differentiated P19 cells. Increased spontaneous activities of the P19-derived neurons were correspondingly recorded. Two to three hours recordings were performed between 17 and 25 days when extracellular signals were stabilised. It was found that P19-derived neurons developed network properties as partially synchronised network activities. P19-derived neurons appeared to give inhomogenous response to the 2 major neurotransmitters, -aminobutyric acid (GABA) and glutamate. The P19-derived neuronal networks obtained from optimised protocol in this thesis were predominantly GABAergic. The reproducible long term extracellular recordings performed showed that neurons derived from P19 embryonal carcinoma cells could be applied as a model for cell based biosensor in corporation with microelectrode arrays.

Signalling mechanisms affecting regulated neurotrophin secretion in hippocampal neurons

Die Neurotrophine aus Säugetiere BDNF und NT-3 sind von Neuronen sekretierte Wachstumsfaktoren. Ferner sind Neurotrophine in verschiedene Formen der aktivitätsabhängigen synaptische Plastizität involviert. Obwohl die Ausschüttung von Neurotrophine aus Synapsen beschrieben worden ist, sind die intrazellulären Signalkaskaden, die die synaptische Ausschüttung von Neurotrophine regulieren, bei weitem nicht verstanden. Deswegen ist die Analyse der Sekretion von Neurotrophine auf subzellulärer Ebene erforderlich, um die genaue Rolle von präsynaptische und postsynaptische NT-Sekretion in der synaptischen Plastizität aufzudecken. In der vorliegenden Arbeit wurden die Kulturen von dissoziierten hippocampalen Neuronen aus Ratten mit grün fluoreszierenden Protein-markierten Konstrukten von BDNF und NT-3 transfiziert und Neurotrophine-enthaltenden Vesikeln durch die Colokalisierung mit dem cotransfizierten postsynaptischen Marker PSD-95-DsRed an glutamatergen Synapsen identifiziert. Depolarisationsinduzierte Sekretion von BDNF und NT-3 wurde per Direktaufnahme am Fluoreszenzmikroskop beobachtet. Die unvermittelte postsynaptische Depolarisation mit erhöhtem Kalium, in Gegenwart von Inhibitoren der synaptischen Transmission, erlaubte die Untersuchung der Signalwege, die am postsynaptischen Sekretionsprozess der Neurotrophinvesikel beteiligt sind. Es konnte gezeigt werden, dass die depolarisationsinduzierte postsynaptische Ausschüttung der Neurotrophine durch Calcium-Einstrom ausgelöst wird, entweder über L-Typ-spannungsabhängige Calcium-Kanäle oder über NMDA-Rezeptoren. Eine anschließende Freisetzung von Calcium aus intrazellulären Speichern über Ryanodin-Rezeptoren ist für den Sekretionsprozess erforderlich. Die postsynaptische Neurotrophinausschüttung wird durch KN-62 und KN-93 gehemmt, was auf eine unmittelbare Abhängigkeit von aktiver alpha-Calcium-Calmodulin-abhängige Proteinkinase II (CaMKII) hinweist. Der Inhibitor der cAMP/Proteinkinase A (PKA), Rp-cAMP-S, sowie der NO-Donor, SNP, minderten die Neurotrophinausschüttung. Hingegen blieben die Erhöhung des intrazellulären cAMP und der NO-Synthase-Inhibitor L-NMMA ohne Wirkung. Mit dem Trk-Inhibitor K252a konnte gezeigt werden, dass autokrine Neurotrophin-induzierte Neurotrophinausschüttung nicht an der synaptischen Freisetzung der Neurotrophine beiträgt und, dass BDNF seine eigene postsynaptische Sekretion nicht auslöst. Freisetzungsexperimente mit dem Fluoreszenz-Quencher Bromphenolblau konnten den Nachweis erbringen, dass asynchrone und anhaltende Fusionsporenöffnung von Neurotrophinvesikeln während der Sekretion stattfindet. Wegen der im Vergleich zum komplexen Sekretionsprozess schnellen Fusionsporenöffnung, scheint die Freisetzungsgeschwindigkeit von Neurotrophine durch ihre Diffusion aus dem Vesikel begrenzt. Zusammenfassend zeigen diese Ergebnisse eine starke Abhängigkeit der aktivitätsabhängigen postsynaptischen Neurotrophinausschüttung vom Calcium-Einstrom, von der Freisetzung von Calcium aus internen Speichern, von der Aktivierung der CaMKII und einem intakten Funktion der PKA, während der Trk-Signalweg, die Aktivierung von Natrium-Kanäle und NO-Signale nicht erforderlich sind.

Turning placenta into brain: placental mesenchymal stem cells differentiate into neurons and oligodendrocytes

We aimed to induce neural stem (NSC) and progenitor cells (NPC) from human placental tissues.

Angiotensinergic and noradrenergic neurons in the rat and human heart

Although the physiological and pharmacological evidences suggest a role for angiotensin II (Ang II) with the mammalian heart, the source and precise location of Ang II are unknown. To visualize and quantitate Ang II in atria, ventricular walls and interventricular septum of the rat and human heart and to explore the feasibility of local Ang II production and function, we investigated by different methods the expression of proteins involved in the generation and function of Ang II. We found mRNA of angiotensinogen (Ang-N), of angiotensin converting enzyme, of the angiotensin type receptors AT(1A) and AT(2) (AT(1B) not detected) as well as of cathepsin D in any part of the hearts. No renin mRNA was traceable. Ang-N mRNA was visualized by in situ hybridization in atrial ganglial neurons. Ang II and dopamine- -hydroxylase (D H) were either colocalized inside the same neuronal cell or the neurons were specialized for Ang II or D H. Within these neurons, the vesicular acetylcholine transporter (VAChT) was neither colocalized with Ang II nor D H, but VAChT-staining was found with synapses en passant encircle these neuronal cells. The fibers containing Ang II exhibited with blood vessels and with cardiomyocytes supposedly angiotensinergic synapses en passant. In rat heart, right atrial median Ang II concentration appeared higher than septal and ventricular Ang II. The distinct colocalization of neuronal Ang II with D H in the heart may indicate that Ang II participates together with norepinephrine in the regulation of cardiac functions: Produced as a cardiac neurotransmitter Ang II may have inotropic, chronotropic or dromotropic effects in atria and ventricles and contributes to blood pressure regulation.