Derivation, Characterization, and Stable Transfection of Induced Pluripotent Stem Cells from Fischer344 Rats

The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species

Behavioral, neuronal, and development consequences of genetically decreased tryptophan hydroxylase 2 activity

Serotonin (5-Hydroxytryptamin, 5-HT) ist ein wichtiger Neurotransmitter im Zentralnervensystem (ZNS). Seine Biosynthese erfolgt unter Beteiligung des Enzyms Tryptophanhydroxylase 2 (TPH2). Polymorphismen im TPH2 Gen beim Menschen sind Risikofaktoren bei der Entstehung von Depressionen und Angstverhalten. Die gängigsten Antidepressiva und Anxiolytika wirken auf das Serotonin System. Unklar ist, ob das komplette oder teilweise Fehlen von Serotonin im Gehirn zu Entwicklungsstörungen und neurochemischen oder psychologischen Veränderungen führt. In dieser Arbeit werden Mauslinien mit unterschiedlichen TPH2 Aktivitäten im ZNS verglichen und der Einfluss verringerter 5-HT Konzentrationen auf Entwicklung und Verhalten der Tiere untersucht. Zentrales Serotonin ist nur für die postnatale Entwicklung notwendig. Das verzögerte Wachstum von Tph2-/- Tieren ist nicht auf eine Störung der Hypothalamus-Hypophysen-Nebennieren-Achse oder auf metabolische Veränderungen zurückzuführen, sondern kann aus verringerter Vokalisation im Ultraschallbereich resultieren. Tph2-/- Mäuse wurden mit generierten Mausmodellen mit niedriger TPH2 Aktivität vergleichen. Die Ergebnisse zeigen, dass 20% weniger zentrales Serotonin nicht ausreichen, um Depression oder Angst-Verhalten herbeizuführen. Möglicherweise greifen kompensatorische Mechanismen wie ein verringerter Serotoninmetabolismus oder eine gesteigerte 5-HT1A-Rezeptorsensitivität. Der komplette Verlust von Serotonin im Gehirn führt zu einem starken depressiven und weniger ängstlich Verhalten, mit erhöhter Aggression - ohne Veränderung in Aktivität, Geruchsinn, Gedächnis und adulter Neurogenese. Fluoxetine Behandlung von Tph2-defizienten Mäusen zeigte einen Serotonin-unabhängigen Effekt dieses Antodepressivums auf Angst-Verhalten und Depression. Fluoxeine reduzieren den Serotoningehalt im Gehirn von Mäusen mit geringen TPH2-Aktivität, was zeigt, dass TPH-Aktivität die Effizienz von Serotonin beeinflussenAntidepressiva bestimmen,Serotonin (5-HT) is a major neurotransmitter in the brain biosynthesis of which is initiated by tryptophan hydroxylase 2 (TPH2). Polymorphisms in the TPH2 gene are suggested as risk factors associated with depression and anxiety in humans. Furthermore, the most frequently prescribed antidepressants and anxiolytics target the serotonergic system. However, the question whether a complete ablation or partial reduction in brain serotonin leads to the developmental, neurochemical, or psychological abnormalities remains unresolved. In this study, I took advantage of mouse lines with various degree of decrease in TPH2 activity in order to dissect the impact of 5-HT loss on development, brain neurochemistry and behavior. Using Tph2-deficient mice I showed that central serotonin is essential for normal postnatal, but not prenatal development. Growth retardation of Tph2-/- mice was not a result of a disruption of the hypothalamo-pituitary-adrenal axis, metabolic abnormalities, or impaired thermoregulation, but could result from reduced ultrasonic vocalization. I tested Tph2-/- mice along with other newly generated mouse models with partial TPH2 reduction, and showed that 20% reduction in central serotonin is not enough to cause changes in anxiety- and depression-like behaviors most likely due to compensatory mechanisms including reduced serotonin metabolism and increased 5-HT1A receptor sensitivity. However, complete loss of central serotonin leads to a depression-like phenotype, reduced anxiety-like behavior, and exaggerated aggression, but no differences in activity, olfaction, memory, and adult neurogenesis. Fluoxetine treatment of Tph2-/- mice revealed serotonin-independent action of this antidepressant on anxiety- and depression-like behavior. Furthermore, fluoxetine drastically reduced the brain 5-HT content in mice with low TPH2 activity indicating that TPH2 activity may determine the efficiency of antidepressants targeting the serotonergic system

Is L-lactate a novel signaling molecule in the brain?

In the brain, L-lactate is produced by both neurons and astrocytes. There is no doubt that neurons use L-lactate as a supplementary fuel although the importance of this energy source is disputed. Irrespective of its caloric value, L-lactate might also have a signaling role in the brain. Here, we review several current hypotheses of L-lactate mediated signaling. Some proposed mechanisms require L-lactate entry into the neurons leading to a shift in ATP/ADP ratio or redox state. Others postulate interaction with either known receptor HCA1 (GPR81) or a novel, yet unidentified receptor. We argue that the sensitivity of any such mechanism has to match the concentration range of extracellular L-lactate, which is less than ~1.5 mmol/L under physiologic conditions. From that point of view, some of the proposed mechanisms require supraphysiologic levels of L-lactate and could be engaged during ischemia or seizures when L-lactate concentration rises dramatically. Currently, we do not know whether L-lactate production in the brain occurs in microdomains, which might create higher than average local concentrations. Nevertheless, it is clear that in the brain, as in the peripheral tissues, L-lactate is not only used as a source of energy but also acts as a signaling molecule