Akute optogenetische Stimulation von serotonergen Neuronen in Raphe-Kernen reduziert Zellproliferation im Gyrus dentatus der Maus

The subgranular zone of the hippocampal dentate gyrus (DG) is known to be replenished with new-born neurons even after fetal and early postnatal development has completed, a phenomenon called adult neurogenesis. Functions of these new-born neurons and regulatory mechanisms of adult neurogenesis are not fully understood. The DG receives various synaptic inputs including those from serotonergic neurons in the brain stem raphe nuclei. Previous studies in mice have suggested that physical activities enhance neurogenesis via serotonergic neurotransmission. In the present study, the influence of serotonin (5-HT) on adult neurogenesis was investigated by directly activating serotonergic neurons in the dorsal and median raphe nucleus (DRN, MRN) using optogenetics. Specifically, cell proliferation was examined by bromodeoxyuridine (BrdU) labeling in the DG after overnight and one-week optogenetic activation of serotonergic neurons in the raphe nuclei in freely behaving tryptophan hydroxylase 2-channelrhodopsin 2 (Tph2-ChR2) transgenic mice. These mice express photo-excitable cation channels, ChR2, under the control of the Tph2 promoter, allowing specific activation of serotonergic neurons. First, specific activation of serotonergic neurons in the DRN or MRN was electrophysiologically confirmed in brain slices and in anesthetized mice. In consequence of the optogenetic activation of serotonergic neurons in the DRN or MRN, a reduction in firing rates was observed in the DG in the anesthetized mice. After overnight stimulation of serotonergic neurons in the freely behaving animals, BrdU-positive cells in the DG were significantly reduced in number compared to non-stimulated control mice. The total distances traveled did not differ significantly between stimulated and non-stimulated mice, indicating that the reduced cell proliferation in the DG was not due to reduced physical movement. Further, as revealed by high-performance liquid chromatography (HPLC), reduced cell proliferation in the DG was accompanied by reduced 5-HT concentrations in both the raphe nuclei and the hippocampus, which may reflect autoinhibitory regulation of 5-HT release. When optogenetic stimulation of serotonergic neurons was extended for 6 nights, reduction in cell proliferation was absent without affecting total distances traveled, although interhemispheric differences in the number of BrdU-positive cells were observed. In sum, acute optogenetic stimulation of serotonergic neurons temporarily decreases cell proliferation in the DG, with the effect disappearing if the stimulation continues over days. This effect could be mediated by an electrophysiologically measured inhibitory influence of 5-HT on the DG and adaptive changes subsequent to long-term optogenetic stimulation.Die subgranuläre Zone des Gyrus dentatus (DG) im Hippocampus wird auch nach Ende der fetalen und frühen postnatalen Entwicklung mit neugeborenen Neuronen aufgefüllt, ein Phänomen, das als adulte Neurogenese bezeichnet wird. Die Funktionen dieser neugeborenen Neuronen und die Regulationsmechanismen der adulten Neurogenese sind nicht völlig verstanden. Der DG erhält verschiedene synaptische Inputs, einschließlich derer von serotonergen Neuronen in den Raphe-Kernen des Hirnstamms. Frühere Studien an Mäusen zeigten, dass körperliche Aktivitäten die Neurogenese mittels serotonerger Neurotransmission verbessern. In der vorliegenden Studie wurde der Einfluss von Serotonin (5-HT) auf adulte Neurogenese durch direkte Aktivierung serotonerger Neuronen im dorsalen und medianen Raphe-Kern (DRN, MRN) mittels Optogenetik untersucht. Konkret wurde die Zellproliferation durch Bromdesoxyuridin (BrdU)-Markierung im DG nach über Nacht und einwöchiger optogenetischer Aktivierung von serotonergen Neuronen in den Raphe-Kernen in sich frei bewegenden transgenen Tryptophan Hydroxylase 2-Channelrhodopsin 2 (Tph2-ChR2)-Mäusen untersucht. Diese Mäuse exprimieren photoanregbare Kationenkanäle, ChR2, unter der Kontrolle des Tph2-Promotors, was eine spezifische Aktivierung von serotonergen Neuronen ermöglicht. Zuerst wurde die spezifische Aktivierung von serotonergen Neuronen im DRN oder MRN in Gehirnschnitten und in anästhesierten Mäusen mit elektrophysiologischen Methoden bestätigt. In Folge der optogenetischen Aktivierung von serotonergen Neuronen im DRN oder MRN wurde eine Senkung der Feuerraten im DG in anästhesierten Mäusen beobachtet. Nach Stimulation von serotonergen Neuronen über Nacht in frei beweglichen Tieren war die Zahl der BrdU-positiven Zellen im DG im Vergleich zu nicht-stimulierten Kontrollen signifikant reduziert. Die reduzierte Zellproliferation war nicht auf reduzierte körperliche Bewegung zurückzuführen, da sich die zurückgelegte Gesamtstrecke zwischen stimulierten und nicht-stimulierten Mäusen nicht unterschied. Hochleistungs-Flüssigkeitschromatographie (HPLC) zeigte, dass die reduzierte Zellproliferation im DG von reduzierten 5-HT-Konzentrationen in den Raphe-Kernen und im Hippocampus begleitet wurde, was eine autoinhibitorische Regulierung der 5-HT-Freisetzung widerspiegeln könnte. Wenn die optogenetische Stimulation serotonerger Neuronen auf 6 Nächte verlängert wurde, wurden zwar interhemisphärische Unterschiede in der Anzahl von BrdU-positiven Zellen beobachtet, doch weder die Zellproliferation noch die zurückgelegte Gesamtstrecke waren beeinflusst. Kurz gefasst verringert die akute optogenetische Stimulation serotonerger Neuronen die Zellproliferation im DG vorübergehend, aber dieser Effekt verschwindet, wenn die Stimulation über Tage andauert. Elektrophysiologisch gemessen könnte dieser Effekt durch den inhibitorischen Einfluss von 5-HT auf den DG und nachfolgende adaptive Veränderungen nach langfristiger optogenetischer Stimulation vermittelt sein

The identification and functional implications of human-specific "fixed" amino acid substitutions in the glutamate receptor family

<p>Abstract</p> <p>Background</p> <p>The glutamate receptors (GluRs) play a vital role in the mediation of excitatory synaptic transmission in the central nervous system. To clarify the evolutionary dynamics and mechanisms of the GluR genes in the lineage leading to humans, we determined the complete sequences of the coding regions and splice sites of 26 chimpanzee GluR genes.</p> <p>Results</p> <p>We found that all of the reading frames and splice sites of these genes reported in humans were completely conserved in chimpanzees, suggesting that there were no gross structural changes in humans after their divergence from the human-chimpanzee common ancestor. We observed low <it>K</it>_<it>A</it>/<it>K</it>_{<it>S </it>}ratios in both humans and chimpanzees, and we found no evidence of accelerated evolution. We identified 30 human-specific "fixed" amino acid substitutions in the GluR genes by analyzing 80 human samples of seven different populations worldwide. Grantham's distance analysis showed that <it>GRIN2C </it>and <it>GRIN3A </it>are the most and the second most diverged GluR genes between humans and chimpanzees. However, most of the substitutions are non-radical and are not clustered in any particular region. Protein motif analysis assigned 11 out of these 30 substitutions to functional regions. Two out of these 11 substitutions, D71G in <it>GRIN3A </it>and R727H in <it>GRIN3B</it>, caused differences in the functional assignments of these genes between humans and other apes.</p> <p>Conclusion</p> <p>We conclude that the GluR genes did not undergo drastic changes such as accelerated evolution in the human lineage after the divergence of chimpanzees. However, there remains a possibility that two human-specific "fixed" amino acid substitutions, D71G in <it>GRIN3A </it>and R727H in <it>GRIN3B</it>, are related to human-specific brain function.</p

Serotonin Deficiency Increases Context-Dependent Fear Learning Through Modulation of Hippocampal Activity

Brain serotonin (5-hydroxytryptamine, 5-HT) system dysfunction is implicated in exaggerated fear responses triggering various anxiety-, stress-, and trauma-related disorders. However, the underlying mechanisms are not well understood. Here, we investigated the impact of constitutively inactivated 5-HT synthesis on context-dependent fear learning and extinction using tryptophan hydroxylase 2 (Tph2) knockout mice. Fear conditioning and context-dependent fear memory extinction paradigms were combined with c-Fos imaging and electrophysiological recordings in the dorsal hippocampus (dHip). Tph2 mutant mice, completely devoid of 5-HT synthesis in brain, displayed accelerated fear memory formation and increased locomotor responses to foot shock. Furthermore, recall of context-dependent fear memory was increased. The behavioral responses were associated with increased c-Fos expression in the dHip and resistance to foot shock-induced impairment of hippocampal long-term potentiation (LTP). In conclusion, increased context-dependent fear memory resulting from brain 5-HT deficiency involves dysfunction of the hippocampal circuitry controlling contextual representation of fear-related behavioral responses

Serotonin Deficiency Increases Context-Dependent Fear Learning Through Modulation of Hippocampal Activity

Brain serotonin (5-hydroxytryptamine, 5-HT) system dysfunction is implicated in exaggerated fear responses triggering various anxiety-, stress-, and trauma-related disorders. However, the underlying mechanisms are not well understood. Here, we investigated the impact of constitutively inactivated 5-HT synthesis on context-dependent fear learning and extinction using tryptophan hydroxylase 2 ( ) knockout mice. Fear conditioning and context-dependent fear memory extinction paradigms were combined with c-Fos imaging and electrophysiological recordings in the dorsal hippocampus (dHip). mutant mice, completely devoid of 5-HT synthesis in brain, displayed accelerated fear memory formation and increased locomotor responses to foot shock. Furthermore, recall of context-dependent fear memory was increased. The behavioral responses were associated with increased c-Fos expression in the dHip and resistance to foot shock-induced impairment of hippocampal long-term potentiation (LTP). In conclusion, increased context-dependent fear memory resulting from brain 5-HT deficiency involves dysfunction of the hippocampal circuitry controlling contextual representation of fear-related behavioral responses.</p

Elektrophysiologische Untersuchung bei zwei Tiermodellen für emotionale Störungen - Serotonin Transporter knockout Mäuse und Tryptophan Hydroxylase 2 knockout Mäuse

Serotonin (5-HT) has been implicated in the regulation of emotions as well as in its pathological states, such as anxiety disorders and depression. Mice with targeted deletion of genes encoding various mediators of central serotonergic neurotransmission therefore provides a powerful tool in understanding contributions of such mediators to homeostatic mechanisms as well as to the development of human emotional disorders. Within this thesis a battery of electrophysiological recordings were conducted in the dorsal raphe nucleus (DRN) and the hippocampus of two murine knockout lines with deficient serotonergic systems. Serotonin transporter knockout mice (5-Htt KO), which lack protein responsible for reuptake of 5-HT from the extracellular space and tryptophan hydroxylase 2 knockout (Tph2 KO) mice, which lack the gene encoding the neuronal 5-HT-synthesising enzyme. First, 5-HT1A receptor-mediated autoinhibition of serotonergic neuron firing in the DRN was assessed using the loose-seal cell-attached configuration. Stimulation of 5-HT1A receptors by a selective agonist, R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT), showed a mild sensitisation and a marked desensitisation of these receptors in Tph2 KO and 5-Htt KO mice, respectively. While application of tryptophan, a precursor of 5-HT and a substrate of Tph2, did not cause autoinhibition in Tph2 KO mice due to the lack of endogenously produced 5-HT, data from 5-Htt KO mice as well as heterozygous mice of both KO mice lines demonstrated the presence of autoinhibitory mechanisms as normal as seen in wildtype (WT) controls. When the Tph2-dependent step in the 5-HT synthesis pathway was bypassed by application of 5-hydroxytryptophan (5-HTP), serotonergic neurons of both Tph2 KO and 5-Htt KO mice showed decrease in firing rates at lower concentrations of 5-HTP than in WT controls. Elevated responsiveness of serotonergic neurons from Tph2 KO mice correspond to mild sensitisation of 5-HT1A receptors, while responses from 5-Htt KO mice suggest that excess levels of extracellular 5-HT, created by the lack of 5-Htt, stimulates 5-HT1A receptors strong enough to overcome desensitisation of these receptors. Second, the whole-cell patch clamp recording data from serotonergic neurons in the DRN showed no differences in basic electrophysiological properties between Tph2 KO and WT mice, except lower membrane resistances of neurons from KO mice. Moreover, the whole-cell patch clamp recording from CA1 pyramidal neurons in the hippocampus of 5-Htt KO mice showed increased conductance both at a steady state and at action potential generation. Lastly, magnitude of long-term potentiation (LTP) induced by the Schaffer collateral/commissural pathway stimulation in the ventral hippocampus showed no differences among Tph2 KO, 5-Htt KO, and WT counterparts. Taken together, lack and excess of extracellular 5-HT caused sensitisation and desensitisation of autoinhibitory 5-HT1A receptors, respectively. However, this may not directly translate to the level of autoinhibitory regulation of serotonergic neuron firing when these receptors are stimulated by endogenously synthesised 5-HT. In general, KO mice studied here showed an astonishing level of resilience to genetic manipulations of the central serotonergic system, maintaining overall electrophysiological properties and normal LTP inducibility. This may further suggest existence of as-yet-unknown compensatory mechanisms buffering potential alterations induced by genetic manipulations.Serotonin (5-HT) ist an der Regulation von der Emotionen, sowie ihrer pathologischen Zustände, wie Angststörungen und Depressionen beteiligt. Mäuse denen, mittels einer zielgerichteteten Deletion von Genen, die verschiedenste Proteine involviert in der zentralen serotonergen Nerotransmission fehlen, dienen daher als ein nützliches Tiermodell, um die Rolle dieser Mediatoren bei Homöostasemechanismen und der Entwicklung emotionaler Störungen beim Menschen zu verstehen. Im Rahmen dieser Thesis wurde eine Batterie von elektrophysiologischen Ableitungen im Hippocampus sowie in der dorsalen Raphe Nucleus (DRN) zweier Knockout-Mauslinien mit einem defizienten serotonergen Systems durchgeführt. Serotonintransporter Knockout-Mäuse (5-Htt KO), denen das Protein zur Wiederaufnahme von 5-HT aus dem extrazellulären Raum fehlt und Tryptophanhydroxylase 2 Knockout-Mäuse (Tph2 KO), denen das Gen für das 5-HT-synthetisierende Enzym im Gehirn fehlt. Zunächst wurde mittels der “loose-seal cell-attached” Aufnahmemethode die Eigenhemmung der serotonergen Neuronen untersucht, die durch 5-HT1A Rezeptoren in der DRN vermittelt wird. Stimulierung der 5-HT1A Rezeptoren durch einen selektiven Agonist, R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT), zeigte eine milde Sensibilisierung und eine deutliche Desensibilisierung dieser Rezeptoren in Tph2 KO bzw. in 5-Htt KO Mäusen. Während die Anwendung von Tryptophan, eine Vorstufe von 5-HT und ein Substrat der Tph2, keine Eigenhemmung, aufgrund des Mangels an endogen produziertem 5-HT, in Tph2 KO Mäusen verursachte, wiesen Daten von 5-Htt KO Mäusen sowie von heterozygoten Mäusen beider KO Mauslinien die Existenz der Eigenhemmungsmechanismen wie in den Wildtypen (WT) nach. Wurde der Tph2-abhängige Schritt im 5-HT Syntheseweg durch Anwendung von 5-Hydroxytryptophan (5-HTP) umgangen, zeigten sowohl Tph2 KO als auch 5-Htt KO Mäuse eine Verminderung der serotonergen neuronalen Feuerungsrate bei niedrigeren Konzentrationen von 5-HTP im Vergleich zu den WT. Die erhöhte Reaktionsfähigkeit der serotonergen Neuronen von Tph2 KO Mäusen entsprechen der milden Sensibilisierung der 5-HT1A Rezeptoren. Stattdessen deuten die Reaktionen der serotonergen Neuronen von 5-Htt KO Mäusen darauf hin, dass das überschüssige Niveau von extrazellularem 5-HT, welches durch den Mangel an 5-Htt verursacht wird, 5-HT1A Rezeptoren stark genug stimuliert, um eine Desensibilisierung dieser Rezeptoren zu überwinden. Zweitens zeigten die Daten der whole-cell Patch Clamp Ableitung von serotonergen Neuronen im DRN keine Unterschiede in grundlegenden elektrophysiologischen Eigenschaften zwischen Tph2 KO und WT, außer niedrigen Membranwiderständen in KO Mäusen. Darüber hinaus zeigte die whole-cell Patch Clamp Ableitungen von CA1 Pyramidenzellen im Hippocampus der 5-Htt KO Mäuse eine erhöhte Leitfähigkeit sowohl bei Ruheständen als auch bei Aktionspotentialerzeugungen. Schließlich zeigte die Stärke der Langzeitpotenzierung (long-term potentiation: LTP) durch die Stimulation der Schaffer-Kollateralen/kommissuralen Fasern im ventralen Hippocampus keine Unterschiede zwischen Tph2 KO, 5-Htt KO, und jeweiligen WT. Zusammengefasst verursachten der Mangel und der Überschuss von extrazellularen 5-HT eine Sensibilisierung bzw. Desensibilisierung der autoinhibitorischen 5-HT1A Rezeptoren. Dies kann jedoch nicht direkt in die Regulierung von serotonergen Neuronen Feuerung umgesetzt werden, wenn die 5-HT1A Rezeptoren durch endogen synthetisiertes 5-HT stimuliert werden. Im Allgemeinen zeigten die hier untersuchten KO Mäuse, ein erstaunliches Maß an Widerstandskraft, die die allgemeinen elektrophysiologischen Eigenschaften und die normale LTP Induzierbarkeit trotz genetischer Manipulationen des zentralen serotonergen Systems aufrechterhielt. Weiterhin deutet dies auf die Existenz noch unbekannter Kompensationsmechanismen hin, die diese potentiellen Veränderungen abzudämpfen scheinen

Targeting brain serotonin synthesis: insights into neurodevelopmental disorders with long-term outcomes related to negative emotionality, aggression and antisocial behaviour

Aggression, which comprises multi-faceted traits ranging from negative emotionality to antisocial behaviour, is influenced by an interaction of biological, psychological and social variables. Failure in social adjustment, aggressiveness and violence represent the most detrimental long-term outcome of neurodevelopmental disorders. With the exception of brain-specific tryptophan hydroxylase-2 (Tph2), which generates serotonin (5-HT) in raphe neurons, the contribution of gene variation to aggression-related behaviour in genetically modified mouse models has been previously appraised (Lesch 2005 Novartis Found Symp. 268, 111–140; Lesch & Merschdorf 2000 Behav. Sci. Law 18, 581–604). Genetic inactivation of Tph2 function in mice led to the identification of phenotypic changes, ranging from growth retardation and late-onset obesity, to enhanced conditioned fear response, increased aggression and depression-like behaviour. This spectrum of consequences, which are amplified by stress-related epigenetic interactions, are attributable to deficient brain 5-HT synthesis during development and adulthood. Human data relating altered TPH2 function to personality traits of negative emotionality and neurodevelopmental disorders characterized by deficits in cognitive control and emotion regulation are based on genetic association and are therefore not as robust as the experimental mouse results. Mouse models in conjunction with approaches focusing on TPH2 variants in humans provide unexpected views of 5-HT's role in brain development and in disorders related to negative emotionality, aggression and antisocial behaviour

Impacts of Brain Serotonin Deficiency following Tph2 Inactivation on Development and Raphe Neuron Serotonergic Specification

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT$_{1A}$ and 5-HT$_{1B}$ receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis