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Pluripotent Stem Cell Based Cultures to Study Key Aspects of Human Cerebral Cortex Development
Get PDFThe human brain is a highly organized structure and the cerebral cortex in particular has expanded massively in size during evolution. The cerebral cortex is arranged into layers of specialized neuron subtypes formed during development by orchestrated stem cell maintenance, expansion, fate commitment and differentiation. The cortical neural stem cells generate billions of neurons in a systematic fashion. The mechanisms and their interplay that control most aspects of human brain development are unclear. This is partially due to the ethical and practical challenges associated with analyzing fetal human development. Recent progress into understanding the formation of the human brain has taken advantage of in vitro modeling of corticogenesis using pluripotent cells. Human pluripotent stem cells and procedures developed for their differentiation provided previously unavailable opportunities to study the mechanisms involved in development of the cerebral cortex. These human cell culture models can be applied to address specific biological questions and have been successfully utilized to investigate mechanisms associated, not only with normal brain
development, but also neuropsychiatric disorders. Here, we review the recent literature that uses these cell culture models to study human corticogenesis. Then, we discuss the challenges and limitations of the current models
Ketamine enhances structural plasticity in human dopaminergic neurons: possible relevance for treatment-resistant depression
Get PDFKetamine increases the expression of GluR1 and GluR2 α-amino-3-hydroxy-5-methy-4-isoxazole propionate receptor subunits in human dopaminergic neurons differentiated from induced pluripotent stem cells
No full textThe mechanisms underlying the prolonged antidepressant effects after a single exposure to ketamine are only partially understood. Converging findings indicate a critical role of structural neuroplasticity, recently also proposed for dopaminergic (DA) neurons known to be involved in a depression core symptom, anhedonia. We recently showed that ketamine induces dendritic outgrowth in human DA neurons differentiated in vitro from iPSC of healthy donors, a phenomenon blocked by the AMPA receptor antagonist NBQX. Since changes of expression of AMPA receptor subunits GluR1 and GluR2 were observed in neuroplasticity of rodent DA neurons, we aimed to explore this phenomenon in human DA neurons. Using specific antibodies against GluR1 and GluR2 AMPA receptor subunits, we demonstrated that GluR1 levels were significantly higher in soma than in dendrites, while for GluR2 levels were significantly higher in dendrites than in soma. One hr exposure to 1 μM ketamine increased the signal of both subunits in dendrites, but only of GluR2 in soma, at 24, 48 and 72 hrs. Non-linear polynomial fitting of dendritic expression indicated that the two curves were significantly different, with a stronger and more sustained effects on GluR2 expression. Both curves showed a relatively rapid building towards higher values when compared with the slow progression of structural plasticity parameters, whose maximal effects were observed at 72 hrs. Overall, these data support a role for GluR1 and GluR2 dendritic upregulation in driving structural plasticity in human DA neurons depending upon ketamine transient exposure, indicating translationally relevant downstream mechanism possibly involved in antidepressant effects
(2 R,6 R)-Hydroxynorketamine promotes dendrite outgrowth in human inducible pluripotent stem cell-derived neurons through AMPA receptor with timing and exposure compatible with ketamine infusion pharmacokinetics in humans
No full textThe mechanisms underlying the prolonged antidepressant effects after a single infusion of ketamine are only partially understood. Ketamine half-life of about 2 hours cannot explain antidepressant effects that last for one week, suggesting the triggering of long lasting neuroplasticity. Recent human pharmacokinetics (PK) data indicate that a ketamine metabolite, (2R,6R)-hydroxynorketamine (HNK), persists in the high submicromolar range for additional 6-12 hours. Since in rodents HNK can induce dendrite outgrowth via AMPA receptor-mediated mechanisms, in this work we aimed to show that HNK produces similar effects in human neurons at concentrations and exposure-time compatible with human PK after ketamine infusion. Human dopaminergic neurons were differentiated in vitro from iPSCs obtained from healthy donors. Exposure to submicromolar HNK for 6 hours produced dendritic outgrowth when measured 3 days after exposure. These neuroplasticity effects were similar to those obtained with exposure to micromolar concentrations of ketamine for 1 or 6 hours and were blocked by pretreatment with the AMPA receptor antagonists NBQX and GYKI 52466, as well as by the mTOR pathway blocker rapamycin. It is reasonable to conclude that the mechanistic similarity of the effects produced by ketamine and HNK and their diachronic brain exposure due to their different plasma PK observed after single therapeutic infusion can contribute to the final sustained antidepressant action
Involvement of DA D3 Receptors in Structural Neuroplasticity of Selected Limbic Brain Circuits: Possible Role in Treatment-Resistant Depression
No full textStructural neuroplasticity in the adult brain is a process involving quantitative changes of the number and size of neurons and of their dendritic arborization, axon branching, spines, and synapses. These changes can occur in specific neural circuits as adaptive response to environmental challenges, exposure to stressors, tissue damage or degeneration. Converging studies point to evidence of structural plasticity in circuits operated by glutamate, GABA, dopamine, and serotonin neurotransmitters, in concert with neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF) or Insulin Growth Factor 1 (IGF1) and a series of modulators that include circulating hormones. Intriguingly, most of these endogenous agents trigger the activation of the PI3K/Akt/mTOR and ERK1/2 intracellular pathways that, in turn, lead to the production of growth-related structural changes, enhancing protein synthesis, metabolic enzyme functions, mitogenesis for energy, and new lipid-bilayer membrane apposition. The dopamine (DA) D3 receptor has been shown to play a specific role by inducing structural plasticity of the DAergic neurons of the nigrostriatal and mesocorticolimbic circuit, where they are expressed in rodents and humans, via activation of the mTORC1 and ERK1/2 pathways. These effects are BDNF-dependent and require the recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to allow the structural changes. Since in mood disorders, depression and anhedonia have been proposed to be associated with impaired neuroplasticity and reduced DAergic tone in brain circuits connecting prefrontal cortex, ventral striatum, amygdala, and ventral mesencephalon, activation of D3 receptors could provide a therapeutic benefit. Sustained improvements of mood and anhedonia were observed in subjects with an unsatisfactory response to serotonin uptake inhibitors (SSRI) when treated with D3-preferential D2/D3 agonists such as pramipexole and ropinirole. The recent evidence that downstream mTOR pathway activation in human mesencephalic DA neurons is also produced by ketamine, probably the most effective antidepressant currently used in subjects with treatment-resistant depression, further supports the rationale for a D3 receptor activation in mood disorders
Ketamine effects on mammalian target of rapamycin signaling in the mouse limbic system depend on functional dopamine D3 receptors
No full textKetamine is a noncompetitive glutamate N-methyl-D-aspartic acid receptor antagonist. When acutely administered to rodents, it produces a rapid antidepressant effect. There is evidence that N-methyl-D-aspartic acid receptor blockade enhances glutamatergic transmission preferentially engaging α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leading to mTOR (mammalian target of rapamycin) pathways activation, thus resulting into downstream neuroadaptive changes in limbic structures. Recent in-vitro data on primary neuronal cultures showed that ketamine activates mTOR also in dopaminergic neurons, and this activation depends on the presence of functional dopamine D3 receptors. The aim of this work was to study the in-vivo relevance of viable D3 receptors in mediating the effects of acute ketamine administration on the mTOR downstream substrate p70 ribosomal S6 kinase (p70S6K), an obligatory substrate for mTOR. We compared the effects of single ketamine 5 mg/kg, 10 mg/kg, or vehicle injection in wild-type and D3 receptor knockout mice. Animals were killed after 60 min, and their brains were processed for p-p70S6K immunohistochemistry. Ketamine increased p70S6K phosphorylation in prefrontal cortex, nucleus accumbens core and shell, ventral tegmental area, substantia nigra, hippocampal CA1, CA2, and CA3, and basolateral amygdala of wild-type mice but not in mutant mice. Our study demonstrates that ketamine-induced p70S6K phosphorylation is dependent on viable D3R expressed in most of limbic structures
Synergic action of L-acetylcarnitine and L-methylfolate in Mouse Models of Stress-Related Disorders and Human iPSC-Derived Dopaminergic Neurons
No full textThe epigenetic agents, L-acetylcarnitine (LAC) and L-methylfolate (MF) are putative candidates as add-on drugs in depression. We evaluated the effect of a combined treatment with LAC and MF in two different paradigms of chronic stress in mice and in human inducible pluripotent stem cells (hiPSCs) differentiated into dopaminergic neurons. Two groups of mice were exposed to chronic unpredictable stress (CUS) for 28 days or chronic restraint stress (CRS) for 21 day, and LAC (30 or 100 mg/kg) and/or MF (0.75 or 3 mg/kg) were administered i.p. once a day for 14 days, starting from the last week of stress. In both stress paradigms, LAC and MF acted synergistically in reducing the immobility time in the forced swim test and enhancing BDNF protein levels in the frontal cortex and hippocampus. In addition, LAC and MF acted synergistically in enhancing type-2 metabotropic glutamate receptor (mGlu2) protein levels in the hippocampus of mice exposed to CRS. Interestingly, CRS mice treated with MF showed an up-regulation of NFκB p65, which is a substrate for LAC-induced acetylation. We could also demonstrate a synergism between LAC and MF in cultured hiPSCs differentiated into dopamine neurons, by measuring dendrite length and number, and area of the cell soma after 3 days of drug exposure. These findings support the combined use of LAC and MF in the treatment of MDD and other stress-related disorders
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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