RESUMO
Generating stable human embryonic stem cells (hESCs) with targeted genetic mutations allows for the interrogation of protein function in numerous cellular contexts while maintaining a relatively high degree of isogenicity. We describe a step-by-step protocol for generating knockout hESC lines with mutations in genes involved in synaptic transmission using CRISPR-Cas9. We describe steps for gRNA design, cloning, stem cell transfection, and clone isolation. We then detail procedures for gene knockout validation and differentiation of stem cells into functional induced neurons.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Células-Tronco Embrionárias Humanas , Neurônios , Humanos , Sistemas CRISPR-Cas/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Edição de Genes/métodos , Diferenciação Celular/genética , Técnicas de Inativação de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Sinapses/metabolismo , Sinapses/genéticaRESUMO
Rapid release of neurotransmitters in synchrony with action potentials is considered a key hardwired property of synapses. Here, in glutamatergic synapses formed between induced human neurons, we show that action potential-dependent neurotransmitter release becomes progressively desynchronized as synapses mature and age. In this solely excitatory network, the emergence of NMDAR-mediated transmission elicits endoplasmic reticulum (ER) stress leading to downregulation of key presynaptic molecules, synaptotagmin-1 and cysteine string protein α, that synchronize neurotransmitter release. The emergence of asynchronous release with neuronal maturity and subsequent aging is maintained by the high-affinity Ca2+ sensor synaptotagmin-7 and suppressed by the introduction of GABAergic transmission into the network, inhibition of NMDARs, and ER stress. These results suggest that long-term disruption of excitation-inhibition balance affects the synchrony of excitatory neurotransmission in human synapses.
Assuntos
Neurônios , Transmissão Sináptica , Humanos , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Sinapses/metabolismo , Neurotransmissores/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Envelhecimento , Cálcio/metabolismoRESUMO
Ketamine produces rapid antidepressant action in patients with major depression or treatment-resistant depression. Studies have identified brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), as necessary for the antidepressant effects and underlying ketamine-induced synaptic potentiation in the hippocampus. Here, we delete BDNF or TrkB in presynaptic CA3 or postsynaptic CA1 regions of the Schaffer collateral pathway to investigate the rapid antidepressant action of ketamine. The deletion of Bdnf in CA3 or CA1 blocks the ketamine-induced synaptic potentiation. In contrast, ablation of TrkB only in postsynaptic CA1 eliminates the ketamine-induced synaptic potentiation. We confirm BDNF-TrkB signaling in CA1 is required for ketamine's rapid behavioral action. Moreover, ketamine application elicits dynamin1-dependent TrkB activation and downstream signaling to trigger rapid synaptic effects. Taken together, these data demonstrate a requirement for BDNF-TrkB signaling in CA1 neurons in ketamine-induced synaptic potentiation and identify a specific synaptic locus in eliciting ketamine's rapid antidepressant effects.