Neuronal transdifferentiation from human primary adult fibroblasts
By onThis is a protocol for the direct conversion of human primary fibroblasts into neurons using a combination of transcription factor- and small molecule-based approach. The majority of converted neurons showing characteristics of cortical glutamatergic neurons.
Molecular heterogeneity in the substantia nigra: A roadmap for understanding PD motor pathophysiology
By onThis article discusses the existing knowledge of DA neuron subtypes and attempts to provide a roadmap for how their distinctive properties can provide novel insights into the motor symptoms of Parkinson's disease (PD).
Unique functional responses differentially map onto genetic subtypes of dopamine neurons
By onGenetic strategies to isolate dopaminergic subtypes established a novel subtype of dopamine neurons within the mouse substantia nigra. The results show that the subtypes each display different neural activity patterns related to locomotion and reward
Sleep-Wake Recording in Dan lab
By onSurgery and Setups for Recording 24-Hour Natural Sleep/Wake Cycles in Mice.
Immunohistochemistry on free-floating and paraffin-embedded tissue sections
By onThis protocol is used for free-floating frozen (30-50 microns) and paraffin-embedded (10 microns) tissue sections.
Cytokine profiling analysis on conditioned medium of human neurons using Luminex multiplex assay
By onThis protocol is used to identify secreted inflammatory factors by cytokine profiling of the conditioned medium from human transdifferentiated neurons of healthy donors and AD patients.
Disrupted sleep-wake regulation in the MCI-Park mouse model of Parkinson’s disease
By onDisrupted sleep has a profound adverse impact on lives of Parkinson's disease (PD) patients and their caregivers. Sleep disturbances are exceedingly common in PD, with substantial heterogeneity in type, timing, and severity. Among the most common sleep-related symptoms reported by PD patients are insomnia, excessive daytime sleepiness, and sleep fragmentation, characterized by interruptions and decreased continuity of sleep. Alterations in brain wave activity, as measured on the electroencephalogram (EEG), also occur in PD, with changes in the pattern and relative contributions of different frequency bands of the EEG spectrum to overall EEG activity in different vigilance states consistently observed. The mechanisms underlying these PD-associated sleep-wake abnormalities are poorly understood, and they are ineffectively treated by conventional PD therapies. To help fill this gap in knowledge, a new progressive model of PD - the MCI-Park mouse- was studied. Near the transition to the parkinsonian state, these mice exhibited significantly altered sleep-wake regulation, including increased wakefulness, decreased non-rapid eye movement (NREM) sleep, increased sleep fragmentation, reduced rapid eye movement (REM) sleep, and altered EEG activity patterns. These sleep-wake abnormalities mirror those identified in PD patients. Thus, this model may help elucidate the circuit mechanisms underlying sleep disruption in PD and identify targets for novel therapeutic approaches.
Sleep-Wake Recording in the Turek Laboratory
By onSurgery and recording protocol for measuring sleep-wake behavior in mice for Turek Laboratory.
Immunostaining of corticostriatal culture on fluid-walled dumbbells
By onThis protocol describes the immunocytochemistry of the iPSC-derived corticostriatal culture on fluid-walled dumbbells.
A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy
By onThe authors built a microfluidic device that mimics neuronal circuits and enables drug screening for effects on neuronal connections.
LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification
By onThe genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element–1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type–specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.
Localised axotomy of human Cortical Neurons (CNs) from induced pluripotent stem cells (iPSCs)
By onThis protocol describes the process followed to perform localised axotomy of iPSC-derived human cortical axons cultured within fluid-walled dumbbells in 6 cm TCT-treated Petri dishes.
Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway
By onThis protocol described the generation of fluid-walled dumbbells for culturing of induced pluripotent stem cell-derived neurons with directional connectivity so as to recapitulate the human corticostriatal connectivity.
Automatic flow in fluid-walled dumbbells driven by Laplace pressure
By onThis protocol describes experiments performed to quantify pressure and volume variations inside fluid-walled dumbbells when a pressure difference is generated between the two chambers.
A role for the subthalamic nucleus in aversive learning
By onThe subthalamic nucleus (STN) is critical for behavioral control; its dysregulation consequently correlates with neurological disorders, including PD. To investigate STN and aversion, affective behavior is addressed using optogenetics in mice.
State-dependent GABAergic regulation of striatal spiny projection neuron excitability: NEURON + Python model of striatal projection neurons
By onThis repository contains a NEURON + Python model of striatal projection neurons (or SPNs) designed to simulate the interaction between GABAergic and glutamatergic synaptic inputs.
Measuring alpha-synuclein concentration in hiPSCs using ELISA
By onThis protocol uses a classical ELISA experimental design with sensitive electrochemical detection to provide a measure of 𝜶-synuclein concentration in the conditioned media of cultured iPSC-derived human cells.
Preparation of tissue for Ribo-Tag/RNAseq analysis
By onThis protocols describes the RiboTag virus vectors injected into mouse brain, tissue dissection, immunoprecipitation and RNA extraction for RNAseq.
Fura-2 a.m. imaging in hiPSCs for measuring intracellular calcium
By onThis protocol outlines Fura-2 imaging of ionomycin response, with and without R568, a CaSR positive modulator, in hiPSCs.
Immunocytochemistry for CASR in iPSc-derived dopaminergic neurons
By onCASR immunocytochemistry protocol to accompany Kilfeather, Khoo et al., 2023: Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease