Human Neuronal Cultures Research Articles

THE EFFECT OF 500 NM DOSAGE OF NIMODIPINE ON NRF2 AND NF-KB EXPRESSION IN NEURON CELL LINE CULTURE SH-SY5Y EXPOSED WITH CHRONIC HYPERGLYCEMIA

Introduction: Metabolic stress and mitochondrial dysfunction in chronic hyperglycemia can cause an increase in Ca2+ cytosol ions that cause dysfunction of NRF2, that will increase the activity of NF-kB which causes an increase in cytokine production, where NF-kB can modulate NRF2 transcription and its activity, so that it has positive or negative effects in the target gene. Giving nimodipine in chronic hyperglycemia conditions can increase levels of NRF2 molecules and reduce levels of NF-kB. Aim: The purpose of this research is to know the effect of treating nimodipine dose 5 μM towards expression of the protein NRF2and NF-kB in SH-SY5Y cell culture induced by chronic hyperglycemia. Method: This study is a true experimental study using randomized posttest only controlled group design in human SH-SY5Y neuron cell culture. Neuron cells were exposed with chronic glucose (25 mM / G25 normoglycemia, 50 mM / G50 hyperglycemia) for 6 days, followed by administration of nimodipine (without nimodipine and nimodipine 500 nM) for 30 minutes. Calculation of NRF2 and NF-kB expressions done by fluorescence method. Results: There were significant differences in the NF-kB neuron cells in the treatment group with controls, both normoglycemia and hyperglycemia (p <0.05). Nimodipine was not able to increase the expression of NRF-2 in the treatmnrnt group, although there were significance differences between these two groups. The Spearman Rank correlation test showed a negative correlation between NRF2 and NF-kB expression in the SH-SY5Y human cell line neuron culture that exposed to chronic hyperglycemia with administration of 500 nM nimodipine. Conclusion: There was a statistically significant decrease in NF-kB expression in SH-SY5Y neuron cells that exposed to chronic hyperglycemia given nimodipine compared to controls group. Nimodipine treatment to SH-SY5Y neuron cells exposed to hyperglycemia not yet increase expression of NRF2.

NitroSynapsin ameliorates hypersynchronous neural network activity in Alzheimer hiPSC models.

Beginning at early stages, human Alzheimer’s disease (AD) brains manifest hyperexcitability, contributing to subsequent extensive synapse loss, which has been linked to cognitive dysfunction. No current therapy for AD is disease-modifying. Part of the problem with AD drug discovery is that transgenic mouse models have been poor predictors of potential human treatment. While it is undoubtedly important to test drugs in these animal models, additional evidence for drug efficacy in a human context might improve our chances of success. Accordingly, in order to test drugs in a human context, we have developed a platform of physiological assays using patch-clamp electrophysiology, calcium imaging, and multielectrode array (MEA) experiments on human (h)iPSC-derived 2D cortical neuronal cultures and 3D cerebral organoids. We compare hiPSCs bearing familial AD mutations vs. their wild-type (WT) isogenic controls in order to characterize the aberrant electrical activity in such a human context. Here, we show that these AD neuronal cultures and organoids manifest increased spontaneous action potentials, slow oscillatory events (~1 Hz), and hypersynchronous network activity. Importantly, the dual-allosteric NMDAR antagonist NitroSynapsin, but not the FDA-approved drug memantine, abrogated this hyperactivity. We propose a novel model of synaptic plasticity in which aberrant neural networks are rebalanced by NitroSynapsin. We propose that hiPSC models may be useful for screening drugs to treat hyperexcitability and related synaptic damage in AD.

Antidotal effects of methylene blue against cyanide neurological toxicity: in vivo and in vitro studies.

The aim of the present study was to determine whether methylene blue (MB) could directly oppose the neurological toxicity of a lethal cyanide (CN) intoxication. KCN, infused at the rate of 0.375 mg/kg/min intravenously, produced 100% lethality within 15 min in unanaesthetized rats (n=12). MB at 10 (n=5) or 20 mg/kg (n=5), administered 3 min into CN infusion, allowed all animals to survive with no sequelae. No apnea and gasping were observed at 20 mg/kg MB (P < 0.001). The onset of coma was also significantly delayed and recovery from coma was shortened in a dose-dependent manner (median of 359 and 737 seconds, respectively, at 20 and 10 mg/kg). At 4 mg/kg MB (n=5), all animals presented faster onset of coma and apnea and a longer period of recovery than at the highest doses (median 1344 seconds, P < 0.001). MB reversed NaCN-induced resting membrane potential depolarization and action potential depression in primary cultures of human fetal neurons intoxicated with CN. MB restored calcium homeostasis in the CN-intoxicated human SH-SY5Y neuroblastoma cell line. We conclude that MB mitigates the neuronal toxicity of CN in a dose-dependent manner, preventing the lethal depression of respiratory medullary neurons and fatal outcome.

Lithium Alters Expression of RNAs in a Type-Specific Manner in Differentiated Human Neuroblastoma Neuronal Cultures, Including Specific Genes Involved in Alzheimer’s Disease

Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple CNS disorders, including Alzheimer’s disease (AD). While perturbation of RNA levels by Li was reported, its effects on the whole-transcriptome remains unexplored. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels.

Optimized Protocol for Robust Differentiation of SH‐SY5Y Cells into Neuronal‐like Cells: An in vitro Model of Human Neuronal Cultures

The SH‐SY5Y cell line, derived from a human glioblastoma, grow and divide indefinitely under normal conditions, but have the potential to differentiate into neuronal‐like cells when treated with retinoic acid (RA). However, the extent of differentiation has been contested, leading to a variety of protocols to induce differentiation. We took a systematic approach to test the various conditions and treatments used in previously published studies to develop an optimized protocol that yields robust differentiation in an efficient manner. In morphological assays, robust and extensive processes from cell bodies and network development between cells in culture were established when brain‐derived neurotrophic factor (BDNF), B‐27, and potassium (K+) were supplemented to RA, that survived up to 10 days in culture. One‐day treatment of RA and BDNF during differentiation was as effective as prolonged post‐differentiation treatment of BDNF, thus reducing the amount of BDNF required. Conversely, supplements of vitamin B12, AraC, and dB‐cAMP did not provide any significant difference in differentiation. In RT‐qPCR analyses, our optimized protocol led to significant increased expression of microtubule associated protein tau (MAPT) and microtubule associated protein 2 (MAP2), both of which are markers of neuronal cells. We are validating the sub‐cellular localization of these proteins via immunohistochemistry. This optimized protocol thus differentiates SH‐SY5Y cells into robust and reliable neuronal‐like cells, which can be used as a model of human neuronal cells and neurodevelopment in a reproducible in vitro system, without the ethical concerns or practical limitations of using fetal cells for human neuronal cultures.Support or Funding InformationOhio Wesleyan University: Department of Botany and Microbiology, Neuroscience Program, Summer Science Research Program, and Theory‐to‐Practice Grant.

Sinomenine inhibits amyloid beta-induced astrocyte activation and protects neurons against indirect toxicity

Amyloid beta is a major constituent of the plaques found in the brains of patients suffering from Alzheimer’s disease (AD). A growing body of research work suggests that neuroinflammation plays important roles in the development of AD. Thus, considerable efforts are directed towards identification of compounds that can reduce or inhibit neuroinflammation. Here, we show that sinomenine, a compound present in a Chinese medicinal plant, Sinomenium acutum, inhibits oligomeric amyloid beta-induced production of reactive oxygen species (ROS), nitric oxide (NO) and inflammation-related molecules from astrocytic cells. The conditioned medium from oligomeric amyloid beta-treated astrocytic cells induces cell death in the hippocampal neuronal cells. Importantly, sinomenine inhibits this cell death. In addition, this compound has inhibitory effects on the production of ROS, NO and inflammation-related factors from oligomeric amyloid-beta treated human astrocytes. Finally, the conditioned medium from oligomeric amyloid beta-treated human astrocytes induces cell death in the primary culture of human neurons, which is inhibited by sinomenine. Thus, sinomenine inhibits amyloid beta-induced production of toxic factors from astrocytes, and confers protection to hippocampal neuronal cells as well as human neurons against indirect toxicity. The results suggest that this compound could provide beneficial effects in AD and other neurodegenerative conditions by reducing inflammation and neuronal cell death.

Plumbagin attenuated oxygen-glucose deprivation/reoxygenation-induced injury in human SH-SY5Y cells by inhibiting NOX4-derived ROS-activated NLRP3 inflammasome.

Plumbagin (PLB), an alkaloid obtained from the roots of the plants of Plumbago genus, is an inhibitor of NADPH oxidase 4 (NOX4). This study aimed to investigate the beneficial effect of PLB against oxygen-glucose deprivation/reoxygenation (OGDR)-induced neuroinjury in human SH-SY5Y neuronal cultures. Our results showed that OGD/R stimulated NOX4 protein expression and reactive oxygen species (ROS) production in SH-SY5Y cells, whereas increased 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) production, resulting in the activation of the NLRP3 inflammasome. And PLB pretreatment reduced the ROS production by regulating the expression of NOX4 and downregulated NF-κB signaling which was induced by OGDR. Furthermore, PLB inhibited OGDR induced NLRP3 inflammasome activation but not PARP1. Overall, PLB improved OGDR induced neuroinjury by inhibiting NOX4-derived ROS-activated NLRP3 inflammasome.

A microfiber scaffold-based 3D in vitro human neuronal culture model of Alzheimer's disease.

Increasing evidence indicates superiority of three-dimensional (3D) in vitro cell culture systems over conventional two-dimensional (2D) monolayer cultures in mimicking native in vivo microenvironments. Tissue-engineered 3D culture models combined with stem cell technologies have advanced Alzheimer's disease (AD) pathogenesis studies. However, existing 3D neuronal models of AD overexpress mutant genes or have heterogeneities in composition, biological properties and cell differentiation stages. Here, we encapsulate patient induced pluripotent stem cell (iPSC) derived neural progenitor cells (NPC) in poly(lactic-co-glycolic acid) (PLGA) microtopographic scaffolds fabricated via wet electrospinning to develop a novel 3D culture model of AD. First, we enhanced cellular infiltration and distribution inside the scaffold by optimizing various process parameters such as fiber diameter, pore size, porosity and hydrophilicity. Next, we compared key neural stem cell features including viability, proliferation and differentiation in 3D culture with 2D monolayer controls. The 3D microfibrous substrate reduces cell proliferation and significantly accelerates neuronal differentiation within seven days of culture. Furthermore, 3D culture spontaneously enhanced pathogenic amyloid-beta 42 (Aβ42) and phospho-tau levels in differentiated neurons carrying familial AD (FAD) mutations, compared with age-matched healthy controls. Overall, our tunable scaffold-based 3D neuronal culture platform serves as a suitable in vitro model that robustly recapitulates and accelerates the pathogenic characteristics of FAD-iPSC derived neurons.

Human Tridimensional Neuronal Cultures for Phenotypic Drug Screening in Inherited Peripheral Neuropathies.

Length-dependent axonal degeneration is the pathologic hallmark of several neurodegenerative disorders, including inherited peripheral neuropathies (Charcot-Marie-Tooth (CMT) disease). CMT is currently an untreatable disorder. This is partially due to lack of translational models suitable for drug discovery. In vitro models of CMT have been hindered by the 2D configuration of neuronal cultures, which limits visualization and orientation of axons. To overcome these limitations, we cultured induced pluripotent stem cell (iPSC)-derived spinal motor neurons as 3D spheroids, which grow axons in a centrifugal fashion when plated. Using these iPSC-derived spinal spheroids, we demonstrate neurofilament deposits in motor neuron axons of three patients with CMT2E, caused by mutations in the NEFL gene. This phenotype is partially reversed by two kinase inhibitors. In summary, we developed a human tridimensional in vitro system that models length-dependent axonopathies, recapitulates key pathophysiologic features of CMT2E, and should facilitate the identification of new therapeutic compounds for CMT.

Graphene oxide-conductive polymer nanocomposite coatings obtained by the EPD method as substrates for neurite outgrowth

The goal of this study was to investigate the influence of graphene oxide (GO) and polyaniline (PANI) nanocomposite coatings deposited on titanium substrates on two types of nerve cells in vitro, namely human neuroblastoma SH-SY5Y cells and primary neuronal cell cultures. Selected physicochemical properties such as morphology, microstructure, surface energy and wettability of the surface of the nanocomposite coatings deposited on a Ti substrate using electrophoretic deposition (EPD) were investigated, followed by biological tests. Moreover, infrared spectroscopy was performed to determine the chemistry of the nanocomposites, while the cathodic charge storage capacity was assessed in the electrochemical characterisation of these coatings.The more sensitive nature of primary cells may have contributed to the observation of significant differences between GO-based coatings, which was not observed with SH-SY5Y cells. Of the four studied coatings, the most promising one was the GO:PANI_16:1 nanocomposite coating. In terms of biocompatibility, cell morphology and neurite length, this coating was the most promising when used in contact with the cell line as well as the primary culture. An interesting phenomenon not seen so far in contact with GO:PANI_16:1 is the tendency of neuronal cell bodies to aggregate in ‘neurosphere-like’ structures.

LncRNA EPIC1 downregulation mediates hydrogen peroxide-induced neuronal cell injury.

Excessive oxidative stress causes neuronal cell injury. Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) is a MYC-interacting LncRNA. Its expression and potential functions in hydrogen peroxide (H2O2)-stimulated neuronal cells are studied. In SH-SY5Y neuronal cells and primary human neuron cultures, H2O2 downregulated Lnc-EPIC1 and key MYC targets (Cyclin A1, CDC20 and CDC45). Ectopic overexpression of Lnc-EPIC1 increased expression of MYC targets and significantly attenuated H2O2-induced neuronal cell death and apoptosis. Contrarily, Lnc-EPIC1 siRNA potentiated neuronal cell death by H2O2. MYC knockout by CRISPR/Cas9 method also facilitated H2O2-induced SH-SY5Y cell death. Significantly, MYC knockout abolished Lnc-EPIC1-induced actions in H2O2-stimulated neuronal cells. Together, these results suggest that Lnc-EPIC1 downregulation mediates H2O2-induced neuronal cell death.

Toxic Tau Oligomers Modulated by Novel Curcumin Derivatives

The pathological aggregation and accumulation of tau, a microtubule-associated protein, is a common feature amongst more than 18 different neurodegenerative diseases that are collectively known as tauopathies. Recently, it has been demonstrated that the soluble and hydrophobic tau oligomers are highly toxic in vitro due to their capacity towards seeding tau misfolding, thereby propagating the tau pathology seen across different neurodegenerative diseases. Modulating the aggregation state of tau oligomers through the use of small molecules could be a useful therapeutic strategy to target their toxicity, regardless of other factors involved in their formation. In this study, we screened and tested a small library of newly synthesized curcumin derivatives against preformed recombinant tau oligomers. Our results show that the curcumin derivatives affect and modulate the tau oligomer aggregation pathways, converting to a more aggregated non-toxic state as assessed in the human neuroblastoma SH-SY5Y cell line and primary cortical neuron cultures. These results provide insight into tau aggregation and may become a basis for the discovery of new therapeutic agents, as well as advance the diagnostic field for the detection of toxic tau oligomers.

Lithium alters expression of RNAs in a type-specific manner in differentiated human neuroblastoma neuronal cultures, including specific genes involved in Alzheimer\u2019s disease

Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple nervous system disorders, including Alzheimer’s disease (AD). While perturbation of RNA levels by Li has been previously reported, its effects on the whole transcriptome has been given little attention. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels. We cultured and differentiated human neuroblastoma (SK-N-SH) cells to neuronal cells with all-trans retinoic acid (ATRA). We exposed cultures for one week to lithium chloride or distilled water, extracted total RNA, depleted ribosomal RNA and performed whole-transcriptome RT-sequencing. We analyzed results by RNA length and type. We further analyzed expression and protein interaction networks between selected Li-altered protein-coding RNAs and common AD-associated gene products. Lithium changed expression of RNAs in both non-specific (inverse to sequence length) and specific (according to RNA type) fashions. The non-coding small nucleolar RNAs (snoRNAs) were subject to the greatest length-adjusted Li influence. When RNA length effects were taken into account, microRNAs as a group were significantly less likely to have had levels altered by Li treatment. Notably, several Li-influenced protein-coding RNAs were co-expressed or produced proteins that interacted with several common AD-associated genes and proteins. Lithium’s modification of RNA levels depends on both RNA length and type. Li activity on snoRNA levels may pertain to bipolar disorders while Li modification of protein coding RNAs may be relevant to AD.

Reconstitution of the Human Nigro-striatal Pathway on-a-Chip Reveals OPA1-Dependent Mitochondrial Defects and Loss of Dopaminergic Synapses

SummaryStem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson’s disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity.

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures.

Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 μM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 μM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 μM 2-BP or 100 μM glutamate for 24 h, while 300 μM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.

Peptide-Based Scaffolds for the Culture and Transplantation of Human Dopaminergic Neurons.

Cell replacement therapy is a promising treatment strategy for Parkinson's disease (PD); however, the poor survival rate of transplanted neurons is a critical barrier to functional recovery. In this study, we used self-assembling peptide nanofiber scaffolds (SAPNS) based on the peptide RADA16-I to support the in vitro maturation and in vivo post-transplantation survival of encapsulated human dopaminergic (DA) neurons derived from induced pluripotent stem cells. Neurons encapsulated within the SAPNS expressed mature neuronal and midbrain DA markers and demonstrated in vitro functional activity similar to neurons cultured in two dimensions. A microfluidic droplet generation method was used to encapsulate cells within monodisperse SAPNS microspheres, which were subsequently used to transplant adherent, functional networks of DA neurons into the striatum of a 6-hydroxydopamine-lesioned PD mouse model. SAPNS microspheres significantly increased the in vivo survival of encapsulated neurons compared with neurons transplanted in suspension, and they enabled significant recovery in motor function compared with control lesioned mice using approximately an order of magnitude fewer neurons than have been previously needed to demonstrate behavioral recovery. These results indicate that such biomaterial scaffolds can be used as neuronal transplantation vehicles to successfully improve the outcome of cell replacement therapies for PD. Impact Statement Transplantation of dopaminergic (DA) neurons holds potential as a treatment for Parkinson's disease (PD), but low survival rates of transplanted neurons is a barrier to successfully improving motor function. In this study, we used hydrogel scaffolds to transplant DA neurons into PD model mice. The hydrogel scaffolds enhanced survival of the transplanted neurons compared with neurons that were transplanted in a conventional manner, and they also improved recovery of motor function by using significantly fewer neurons than have typically been transplanted to see functional benefits. This cell transplantation technology has the capability to improve the outcome of neuron transplantation therapies.

A combinational treatment of carotenoids decreases Aβ secretion in human neurons via β-secretase inhibition

Alzheimer’s disease (AD) is the most common cause of dementia and is characterized neuropathologically by the presence of amyloid plaques and neurofibrillary tangles. Amyloid-β (Aβ) peptides, major components of amyloid plaques and crucial pathogenic molecules in terms of the amyloid hypothesis, are derived from successive proteolytic processing of amyloid-β precursor protein (APP). In this study, we established a human neuronal culture system using induced pluripotent stem cells (iPSCs) to evaluate the possible effects of natural compounds on the amyloid phenotype. Unexpectedly, we found that combinational treatment of carotenoids, but not docosahexaenoic acid, significantly decreased Aβ secretion from iPSC-derived human cortical neurons. Importantly, the effects of the carotenoids resulted from specific inhibition of BACE1 activity and not from expression changes in APP or BACE1. Therefore, these results indicate a novel beneficial function of carotenoids in the anti-amyloidogenic processing of APP. Collectively, this study will shed light on neuronal protection by a novel mechanism during the pathogenesis of AD.

Proteomics of the human neuronal cell culture model of Alzheimer's disease

Proteomics of the human neuronal cell culture model of Alzheimer's disease

Adenosine 5'-Monophosphate Protects from Hypoxia by Lowering Mitochondrial Metabolism and Oxygen Demand.

Ischemia and reperfusion injury following severe trauma or cardiac arrest are major causes of organ damage in intensive care patients. The brain is particularly vulnerable because hypoxia rapidly damages neurons due to their heavy reliance on oxidative phosphorylation. Therapeutic hypothermia can reduce ischemia-induced brain damage, but cooling procedures are slow and technically difficult to perform in critical care settings. It has been previously reported that injection of naturally occurring adenosine 5'-monophosphate (AMP) can rapidly induce hypothermia in mice. We studied the underlying mechanisms and found that AMP transiently reduces the heart rate, respiratory rate, body temperature, and the consciousness of adult male and female C57BL/6J mice. Adding AMP to mouse or human neuronal cell cultures dose-dependently reduced the membrane potential (ΔΨm) and Ca signaling of mitochondria in these cells. AMP treatment increased intracellular AMP levels and activated AMP-activated protein kinase, which resulted in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and of mitochondrial and cytosolic Ca signaling in resting and stimulated neurons. Pretreatment with an intraperitoneal injection of AMP almost doubled the survival time of mice under hypoxic (6% O2) or anoxic (<1% O2) conditions when compared to untreated mice. These findings suggest that AMP induces a hypometabolic state that slows mitochondrial respiration, reduces oxygen demand, and delays the processes that damage mitochondria in the brain and other organs following hypoxia and reperfusion. Further examination of these mechanisms may lead to new treatments that preserve organ function in critical care patients.

Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that—in many aspects—recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that—unlike two- or three-lens light-sheet microscopes—it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.