Neuroepithelial organoid patterning is mediated by a neighborhood watch mechanism.

During epithelial tissue patterning, morphogens operate across multiple length scales to instruct cell identities. However, how cell fate changes are coordinated over these scales to establish spatial organization remains poorly understood. Here, we use human neural tube organoids as models of epithelial patterning and develop an in silico approach to define conditions permissive to patterning. By systematically varying morphogen position, diffusivity, and fate-inducing concentration levels, we show that cells follow a "neighborhood watch" (NW) mechanism that is deterministically dictated by initial morphogen source positions, reflecting scale-invariant in vitro phenotypes. We define how the frequency and local bias of morphogen sources stabilize pattern orientation. The model predicts enhanced patterning through floor plate inhibition, and receptor-ligand interaction analysis of single-cell RNA sequencing (scRNA-seq) data identifies wingless-related integration site (WNT) and bone morphogenic protein (BMP) as inhibition modulators, which we validate in vitro. These results suggest that developing neuroepithelia employ NW-based mechanisms to organize morphogen sources, define cellular identity, and establish patterns.

Large-scale perfused tissues via synthetic 3D soft microfluidics.

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing neural and liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Modulation of ATP-induced LTP by cannabinoid receptors in rat hippocampus.

Cannabinoids exert powerful action on various forms of synaptic plasticity. These retrograde messengers modulate GABA and glutamate release from presynaptic terminals by acting on presynaptic CB1 receptors. In particular, they inhibit long-term potentiation (LTP) elicited by electrical stimulation of excitatory pathways in rat hippocampus. Recently, LTP of the field excitatory postsynaptic potential (fEPSP) induced by exogenous ATP has been thoroughly explored. The present study demonstrates that cannabinoids inhibit ATP-induced LTP in hippocampal slices of rat. Administration of 10 µM of ATP led to strong inhibition of fEPSPs in CA1/CA3 hippocampal synapses. Within 40 min after ATP removal from bath solution, robust LTP was observed (fEPSP amplitude comprised 130.1 ± 3.8% of control, n = 10). This LTP never appeared when ATP was applied in addition to cannabinoid receptor agonist WIN55,212-2 (100 nM). Selective CB1 receptor antagonist, AM251 (500 nM), completely abolished this effect of WIN55,212-2. Our data indicate that like canonical LTP elicited by electrical stimulation, ATP-induced LTP is under control of CB1 receptors.

Engineering neurovascular organoids with 3D printed microfluidic chips.

The generation of tissue and organs requires close interaction with vasculature from the earliest moments of embryonic development. Tissue-specific organoids derived from pluripotent stem cells allow for the in vitro recapitulation of elements of embryonic development. However, they are not intrinsically vascularized, which poses a major challenge for their sustained growth, and for understanding the role of vasculature in fate specification and morphogenesis. Current organoid vascularization strategies do not recapitulate the temporal synchronization and spatial orientation needed to ensure in vivo-like early co-development. Here, we developed a human pluripotent stem cell (hPSC)-based approach to generate organoids which interact with vascular cells in a spatially determined manner. The spatial interaction between organoid and vasculature is enabled by the use of a custom designed 3D printed microfluidic chip which allows for a sequential and developmentally matched co-culture system. We show that on-chip hPSC-derived pericytes and endothelial cells sprout and self-assemble into organized vascular networks, and use cerebral organoids as a model system to explore interactions with this de novo generated vasculature. Upon co-development, vascular cells physically interact with the cerebral organoid and form an integrated neurovascular organoid on chip. This 3D printing-based platform is designed to be compatible with any organoid system and is an easy and highly cost-effective way to vascularize organoids. The use of this platform, readily performed in any lab, could open new avenues for understanding and manipulating the co-development of tissue-specific organoids with vasculature.

Neural cell adhesion molecule-associated polysialic acid regulates synaptic plasticity and learning by restraining the signaling through GluN2B-containing NMDA receptors.

The neural cell adhesion molecule (NCAM) is the predominant carrier of alpha2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated with schizophrenia in humans and cause deficits in hippocampal synaptic plasticity and contextual fear conditioning in mice. Here, we show that PSA inhibits opening of recombinant NMDA receptors composed of GluN1/2B (NR1/NR2B) or GluN1/2A/2B (NR1/NR2A/NR2B) but not of GluN1/2A (NR1/NR2A) subunits. Deficits in NCAM/PSA increase GluN2B-mediated transmission and Ca(2+) transients in the CA1 region of the hippocampus. In line with elevation of GluN2B-mediated transmission, defects in long-term potentiation in the CA1 region and contextual fear memory in NCAM/PSA-deficient mice are abrogated by application of a GluN2B-selective antagonist. Furthermore, treatment with the glutamate scavenger glutamic-pyruvic transaminase, ablation of Ras-GRF1 (a mediator of GluN2B signaling to p38 MAPK), or direct inhibition of hyperactive p38 MAPK can restore impaired synaptic plasticity in brain slices lacking PSA/NCAM. Thus, PSA carried by NCAM regulates plasticity and learning by inhibition of the GluN2B-Ras-GRF1-p38 MAPK signaling pathway. These findings implicate carbohydrates carried by adhesion molecules in modulating NMDA receptor signaling in the brain and demonstrate reversibility of cognitive deficits associated with ablation of a schizophrenia-related adhesion molecule.

Engineering Organoid Vascularization.

The development of increasingly biomimetic human tissue analogs has been a long-standing goal in two important biomedical applications: drug discovery and regenerative medicine. In seeking to understand the safety and effectiveness of newly developed pharmacological therapies and replacement tissues for severely injured non-regenerating tissues and organs, there remains a tremendous unmet need in generating tissues with both functional complexity and scale. Over the last decade, the advent of organoids has demonstrated that cells have the ability to reorganize into complex tissue-specific structures given minimal inductive factors. However, a major limitation in achieving truly in vivo-like functionality has been the lack of structured organization and reasonable tissue size. In vivo, developing tissues are interpenetrated by and interact with a complex network of vasculature which allows not only oxygen, nutrient and waste exchange, but also provide for inductive biochemical exchange and a structural template for growth. Conversely, in vitro, this aspect of organoid development has remained largely missing, suggesting that these may be the critical cues required for large-scale and more reproducible tissue organization. Here, we review recent technical progress in generating in vitro vasculature, and seek to provide a framework for understanding how such technologies, together with theoretical and developmentally inspired insights, can be harnessed to enhance next generation organoid development.

Measurement of intracellular concentration of fluorescently-labeled targets in living cells.

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.

Protective cap over CA1 synapses: extrasynaptic glutamate does not reach the postsynaptic density.

Numerous data indicate that nonsynaptic release of glutamate occurs both in normal and pathophysiological conditions. When reaching receptors in the postsynaptic density (PSD), glutamate (Glu) could affect the synaptic transmission. We have tested this possibility in the hippocampal CA1 synapses of rats, either by applying exogenous Glu to the CA1 neurons or by disruption of Glu transporter activity. L-Glu (400 microM) was directly applied to the hippocampal slices acutely isolated from the rats. It produced a strong inhibition of both ortho- and antidromically elicited action potentials fired by CA1 neurons while the excitatory postsynaptic current (EPSC) measured in these neurons remained totally unaffected. The optical isomer D-Glu which is not transported by the systems of Glu uptake inhibited not only orthodromic and antidromic spikes, but also EPSC. Non-specific glutamate transporter inhibitor DL-threo-beta-hydroxyaspartic acid (THA, 400 microM) mimicked the effects of exogenous Glu and produced strong inhibition of both orthodromic and antidromic spikes, without any influence on the amplitude of EPSCs. Dihydrokainate (DHK, 300 microM), selective inhibitor of GLT-1 subtype of glutamate transporter, exerted a significant inhibitory action on the orthodromically evoked spikes and also on the EPSC. Our results indicate that extrasynaptic and PSD membranes of CA1 neurons form separate compartments differing in the mechanisms and efficiency of external Glu processing: the protection of PSD markedly prevails.

Post-synaptic N-methyl-d-aspartate signalling in hippocampal neurons of rat: spillover increases the impact of each spike in a short burst discharge.

High-frequency burst discharges in hippocampus typically consist of less than ten spikes fired at frequencies too high to be followed by a post-synaptic neuron. How significant are these numbers for synaptic signalling? We have measured the N-methyl-d-aspartate (NMDA) component of the excitatory post-synaptic current (EPSC(NMDA)) in hippocampal CA1 neurons of rat after burst discharge of variable duration. The synaptic facilitation is accompanied by a slow-down of the EPSC(NMDA) which develops on a spike-to-spike basis. Consequently the charge transferred by the after-burst EPSC(NMDA) is increased with each spike. The phenomenon is most probably due to the spillover-mediated recruitment of extrasynaptic NMDA receptors. In terms of post-synaptic signalling it dramatically increases the impact of each spike in a short burst discharge.

Backpropagating action potentials enable detection of extrasynaptic glutamate by NMDA receptors.

Synaptic NMDA receptors (NMDARs) are crucial for neural coding and plasticity. However, little is known about the adaptive function of extrasynaptic NMDARs occurring mainly on dendritic shafts. Here, we find that in CA1 pyramidal neurons, back-propagating action potentials (bAPs) recruit shaft NMDARs exposed to ambient glutamate. In contrast, spine NMDARs are "protected," under baseline conditions, from such glutamate influences by peri-synaptic transporters: we detect bAP-evoked Ca(2+) entry through these receptors upon local synaptic or photolytic glutamate release. During theta-burst firing, NMDAR-dependent Ca(2+) entry either downregulates or upregulates an h-channel conductance (G(h)) of the cell depending on whether synaptic glutamate release is intact or blocked. Thus, the balance between activation of synaptic and extrasynaptic NMDARs can determine the sign of G(h) plasticity. G(h) plasticity in turn regulates dendritic input probed by local glutamate uncaging. These results uncover a metaplasticity mechanism potentially important for neural coding and memory formation.

Extrasynaptic NR2B and NR2D subunits of NMDA receptors shape 'superslow' afterburst EPSC in rat hippocampus.

In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA receptor-mediated EPSCs (EPSC(NMDA)). Such a time course has been attributed to the cooperation of synapses through glutamate spillover. Imitating a natural pattern of activity, we have applied short bursts (2-7 stimuli) of high-frequency stimulation and observed a spike-to-spike slow-down of the EPSC(NMDA) kinetics, which accompanied synaptic facilitation. It was found that the early component of the EPSC(NMDA) and the burst-induced late component of the EPSC(NMDA) have distinct pharmacological properties. The competitive NMDA antagonist R-(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP), which has higher affinity to NR2A than to NR2B subunits and lowest affinity at NR2D subunits, significantly slowed down the decay rate of the afterburst EPSC while leaving the kinetics of the control current unaffected. In contrast, ifenprodil, a highly selective NR2B antagonist, and [+/-]-cis-1-[phenanthren-2yl-carbonyl]piperazine-2,3-dicarboxylic acid (PPDA), a competitive antagonist that is moderately selective for NR2D subunits, more strongly inhibited the late component of the afterburst EPSC(NMDA). The receptors formed by NR2B and (especially) NR2D subunits are known to have higher agonist sensitivity and much slower deactivation kinetics than NR2A-containing receptors. Furthermore, NR2B is preferentially and NR2D is exclusively located on extrasynaptic membranes. As the density of active synapses increases, the confluence of released glutamate makes EPSC decay much longer by activating more extrasynaptic NR2B- and NR2D-subunit-containing receptors. Long-term potentiation (LTP) induced by successive rounds of burst stimulation is accompanied by a long-term increase in the contribution of extrasynaptic receptors in the afterburst EPSC(NMDA.)