Generation of Functional Human 3D Cortico-Motor Assembloids.

Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Maturation and circuit integration of transplanted human cortical organoids.

Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.

Dynamic Gain Decomposition Reveals Functional Effects of Dendrites, Ion Channels, and Input Statistics in Population Coding.

Modern, high-density neuronal recordings reveal at ever higher precision how information is represented by neural populations. Still, we lack the tools to understand these processes bottom-up, emerging from the biophysical properties of neurons, synapses, and network structure. The concept of the dynamic gain function, a spectrally resolved approximation of a population's coding capability, has the potential to link cell-level properties to network-level performance. However, the concept is not only useful but also very complex because the dynamic gain's shape is co-determined by axonal and somato-dendritic parameters and the population's operating regime. Previously, this complexity precluded an understanding of any individual parameter's impact. Here, we decomposed the dynamic gain function into three components corresponding to separate signal transformations. This allowed attribution of network-level encoding features to specific cell-level parameters. Applying the method to data from real neurons and biophysically plausible models, we found: (1) The encoding bandwidth of real neurons, approximately 400 Hz, is constrained by the voltage dependence of axonal currents during early action potential initiation. (2) State-of-the-art models only achieve encoding bandwidths around 100 Hz and are limited mainly by subthreshold processes instead. (3) Large dendrites and low-threshold potassium currents modulate the bandwidth by shaping the subthreshold stimulus-to-voltage transformation. Our decomposition provides physiological interpretations when the dynamic gain curve changes, for instance during spectrinopathies and neurodegeneration. By pinpointing shortcomings of current models, it also guides inference of neuron models best suited for large-scale network simulations.

Reliability of human cortical organoid generation.

The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization.

BACKGROUND: Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s METHODS: Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. RESULTS: Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. CONCLUSIONS: Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate.

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention.

BACKGROUND: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. METHODS: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na+/K+ pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. RESULTS: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na+/K+ ATPase elicits SD. Elevated K+ or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. CONCLUSIONS: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.

Dynamic Gain Analysis Reveals Encoding Deficiencies in Cortical Neurons That Recover from Hypoxia-Induced Spreading Depolarizations.

Cortical regions that are damaged by insults, such as ischemia, hypoxia, and trauma, frequently generate spreading depolarization (SD). At the neuronal level, SDs entail complete breakdown of ionic gradients, persisting for seconds to minutes. It is unclear whether these transient events have a more lasting influence on neuronal function. Here, we describe electrophysiological changes in cortical neurons after recovery from hypoxia-induced SD. When examined with standard measures of neuronal excitability several hours after recovery from SD, layer 5 pyramidal neurons in brain slices from mice of either sex appear surprisingly normal. However, we here introduce an additional parameter, dynamic gain, which characterizes the bandwidth of action potential encoding by a neuron, and thereby reflects its potential efficiency in a multineuronal circuit. We find that the ability of neurons that recover from SD to track high-frequency inputs is markedly curtailed; exposure to hypoxia did not have this effect when SD was prevented pharmacologically. Staining for Ankyrin G revealed at least a fourfold decrease in the number of intact axon initial segments in post-SD slices. Since this effect, along with the effect on encoding, was blocked by an inhibitor of the Ca2+-dependent enzyme, calpain, we conclude that both effects were mediated by the SD-induced rise in intracellular Ca2+ Although effects of calpain activation were detected in the axon initial segment, changes in soma-dendritic compartments may also be involved. Whatever the precise molecular mechanism, our findings indicate that in the context of cortical circuit function, effectiveness of neurons that survive SD may be limited.SIGNIFICANCE STATEMENT Spreading depolarization, which commonly accompanies cortical injury, entails transient massive breakdown of neuronal ionic gradients. The function of cortical neurons that recover from hypoxia-induced spreading depolarization is not obviously abnormal when tested for usual measures of neuronal excitability. However, we now demonstrate that they have a reduced bandwidth, reflecting a significant impairment of their ability to precisely encode high-frequency components of their synaptic input in output spike trains. Thus, neurons that recover from spreading depolarizations are less able to function normally as elements in the multineuronal cortical circuitry. These changes are correlated with activation of the calcium-dependent enzyme, calpain.

The earliest neuronal responses to hypoxia in the neocortical circuit are glutamate-dependent.

Soon after exposure to hypoxia or ischemia, neurons in cortical tissues undergo massive anoxic depolarization (AD). This precipitous event is preceded by more subtle neuronal changes, including enhanced excitatory and inhibitory synaptic transmitter release. Here, we have used patch-in-slice techniques to identify the earliest effects of acute hypoxia on the synaptic and intrinsic properties of Layer 5 neurons, to determine their time course and to evaluate the role of glutamate receptors in their generation. Coronal slices of mouse somatosensory cortex were maintained at 36°C in an interface chamber and challenged with episodes of hypoxia. In recordings with cell-attached electrodes, the open probability of Ca(2+)-dependent BK channels began to increase within seconds of hypoxia onset, indicating a sharp rise in [Ca(2+)]i just beneath the membrane. By using a high concentration of K(+) in the pipette, we simultaneously monitored the membrane potential and showed that the [Ca(2+)]i rise was not associated with membrane depolarization. The earliest hypoxia-induced synaptic disturbance was a marked increase in the frequency of sPSCs, which also began soon after the removal of oxygen and long before AD. This synaptic effect was accompanied by depletion of the readily releasable transmitter pools, as demonstrated by a decreased response to hyperosmotic solutions. The early [Ca(2+)]i rise, the early increase in transmitter release and the subsequent AD itself were all prevented by bathing in a cocktail containing blockers of ionotropic glutamate receptors. We found no evidence for involvement of pannexin hemichannels or TRPM7 channels in the early responses to hypoxia in this experimental preparation. Our data indicate that the earliest cellular consequences of cortical hypoxia are triggered by activation of glutamate-gated channels.

Host circuit engagement of human cortical organoids transplanted in rodents.

Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell-derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30-60 days, and the transplantation procedure itself requires ~0.5-1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron-host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages.

Engineering brain assembloids to interrogate human neural circuits.

The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.

Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome.

Defects in interneuron migration can disrupt the assembly of cortical circuits and lead to neuropsychiatric disease. Using forebrain assembloids derived by integration of cortical and ventral forebrain organoids, we have previously discovered a cortical interneuron migration defect in Timothy syndrome (TS), a severe neurodevelopmental disease caused by a mutation in the L-type calcium channel (LTCC) Cav1.2. Here, we find that acute pharmacological modulation of Cav1.2 can regulate the saltation length, but not the frequency, of interneuron migration in TS. Interestingly, the defect in saltation length is related to aberrant actomyosin and myosin light chain (MLC) phosphorylation, while the defect in saltation frequency is driven by enhanced γ-aminobutyric acid (GABA) sensitivity and can be restored by GABA-A receptor antagonism. Finally, we describe hypersynchronous hCS network activity in TS that is exacerbated by interneuron migration. Taken together, these studies reveal a complex role of LTCC function in human cortical interneuron migration and strategies to restore deficits in the context of disease.

Generation of human striatal organoids and cortico-striatal assembloids from human pluripotent stem cells.

Cortico-striatal projections are critical components of forebrain circuitry that regulate motivated behaviors. To enable the study of the human cortico-striatal pathway and how its dysfunction leads to neuropsychiatric disease, we developed a method to convert human pluripotent stem cells into region-specific brain organoids that resemble the developing human striatum and include electrically active medium spiny neurons. We then assembled these organoids with cerebral cortical organoids in three-dimensional cultures to form cortico-striatal assembloids. Using viral tracing and functional assays in intact or sliced assembloids, we show that cortical neurons send axonal projections into striatal organoids and form synaptic connections. Medium spiny neurons mature electrophysiologically following assembly and display calcium activity after optogenetic stimulation of cortical neurons. Moreover, we derive cortico-striatal assembloids from patients with a neurodevelopmental disorder caused by a deletion on chromosome 22q13.3 and capture disease-associated defects in calcium activity, showing that this approach will allow investigation of the development and functional assembly of cortico-striatal connectivity using patient-derived cells.

Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

Human 3D cellular model of hypoxic brain injury of prematurity.

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.

Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures.

Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.

The Outwardly Rectifying Current of Layer 5 Neocortical Neurons that was Originally Identified as "Non-Specific Cationic" Is Essentially a Potassium Current.

In whole-cell patch clamp recordings from layer 5 neocortical neurons, blockade of voltage gated sodium and calcium channels leaves a cesium current that is outward rectifying. This current was originally identified as a "non-specific cationic current", and subsequently it was hypothesized that it is mediated by TRP channels. In order to test this hypothesis, we used fluorescence imaging of intracellular sodium and calcium indicators, and found no evidence to suggest that it is associated with influx of either of these ions to the cell body or dendrites. Moreover, the current is still prominent in neurons from TRPC1-/- and TRPC5-/- mice. The effects on the current of various blocking agents, and especially its sensitivity to intracellular tetraethylammonium, suggest that it is not a non-specific cationic current, but rather that it is generated by cesium-permeable delayed rectifier potassium channels.