Microglia enable mature perineuronal nets disassembly upon anesthetic ketamine exposure or 60-Hz light entrainment in the healthy brain.

Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.

Mechanism behind the neuronal ephaptic coupling during synchronizing by specific brain-triggered wave as neuronal motor toolkit.

Probable mechanism behind the neuronal ephaptic coupling is investigated based on the introduction of "Brain"-triggered potential excitation signal smartly with a specific very low frequency (VLF) waves as a neuronal motor toolkit. Detection of this electric motor toolkit is attributed to in-vitro precise analyses of a neural network of snail, along to the disconnected snail's neuronal network as a control. This is achieved via rapid (real-time) electrical signals acquisition by blind patch-clamp method during micro-electrode implanting in the neurons at the gigaseal conditions by the surgery operations. This process is based on its waveform (potential excitation signal) detection by mathematical curve fitting process. The characterized waveform of this electrical signal is "Saw Tooth" that is smartly stimulated, alternatively, by the brain during triggering the action potential's (AP's) hyperpolarization zone at a certain time interval at the several µs levels. Triggering the neuron cells results in (1) observing a positive shift (10.0%, depending on the intensity of the triggering wave), and (2) major promotion in the electrical current from sub nano (n) to micro (µ) amper (nA, µA) levels. Direct tracing the time domain (i.e., electrical signal vs. time) and estimation of the frequency domain (diagram of electrical response vs. the applied electrical frequencies) by the "Discrete Fast Fourier Transform" algorithm approve the presence of bilateral and reversible electrical currents between axon and dendrite. This mechanism therefore opens a novel view about the neuronal motor toolkit mechanism, versus the general knowledge about the unilateral electrical current flow from axon to dendrite operations in as neural network. The reliability of this mechanism is evaluated via (1) sequential modulation and demodulation of the snail's neuron network by a simulation electrical functions and sequentially evaluation of the neuronal current sensitivity between pA and nA (during the promotion of the signal-to-noise ratio, via averaging of 30 ± 1 (n = 15) and recycling the electrical cycles before any neuronal response); and (2) operation of the process on the differentiated stem cells. The interstice behavior is attributed to the effective role of Ca2+ channels (besides Na+ and K+ ionic pumping), during hyper/hypo calcium processes, evidenced by inductively coupled plasma as the selected analytical method. This phenomenon is also modeled during proposing quadrupole well potential levels in the neuron systems. This mechanism therefore points to the microprocessor behavior of neuron networks. Stimulation of the neuronal system based on this mechanism, not only controls the sensitivity of neuron electrical stimulation, but also would open a light window for more efficient operating the neuronal connectivity during providing interruptions by phenomena such as neurolysis as well as an efficient treatment of neuron-based disorders.

Blue light exposure enhances neural efficiency of the task positive network during a cognitive interference task.

Exposure to light, particularly blue-wavelength light, has been shown to acutely increase brain activation, alertness, and some elementary aspects of cognitive performance such as working memory and emotional anticipation. Whether blue light exposure can have effects on brain activation and performance during more complex cognitive control tasks up to 30 min after light cessation is unknown. In a sample of 32 healthy adults, we examined the effects of a 30 min exposure to either blue (n = 16) or amber control (n = 16) light on subsequent brain activation and performance during the Multi-Source Interference Task (MSIT) measured a half-hour after light exposure. Performance on the MSIT did not differ between the blue and amber conditions. However, brain activation within the task positive network (TPN) to the interference condition was significantly lower in the blue relative to the amber condition, while no group differences were observed for suppression of the default mode network (DMN). These findings suggest that, compared to control, a single exposure to blue light was associated with enhanced neural efficiency, as demonstrated by reduced TPN activation to achieve the same level of performance. Blue light may be an effective method for optimizing neurocognitive performance under some conditions.

Novel in vitro assay to investigate radiation induced changes in the functionality of human embryonic stem cell-derived neurospheres.

Ionizing radiation (IR) is increasingly used for diagnostics and therapy of severe brain diseases. However, IR also has adverse effects on the healthy brain tissue, particularly on the neuronal network. This is true for adults but even more pronounced in the developing brain of unborn and pediatric patients. Epidemiological studies on children receiving radiotherapy showed an increased risk for cognitive decline ranging from mild deficits in academic functioning to severe late effects in intellectual ability and language as a consequence of altered neuronal development and connectivity. To provide a comprehensive approach for the analysis of radiation-induced alterations in human neuronal functionality, we developed an in vitro assay by combining microelectrode array (MEA) analyses and human embryonic stem cell (hESC) derived three-dimensional neurospheres (NS). In our proof of principle study, we irradiated hESC with 1 Gy X-rays and let them spontaneously differentiate into neurons within NS. After the onset of neuronal activity, we recorded and analyzed the activity pattern of the developing neuronal networks. The network activity in NS derived from irradiated hESC was significantly reduced, whereas no differences in molecular endpoints such as cell proliferation and transcript or protein expression analyses were found. Thus, the combination of MEA analysis with a 3D model for neuronal functionality revealed radiation sequela that otherwise would not have been detected. We therefore strongly suggest combining traditional biomolecular methods with the new functional assay presented in this work to improve the risk assessment for IR-induced effects on the developing brain.

Spatiotemporal constraints on optogenetic inactivation in cortical circuits.

Optogenetics allows manipulations of genetically and spatially defined neuronal populations with excellent temporal control. However, neurons are coupled with other neurons over multiple length scales, and the effects of localized manipulations thus spread beyond the targeted neurons. We benchmarked several optogenetic methods to inactivate small regions of neocortex. Optogenetic excitation of GABAergic neurons produced more effective inactivation than light-gated ion pumps. Transgenic mice expressing the light-dependent chloride channel GtACR1 produced the most potent inactivation. Generally, inactivation spread substantially beyond the photostimulation light, caused by strong coupling between cortical neurons. Over some range of light intensity, optogenetic excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks ('paradoxical effect'). The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, limiting temporal resolution. Our data offer guidance for the design of in vivo optogenetics experiments.

Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity.

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, we assayed behavior as well as molecular rhythms within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. We also generated CRISPR-mediated neuron-specific circadian clock knockouts. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.

Integration of Neurons Derived from Transplanted BDNF-Overexpressing Neural Stem Cells into Circuit of the Irradiated Hippocampus.

Stem cell transplantation is thought to be an effective method for radiation-induced cognitive dysfunction. However, there have been few studies performed to determine whether transplanted stem cells can integrate into hippocampus circuits. Brain-derived neurotrophic factor (BDNF) plays a critical role in brain development. Therefore, we investigated the differentiation and integration of brain-derived neurotrophic factor overexpressing neural stem cells (NSCs). We observed that these transplanted cells migrated to the subgranular zone of irradiated rats at 4 weeks after transplantation. However, control neural stem cells were disordered, distributing in the irradiated hippocampus, and showed greater astroglia differentiation tendency. Retrograde monosynaptic tracing showed that neurons derived from transplanted brain-derived neurotrophic factor overexpressing neural stem cells integrated into the circuit better than those from control cells. Brain-derived neurotrophic factor overexpressing neural stem cells s promoted the expression of brain-derived neurotrophic factor and nerve growth factor and reduced the number of activated microglia caused by radiation. Transplanted brain-derived neurotrophic factor overexpressing neural stem cells failed to improve radiation-induced cognitive dysfunction. These results indicate that brain-derived neurotrophic factor overexpressing neural stem cells suffered less from changed microenvironment after irradiation and possessed the ability to improve the host niche. Neurons derived from Brain-derived neurotrophic factor overexpressing neural stem cells showed the integration potency in the irradiated hippocampus.

Multi-site dynamic recording for Aß oligomers-induced Alzheimer's disease in vitro based on neuronal network chip.

Alzheimer's disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-ß oligomers (AßOs) and the intracellular accumulation of neurofibrillary tangles. However, due to the lack of effective method and experimental models to study the cognitive decline, communication at cell resolution and the implementation of interventions, the diagnosis and treatment on AD still progress slowly. In this paper, we established a pathological model of AD in vitro based on AßOs-induced hippocampal neuronal network chip for multi-site dynamic analysis of the neuronal electrical activity and network connection. The multiple characteristic parameters, including positive and negative spike intervals, firing rate and peak-to-peak values, were extracted through the analysis of spike signals, and two firing patterns from the interneurons and pyramidal neurons were recorded. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. Moreover, an electrical stimulation with frequency at 40 Hz was exerted to preliminarily explore the therapeutic effect on the pathological model of AD. This neuronal network chip enables the implementation of AD models in vitro for studying basic mechanisms of neurodegeneration within networks and for the parallel testing of various potential therapies. It can be a novel technique in the research of AD pathological model in vitro.

Inhibition of cortical neural networks using infrared laser.

The aim of the present study is to optimize parameters for inhibiting neuronal activity safely and investigating thermal inhibition of rat cortex neural networks in vitro by continuous infrared (IR) laser. Rat cortex neurons were cultured on multi-electrode arrays until neural networks were formed with spontaneous neural activity. Neurons were then irradiated to inhibit the activity of the networks using different powers of 1550 nm IR laser light. A finite element heating model, calibrated by the open glass pipette method, was used to calculate temperature increases at different laser irradiation intensities. A damage signal ratio (DSR) was evaluated to avoid excessive heating that may damage cells. The DSR predicted that cortex neurons should be safe at temperatures up to 49.6°C for 30 seconds, but experiments suggested that cortex neurons should not be exposed to temperatures over 46°C for 30 seconds. Neural response experiments showed that the inhibition of neural activity is temperature dependent. The normal neural activity could be inhibited safely with an inhibition degree up to 80% and induced epileptiform activity could be suppressed. These results show that continuous IR laser radiations provide a possible way to safely inhibit the neural network activity.

Modulation of resting-state brain functional connectivity by exposure to acute fourth-generation long-term evolution electromagnetic field: An fMRI study.

By now, the neurophysiological effect of electromagnetic field (EMF) exposure and its underlying regulating mechanisms are not well manifested. In this study, we aimed to investigate whether acute long-term evolution (LTE) EMF exposure could modulate brain functional connectivity using regional homogeneity (ReHo) method and seed-based analysis on resting-state functional magnetic resonance imaging (fMRI). We performed the LTE-EMF exposure experiment and acquired the resting-state brain activities before and after EMF exposure. Then we applied ReHo index to characterize the localized functional connectivity and seed-based method to evaluate the inter-regional functional connectivity. Statistical comparisons were conducted to identify the possible evidence of brain functional connectivity modulation induced by the acute LTE-EMF exposure. We found that the acute LTE-EMF exposure modulated localized intra-regional connectivity (p < 0.05, AlphaSim corrected, voxel size ≥ 18) and inter-regional connectivity in some brain regions (p < 0.05, AlphaSim corrected, voxel size ≥ 18). Our results may indicate that the approaches relying on network-level inferences could provide deeper insight into the acute effect on human functional activity induced by LTE-EMF exposure. Bioelectromagnetics. 40:42-51, 2019. © 2018 Wiley Periodicals, Inc.

Astrocytes integrate and drive action potential firing in inhibitory subnetworks.

Many brain functions depend on the ability of neural networks to temporally integrate transient inputs to produce sustained discharges. This can occur through cell-autonomous mechanisms in individual neurons and through reverberating activity in recurrently connected neural networks. We report a third mechanism involving temporal integration of neural activity by a network of astrocytes. Previously, we showed that some types of interneurons can generate long-lasting trains of action potentials (barrage firing) following repeated depolarizing stimuli. Here we show that calcium signaling in an astrocytic network correlates with barrage firing; that active depolarization of astrocyte networks by chemical or optogenetic stimulation enhances; and that chelating internal calcium, inhibiting release from internal stores, or inhibiting GABA transporters or metabotropic glutamate receptors inhibits barrage firing. Thus, networks of astrocytes influence the spatiotemporal dynamics of neural networks by directly integrating neural activity and driving barrages of action potentials in some populations of inhibitory interneurons.

Orchestrating cells on a chip: Employing surface acoustic waves towards the formation of neural networks.

For the investigation of cell-cell interaction in general and for neural communication and future applications of neural networks, a controllable and well-defined network structure is crucial. We here propose the implementation of an acoustically driven system for tunable and deliberate stimulation and manipulation of cell growth on a chip. This piezoelectric chip allows us to generate a checkerboard-like standing surface acoustic wave pattern coupled to a fluid layer in a microfluidic chamber on top. Such a dynamically induced patterning lattice is shown to allow for the active positioning of the neurons and subsequent guided neurite outgrowth, thus finally overcoming the limitations of static approaches. After thorough characterization of the resulting tunable potential landscape, we successfully demonstrate cell adhesion and even growth of the such positioned cells within the well-defined pressure nodes. We demonstrate neuron growth at predetermined positions and observe a subsequent neurite outgrowth, even being correlated with the artificial potential landscape. For the very delicate and sensitive primary neural cells, this is a change of paradigm! Our experimental findings give us confidence that our hybrid lab-on-a-chip system in the near future will allow researchers to study cell-cell interaction of primary neurons. If scaled to a true network level, it will enable us to control and study how neural networks connect, interact, and communicate.

Focused ultrasound induced opening of the blood-brain barrier disrupts inter-hemispheric resting state functional connectivity in the rat brain.

Focused ultrasound (FUS) is a technology capable of delivering therapeutic levels of energy through the intact skull to a tightly localized brain region. Combining the FUS pressure wave with intravenously injected microbubbles creates forces on blood vessel walls that open the blood-brain barrier (BBB). This noninvasive and localized opening of the BBB allows for targeted delivery of pharmacological agents into the brain for use in therapeutic development. It is possible to use FUS power levels such that the BBB is opened without damaging local tissues. However, open questions remain related to the effects that FUS-induced BBB opening has on brain function including local physiology and vascular hemodynamics. We evaluated the effects that FUS-induced BBB opening has on resting state functional magnetic resonance imaging (rs-fMRI) metrics. Data from rs-fMRI was acquired in rats that underwent sham FUS BBB vs. FUS BBB opening targeted to the right primary somatosensory cortex hindlimb region (S1HL). FUS BBB opening reduced the functional connectivity between the right S1HL and other sensorimotor regions, including statistically significant reduction of connectivity to the homologous region in the left hemisphere (left S1HL). The effect was observed in all three metrics analyzed: functional connectivity between anatomically defined regions, whole brain voxel-wise correlation maps based on anatomical seeds, and spatial patterns from independent component analysis. Connectivity metrics for other regions where the BBB was not perturbed were not affected. While it is not clear whether the effect is vascular or neuronal in origin, these results suggest that even safe levels of FUS BBB opening have an effect on the physiological processes that drive the signals measured by BOLD fMRI. As such these effects must be accounted for when carrying out studies using fMRI to evaluate the effects of pharmacological agents delivered via FUS-induced BBB opening.

Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice.

Of the many perils associated with deep space travel to Mars, neurocognitive complications associated with cosmic radiation exposure are of particular concern. Despite these realizations, whether and how realistic doses of cosmic radiation cause cognitive deficits and neuronal circuitry alterations several months after exposure remains unclear. In addition, even less is known about the temporal progression of cosmic radiation-induced changes transpiring over the duration of a time period commensurate with a flight to Mars. Here we show that rodents exposed to the second most prevalent radiation type in space (i.e. helium ions) at low, realistic doses, exhibit significant hippocampal and cortical based cognitive decrements lasting 1 year after exposure. Cosmic-radiation-induced impairments in spatial, episodic and recognition memory were temporally coincident with deficits in cognitive flexibility and reduced rates of fear extinction, elevated anxiety and depression like behavior. At the circuit level, irradiation caused significant changes in the intrinsic properties (resting membrane potential, input resistance) of principal cells in the perirhinal cortex, a region of the brain implicated by our cognitive studies. Irradiation also resulted in persistent decreases in the frequency and amplitude of the spontaneous excitatory postsynaptic currents in principal cells of the perirhinal cortex, as well as a reduction in the functional connectivity between the CA1 of the hippocampus and the perirhinal cortex. Finally, increased numbers of activated microglia revealed significant elevations in neuroinflammation in the perirhinal cortex, in agreement with the persistent nature of the perturbations in key neuronal networks after cosmic radiation exposure. These data provide new insights into cosmic radiation exposure, and reveal that even sparsely ionizing particles can disrupt the neural circuitry of the brain to compromise cognitive function over surprisingly protracted post-irradiation intervals.

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full-field light of various intensities. Light-evoked membrane potential changes (ΔVm ) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark-adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage-gated Ca2+ influx (QCa ) and consequent glutamate release. Stimulus-evoked glutamate release was quantified by the increase in membrane capacitance (ΔCm ). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔVm /QCa ratio equally at a given light intensity and inhibition did not alter the overall relation between QCa and ΔCm . However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔCm unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non-linear relationship between QCa and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.

Magnetothermal genetic deep brain stimulation of motor behaviors in awake, freely moving mice.

Establishing how neurocircuit activation causes particular behaviors requires modulating the activity of specific neurons. Here, we demonstrate that magnetothermal genetic stimulation provides tetherless deep brain activation sufficient to evoke motor behavior in awake mice. The approach uses alternating magnetic fields to heat superparamagnetic nanoparticles on the neuronal membrane. Neurons, heat-sensitized by expressing TRPV1 are activated with magnetic field application. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. This approach provides genetically and spatially targetable, repeatable and temporarily precise activation of deep-brain circuits without the need for surgical implantation of any device.

Altered Network Topology in Patients with Primary Brain Tumors After Fractionated Radiotherapy.

Radiation therapy (RT) is a critical treatment modality for patients with brain tumors, although it can cause adverse effects. Recent data suggest that brain RT is associated with dose-dependent cortical atrophy, which could disrupt neocortical networks. This study examines whether brain RT affects structural network properties in brain tumor patients. We applied graph theory to MRI-derived cortical thickness estimates of 54 brain tumor patients before and after RT. Cortical surfaces were parcellated into 68 regions and correlation matrices were created for patients pre- and post-RT. Significant changes in graph network properties were tested using nonparametric permutation tests. Linear regressions were conducted to measure the association between dose and changes in nodal network connectivity. Increases in transitivity, modularity, and global efficiency (n = 54, p < 0.0001) were all observed in patients post-RT. Decreases in local efficiency (n = 54, p = 0.007) and clustering coefficient (n = 54, p = 0.005) were seen in regions receiving higher RT doses, including the inferior parietal lobule and rostral anterior cingulate. These findings demonstrate alterations in global and local network topology following RT, characterized by increased segregation of brain regions critical to cognition. These pathological network changes may contribute to the late delayed cognitive impairments observed in many patients following brain RT.

Selective Modulation of the Pupil Light Reflex by Microstimulation of Prefrontal Cortex.

The prefrontal cortex (PFC) is thought to flexibly regulate sensorimotor responses, perhaps through modulating activity in other circuits. However, the scope of that control remains unknown: it remains unclear whether the PFC can modulate basic reflexes. One canonical example of a central reflex is the pupil light reflex (PLR): the automatic constriction of the pupil in response to luminance increments. Unlike pupil size, which depends on the interaction of multiple physiological and neuromodulatory influences, the PLR reflects the action of a simple brainstem circuit. However, emerging behavioral evidence suggests that the PLR may be modulated by cognitive processes. Although the neural basis of these modulations remains unknown, one possible source is the PFC, particularly the frontal eye field (FEF), an area of the PFC implicated in the control of attention. We show that microstimulation of the rhesus macaque FEF alters the magnitude of the PLR in a spatially specific manner. FEF microstimulation enhanced the PLR to probes presented within the stimulated visual field, but suppressed the PLR to probes at nonoverlapping locations. The spatial specificity of this effect parallels the effect of FEF stimulation on attention and suggests that FEF is capable of modulating visuomotor transformations performed at a lower level than was previously known. These results provide evidence of the selective regulation of a basic brainstem reflex by the PFC.SIGNIFICANCE STATEMENT The pupil light reflex (PLR) is our brain's first and most fundamental mechanism for light adaptation. Although it is often described in textbooks as being an immutable reflex, converging evidence suggests that the magnitude of the PLR is modulated by cognitive factors. The neural bases of these modulations are unknown. Here, we report that microstimulation in the prefrontal cortex (PFC) modulates the gain of the PLR, changing how a simple reflex circuit responds to physically identical stimuli. These results suggest that control structures such as the PFC can add complexity and flexibility to even a basic brainstem circuit.

Cell diversity and network dynamics in photosensitive human brain organoids.

In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.